The mul ikinase inhibi o So a enib
enhances glycolysis and syne gizes wi h
glycolysis blockade o cance cell killing
Valen ina Teso i
1
, Anna Chia a Piscaglia
1
, Daniela Samengo
2
, Ma a Ba ba
3
, Camilla Be na dini
3
,
Robe o Sca ena
4
, Alessand o Pon oglio
4
, Lau a Cas ellini
5
, Johannes N. Spelb ink
6
, Giuseppe Maulucci
7
,
Ma ia Ausilia ice Puglisi
1
, Gio amba is a Pani
2
& An onio Gasba ini
1
1
Ins i u e o In e nal Medicine and Gas oen e ology, Ca holic Uni e si y o he Sac ed Hea School o Medicine,
2
Ins i u e o
Gene al Pa hology, Labo a o y o Cell Signaling, Ca holic Uni e si y o he Sac ed Hea School o Medicine,
3
Ins i u e o Human
Ana omy and Cell Biology, Ca holic Uni e si y o he Sac ed Hea School o Medicine,
4
Ins i u e o Biochemis y and Clinical
Biochemis y, Ca holic Uni e si y o he sac ed Hea School o Medicine,
5
Depa men o Radia ion Oncology, Cen e o Clinical
Sciences Resea ch, S an o d Uni e si y School o Medicine, S an o d, CA 94305, USA,
6
Depa men o Pedia ics, Nijmegen Cen e
o Mi ochond ial Diso de s, Radboud Uni e si y Medical Cen e, Gee G oo eplein 10, P.O. Box 9101, 6500 HB Nijmegen, The
Ne he lands; Ins i u e o Biomedical Technology & Tampe e Uni e si y Hospi al, Pi kanmaa Hospi al Dis ic , Uni e si y o Tampe e,
FI-33014, Finland,
7
Ins i u e o Physics, Ca holic Uni e si y o he Sac ed Hea School o Medicine.
Al hough he only e ec i e d ug agains p ima y hepa oca cinoma, he mul ikinase inhibi o So a enib
(SFB) usually ails o e adica e li e cance . Since SFB a ge s mi ochond ia, cell me abolic ep og amming
may unde lie in insic umo esis ance. To cha ac e ize cance cell me abolic esponse o SFB, wemeasu ed
oxygen consump ion, gene a ion o eac i e oxygen species (ROS) and ATP con en in a LCSC (Li e
Cance S em Cells) -2 cells exposed o he d ug. Genome wide analysis o gene exp ession was pe o med by
A yme ix echnology. SFB cy o oxici y was e alua ed by mul iple assays in he p esence o absence o
me abolic inhibi o s, o in cells gene ically deple ed o mi ochond ia. We ound ha low concen a ions
(2.5–5 mM) o SFB had a ela i ely modes e ec on LCSC-2 o 293 T cell g ow h, bu damaged
mi ochond ia and inc eased in acellula ROS. Gene exp ession p o iling o SFB- ea ed cells was consis en
wi h a shi owa d ae obic glycolysis and, acco dingly, SFB cy o oxici y was d ama ically inc eased by
glucose wi hd awal o he glycoly ic inhibi o 2-DG. Unde me abolic s ess, ac i a ion o he AMP
dependen P o ein Kinase (AMPK), bu no ROS blockade, p o ec ed cells om dea h. We conclude ha
mi ochond ial damage and ROS d i e cell killing by SFB, while glycoly ic cell ep og amming may ep esen
a esis ance s a egy po en ially a ge able by combina ion he apies.
Ae obic glycolysis (‘‘Wa bu g e ec ’’) ep esen s one o he dis inc i e ac s (‘‘hallma ks’’) o he malignan
pheno ype
1–4
. Al hough ene ge ically less e icien han espi a ion, e men a i e me abolism is ad an -
ageous o cell g ow h due o he inc eased a ailabili y o anabolic in e media es and he educed cell
dependence on oxygen; mo eo e , by inc easing in acellula educing equi alen s (NADPH and glu a hione)
and dec easing mi ochond ia-de i ed ROS, glycolysis p o ec s malignan cells om oxidan -induced senescence
and apop osis
5
and con ibu es o he su i al o Cance S em Cell (CSC)
6
. Biochemical di e ences be ween
cance ous and no mal cells may help di ec ing a ge ed he apies agains malignan elemen s. Fo ins ance, umo
cells a e o en s ongly dependen on glucose (‘‘glucose-addic ed’’) and he e o e exquisi ely sensi i e o he
glycoly ic inhibi o 2-deoxyglucose (2DG)
7
. No ably, he link be ween me abolism, oxida i e s ess and cance
may be pa icula ly ele an o he li e
8
, ha plays a pi o al ole in he egula ion o glucose homeos asis. Hence
li e cance cells, like he hepa ocholangioca cinoma cell line LCSC-2 we’ e ecen ly de i ed om a no el model
o ca cinogenesis in a s
9
, appea ideally sui ed o in es iga e biochemical mechanisms and he apeu ic implica-
ions o cance cell me abolic ep og amming.
The mul ikinase inhibi o So a enib (SFB) (Nexa a , BAY 43-9006) cu en ly ep esen s he p ima y ea men
op ion o ad anced hepa ocellula ca cinoma
10
; SFB p e e en ially inhibi s he cance - associa ed V600E mu an
o he se ine- h eonine kinase and Ras-e ec o BRAF, while he wild ype enzyme is pa adoxically ac i a ed by
he d ug in he p esence o ac i e Ras signaling
11
; SFB also a ge s, a concen a ions in he high nanomola ange,
a numbe o Recep o Ty osine Kinases (RTK
S
) including, Pla ele De i ed G ow h Fac o – b(PDGFR-b),
OPEN
SUBJECT AREAS:
PRECLINICAL RESEARCH
TARGETED THERAPIES
NUTRIENT SIGNALLING
STRESS SIGNALLING
Recei ed
11 Augus 2014
Accep ed
20 Feb ua y 2015
Published
17 Ma ch 2015
Co espondence and
eques s o ma e ials
should be add essed o
G.P. (gpani@ m.
unica .i )
SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 1
Vascula Endo helial G ow h Fac o -2 (VEGFR-2), and Vascula
Endo helial G ow h Fac o -2 (VEGFR-3)
12
. Howe e , addi ional
mechanisms likely con ibu e o he ele a ed an icance ac i i y o
his compound, and may ha e by ex ension a ole in he equen
eme gence o speci ic chemo esis ance
13
.
Ini ial e idence poin o mi ochond ial damage and oxida i e
s ess as addi ional, kinase-independen mechanisms unde lying cell
esponse o So a enib. In no mal ca diomyocy es, o ins ance, SFB
was epo ed o inhibi mi ochond ial espi a ion and o dec ease
in acellula ATP le els
14
. Along simila lines, SFB has been shown o
inc ease he p oduc ion o mi ochond ial ROS (mROS), dec ease
educed Glu a hione le els (GSH) and induce cell dea h in HepG2
human hepa oma cells
15
, and se um le els o ad anced oxida ion
p o ein p oduc s in So a enib- ea ed HCC pa ien s co ela e wi h
clinical e ec i eness o he d ug
16
. Addi ionally, in human panc ea ic
cell lines SFB elici s MEK/ERK independen apop osis, h ough he
down egula ion o he mi ochond ial an iapop o ic p o ein Mcl-1
17
.
P omp ed by hese e idence and by he eme ging in e es owa ds
me abolism- a ge ed an icance he apies, we sough o in es iga e
he e ec o So a enib on mi ochond ial ac i i y and oxida i e me a-
bolism in a hepa ocolangioc cinoma LCSC-2 cells, in sea ch o
no el mechanisms o esponse and/o esis ance o li e cance cells
o his inc easingly used d ug.
Resul s
So a enib inc eases in acellula ROS and inhibi s espi a ion in
LCSC-2 cells.Sensi i i y o umo cell lines o RTKs inhibi o s is
highly a iable, in pa depending on he mu a ional s a us o RAS
and RAF amily membe s
18
. Exposu e o a hepa ocolangioca cinoma
LCSC-2 cells, ha lack B-RAF ac i a ing mu a ions, o SFB had a
modes g ow h inhibi o y e ec as assessed by P opidium Iodide
(PI) exclusion o colony o ma ion assay (Fig. 1, a, b and c),
especially in he p esence o e al bo ine se um: in ac , unlike
epo ed o higly sensi i e cell lines
12
, 50% inhibi ion was a ained
in he low mic omola , a he han nanomola ange. O no e, in his
ange no ob ious educ ion o phospho yla ed (ac i e) ERK and
AkT, was obse ed unde cell s imula ion wi h Hepa ocy e g ow h
Fac o (HGF), sugges ing a g ow h inhibi o y mechanism dis inc
om RTK o ERK blockade (Fig. 1, d).
Consis en wi h p e ious epo s
15,16
, low cy ome y analysis o cells
loaded wi h he edox-sensi i e dye H2-DCF-DA e ealed a ma ked
and dose dependen inc ease o ROS 12 hou s a e exposu e o 2,5 o
5mM SFB (Fig. 1 e and 1 g); LCSC-2 cells o en appea ed dis ibu ed
in wo dis inc subpopula ion peaks based on DCF-DA luo escence
in ensi y, bo h o which we e shi ed o he igh (inc eased oxida ion)
upon exposu e o he d ug (Fig. 1e). Impo an ly, he p o-oxidan
e ec o SFB was cancelled by he cell pe mean hyd ogen pe oxide
sca enge and GPX mime ic Ebselen (EBS) (Fig. 1 ).
Since mi ochond ia ep esen he main sou ces o ROS in no mal
and in umo cells, we easoned ha ROS inc ease may e lec an e ec
o SFB on hese o ganelles. Acco dingly, baseline oxygen consump ion
( ou ine espi a ion) measu ed by High Resolu ion Respi ome y was
signi ican ly educed a e 12 hou s incuba ion wi h SFB, compa ed o
un ea ed con ols (Fig. 2 a), and a simila di e ence was obse ed
unde maximum elec on low, as elici ed by he mi ochond ial p o-
onopho e Ca bonyl cyanide p-( i- luo ome hoxy) phenyl-hyd azone
(FCCP), (Fig. 2 b).
In o de o add ess whe he SFB ac s di ec ly on mi ochond ia, we
isola ed he o ganelles om a li e and assessed hei ansmem-
b ane po en ial in he p esence o he d ug, using he ca ionic luo -
escen dye JC-1 ollowed by low cy ome y. As shown in Fig. 2, c,
SFB was able o depola ize mi ochond ia in i o, as e ealed by an
e iden educ ion o JC-1 luo escence in he FL2 ( ed) channel.
Taken oge he , hese indings con i m ha in LCSC-2 cells, as in
o he cell models, SFB in e e es wi h mi ochond ial unc ion, and
sugges ha his e ec is due, a leas in pa , o a di ec in e ac ion o
he d ug wi h he o ganelle.
So a enib a ec s ene gy me abolism o LCSC2 cells.To u he
cha ac e ize he impac o SFB on cellula ene gy me abolism, we
i s measu ed in acellula ATP a e wel e hou s cell exposu e o
he d ug. ATP le els we e ma kedly educed (.50%) in SFB- ea ed
cells compa ed o un ea ed con ols (Fig. 3 a), indica ing ha LCSC-2
cells ac i ely u ilize mi ochond ia o hei ene gy supply. In keeping
wi h he abo e inding, SFB elici ed he phospho yla ion on Th eonine
172 o he AMP ac i a ed p o ein kinase (AMPK), an ene gy senso
ha de ec s changes in he in acellula AMP/ATP and igge s
me abolic cell adap a ions aimed a es o ing ATP le els a expense
o anabolic p ocesses (Fig. 3 b). SFB phospho yla ion o AMPK was
po en ia ed by he glycoly ic inhibi o 2DG (Fig. 3 c and Supplemen a y
Figu e S5), and was no inhibi ed by Ebselen (Fig. 3 c), al hough AMPK
ac i a ion may in some con ex s espond o ROS
19
.
To gain insigh in he global me abolic cell esponse o So a enib, we
pe o med mic oa ay analysis o gene exp ession. A o al o 322 di -
e en ially-exp essed genes wi h $1,5 old change we e iden i ied in
LCSC-2 exposed o he d ug. Among hese, 174 genes esul ed up-
egula ed and 148 genes we e down- egula ed in LCSC-2 ea ed wi h
SFB compa ed o un ea ed con ols (da a accessible a h p://www.
ncbi.nlm.nih.go /geo/ unde he ID numbe GSE43053). O no e, mono-
sacca ide me abolism (p 50.00462) and cell p oli e a ion (p 5
0.00631) we e among he highes anking biological p ocesses en iched
o di e en ly exp essed genes (Supplemen a y Fig. S1 on line).
We ocused on genes in ol ed in glucose me abolism. In e es ingly,
he exp ession o h ee genes di ec ly in ol ed in glycolysis [ he solu e
ca ie amily 2 (slc2a3), Enolase 2 (eno2), and he pla ele phospho-
uc okinase (p kp)], was signi ican ly induced by SFB (Fig. 3 d); con-
e sely, SFB dec eased he exp ession o acquapo in 9 (aqp9), membe
o a amily o wa e -selec i e memb ane channels, and o he mi-
ochond ial enzyme py u a e dehyd ogenase (lipoamide) alpha 1
(pdha1), which ca alyzes he i e e sible con e sion o py u a e o
ace yl-CoA, hus linking ae obic glycolysis wi h he ica boxylic acid
(TCA) cycle in mi ochond ia. Di e ences in he exp ession le els o
selec ed genes we e u he alida ed by quan i a i e eal ime PCR
(qPCR) (Fig. 3 e). Mo eo e , consis en wi h me abolic shi owa ds
glycolysis in esponse o SFB, sugges ed by he abo e ansc ip ional
changes, we obse ed enhanced up ake o he luo escen glucose
analog 6NDBG, and inc eased glucose consump ion and L-lac a e
elease by LCSC-2 cells exposed o he d ug (Fig. 3 , g and h).
So a enib oxici y is inc eased by he glycoly ic inhibi o 2-deoxy-
glucose.The abo e indings p op ed us o es whe he he e ec s o
SFB could be enhanced by a glycolysis inhibi o , such as he non-
me abolizable glucose analogue 2-deoxy-glucose (2DG).
In oxici y assays (PI exclusion and MTT), 2DG d ama ically
inc eased cell killing by SFB bo h in LCSC-2 cells (Fig. 4 a and b)
and in he highly malignan mu ine melanoma cell line B16F10
(Fig. 4 c); a simila e ec was elici ed by glucose wi hd awal om
he cul u e medium (Supplemen a y Fig. S2 on line). O no e, he
MEK inhibi o PD98059 had ma ginal e ec s on LCSC-2 cells, ha
was no enhanced by glucose wi hd awal (Supplemen a y Fig S2 on
line). Ins ead, in B16F10 cells, he e ec o So a enib was mimicked
by he mi ochond ial Complex I inhibi o Ro enone
20
(Fig. 4 c);
mo eo e , deple ion o mi ochond ial DNA in a HEK293 cell line
a ian exp essing a doxycycline inducible dominan nega i e mu an
o he mi ochond ial DNA polyme ase gamma 1 (POLG1 D890N)
21
nea ly ab oga ed sensi i i y o SFB ( ig. 4d), u he poin ing o a
mi ochond ial ac ion o he d ug. The syne gis ic e ec o SFB and
2DG o cell killing was also con i med in a numbe o addi ional
human and mu ine cance cell lines, including Ras- ans o med
Mouse Emb yonic Fib oblas s (MEF) lacking p53 (Supplemen a y
Fig. S3 on line).
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 2
AMPK links me abolic damage by SFB o he mTOR/au ophagy
cascade.To u he cha ac e ize he biochemical e en s elici ed
by SFB and i s combina ion wi h 2DG, we in e oga ed nu ien
signalling e en s along he AMPK kinase pa hway. In LCSC2 (Fig. 5
a and Supplemen a y Fig. S5) and B16F10 cells (Supplemen a y Fig.
S4) SFB and 2DG combina o ially and dose-dependen ly inc eased he
phospho yla ion o AMPK on Se 172, consis en wi h p o ound cell
de-ene giza ion. E en mo e d ama ically he combina ion inhibi ed
he phospho yla ion o he ibosomal S6 p o ein, a downs eam
e ec o o he mTOR (mammalian Ta ge o Rapamycin)/S6 Kinase
cascade ha is nega i ely egula ed by AMPK unde nu ien
dep i a ion
22
. Mo eo e , Simila s udies pe o med on HEK293-
POLG1 (D890N) cells showed ha AMPK phospho yla ion by SFB
is a ou ed unde glycolysis blockade by ei he 2DG o glucose
dep i a ion, and ha i equi es ac i e mi ochond ia, as indica ed by
he lack o esponse o he d ug (o he p esence o an opposi e,
inhibi o y ac ion) in m DNA-deple ed cells (Fig. 5 d).
No iceably, as an addi ional ou pu o AMPK/mTOR signalling
modula ion in LCSC2 cells, SFB and SFB 12DG igge ed cell au op-
hagy, a sel -ea ing p ocess aimed a cell su i al unde s a a ion
23
;
his was e ealed, biochemically, by inc eased LC3B elec opho e ic
mobili y (due o co alen conjuga ion o his phagosome-associa ed
p o ein wi h phospha idyl-e hanolamine), and deg ada ion o he
au ophagy subs a e p62 (Fig. 5 a and Supplemen a y Fig. S5), and,
unc ionally, by an accele a ed au ophagic lux (i.e. au ophagosome-
au olysosome ansi ion), moni o ed by con ocal analysis o LCSC-2
cells ansien ly ans ec ed wi h a mRFP-GFP andem luo escen -
agged LC3
24
(Fig. 5 b and 5 c).
Figu e 1
|
G ow h inhibi ion and gene a ion o ROS in LCSC-2 cells exposed o So a enib. (a), (b) and (c) P opidium Iodide exclusion assay (a) and
colony o ma ion assay (b and c) showing dose and ime-dependen g ow h-inhibi o y e ec o SFB on LCSC-2 cells. FCS 5 e al cal se um. In b, ba s
ep esen pla ing e iciency (nucolonies/nuo pla ed cells); c: ep esen a i e pic u e o colonies s ained wi h Giemsa. In a and b ba s a e mean 6SD o
duplica e o iplica e samples. Panels ep esen a i e o se e al independen expe imen s. (d). Wes e n Blo analysis o Ak (Se 473) and p44-42 MAP
Kinase (ERK,Th 202/Ty 204) phospho yla ion a e 12 hou s incuba ion wi h he indica ed combina ions o Hepa ocy e G ow h ac o (HGF,
50 ng/ml) and SFB. An i o al ERK immune-s aining con i ms equal p o ein loading h oughou he lanes. Rele an bands a e indica ed by a ows.
(e). Rep esen a i e low cy ome y plo e ealing b oad dis ibu ion o H2-DCFDA luo escence in LCSC-2 cells and inc eased signal (oxida ion) in
esponse o 2.5 o 5 mM SFB. ( ). E ec o an ioxidan s and GPX mime ic Ebselen on SFB-induced ROS. A shi o cell luo escence p o ile o he le
con i ms e ec i e ROS sca enging by he compound. Plo s ep esen a i e o se e al independen analyses. (g). Quan i a ion o ROS inc ease in LCSC-2
cells exposed o 2.5 mM SFB o 12 hou s. Values a e Mean 6SD o mean luo escence a ios (SFB/Dmso) o e n 56 independen expe imen s. p
calcula ed on aw luo escence alues by pai ed wo- ailed - es .
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 3
LCSC-2 cell killing by SFB in ol es ROS and is coun e ac ed by
AMPK.In o de o cla i y he mechanis ic ole o ROS and phospho-
AMPK, bo h elici ed by SFB, in he cy o oxic ac i i y o he d ug, we
i s e alua ed killing e iciency in LCSC2 cells p e- ea ed wi h he
ROS sca enge Ebselen. The an ioxidan signi ican ly educed cell
dea h in he p esence o 5 and 10 mM SFB (p ,0.01) (Fig. 6 a and
Supplemen a y Fig. 6) con i ming SFB-induced oxida i e damage as
a necessa y con ibu o o he d ug cy o oxic ac ion. Acco dingly,
when LCSC-2 wi h high and low baseline le els o ROS (as e ealed
by he luo escen dye DCF-DA) we e sepa a ed by luo escence-
ac i a ed cell so ing, he wo popula ion displayed di e en sensi i-
i y (sensi i e he o me , esis an he la e ) o he d ug (Fig. 6 b and
c). In e es ingly, howe e , Ebselen had no e ec on massi e cell killing
by he SFB 12DG combina ion (Fig. 6A and Supplemen a y Fig. 6),
implying a ROS independen oxic mechanisms (ene gy deple ion?)
o he la e syne gis ic e ec . On he o he hand, AMPK deple ion by
siRNA echnology (Fig. 6 d and e) po en ia ed cell killing by 5 mMSFB
bo h alone and in combina ion wi h 2DG (p ,0.05 and p ,0.01,
espec i ely), in pa allel wi h impai ed inhibi ion o mTOR/S6K
signaling (Fig. 6 e). A cell p o ec i e ole o AMPK in esponse o
SFB was u he co obo a ed by pha macological modula ion o
he kinase wi h he cell pe mean ac i a o 5-Aminoimidazole-4-
ca boxamide 1-b-D- ibo u anoside (AICAR, 5 mM, p ,0.05),
and he speci ic inhibi o Compound C (20 mM, p ,0.01). Thus,
AMPK ac i a ion likely con ibu es o esis ance o So a enib a leas
in LCSC-2 cells.
Discussion
Cance cell me abolism elies on a delica e balance be ween he
glycoly ic pa hway, conside ably ampli ied o p o ide o he needs
o a apid and in asi e g ow h, and oxida i e phospho yla ion,
which emains ac i e, o con ibu e o he umo ene gy needs
1,25
.
In e e ence wi h his unique me abolic se ing may open new pos-
sibili ies o a ge ed an icance he apy.
We ha e he e in es iga ed he me abolic e ec s o So a enib, a
mul ikinase inhibi o , epo ed o p e e en ially a ge mu an
(V600E) BRAF and a numbe o cance - ele an y osine kinase
ecep o s
12
. These e ec s, ha include inhibi ion o mi ochond ial
espi a ion, educed ATP p oduc ion, and ele a ion o in acellula
ROS, a e in many cell lines, including he a hepa ocolangioca ci-
noma cell line LCSC-2, la gely insu icien o d i e cell dea h; we he e
sugges ha such ine ec i eness is due, o an e icien cell ep og am-
ming owa ds ae obic glycolysis. The d ama ic inc ease o SFB cy o-
oxici y by glucose wi hd awal o he glycoly ic inhibi o 2DG clea ly
suppo s his iew, opening o he possibili y o employing SFB in
no el me abolism-based combina ion he apies. Impo an ly, SFB
po en ia ion by glycolysis blockade was no limi ed o li e -de i ed
cells, bu also ex ended o melanoma cells and o he cell ypes, and is
Figu e 2
|
So a enib inhibi s cellula espi a ion and depola izes isola ed mi ochond ia. (a) and (b). Respi ome ic Analysis o LCSC-2 cells exposed o
2, 6 and 12 hou s o 2.5 mM SFB o Dmso as ehicle con ol. The wo his og ams illus a e he inhibi o y e ec o he d ug on Rou ine Respi a ion (A),
and uncoupled espi a ion (B) (see me hods o expe imen al de ails). As e isks deno e s a is ical signi icance ( wo- ailed S uden - es , compa ed o
un ea ed con ol). (c). Flow cy ome y analysis o mi ochond ial s aining wi h he po en iome ic dye JC-1. Dec eased ed (FL-2) luo escence o
mi ochond ia exposed o SFB indica es loss o ans-memb ane po en ial (y). Plo ep esen a i e o wo independen expe imen s.
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 4
likely o be independen om he umo supp esso p53 (Supple-
men a y Fig. S3).
No ewo hy, signaling s udies (Fig. 1 d) indica e ha me abolic
ac ions o So a enib a e independen om inhibi ion o RTK signal-
ing, sugges ing ins ead a di ec in e ac ion o he compound wi h
mi ochond ia. Since mi ochond ia ep esen a no el p omising sub-
s a e o a ge ed d ugs
26
, molecula in e ac ions o SFB wi h his
o ganelle ce ainly dese e o be u he in es iga ed.
In keeping wi h he idea ha SFB me abolically ep og ams a ge
cells, mic oa ay da a a e consis en wi h a cell shi owa ds ae obic
glycolysis, wi h a signi ican up- egula ion o genes in ol ed in he
glycoly ic pa hway, such as scl2a3 (also known as GLUT-3, a glucose
anspo e
27
), p kp (Phospho uc okinase, pla ele iso o m
28
) and
eno2 (Enolase 2, a pa o he phosphopy u a e hyd a ase complex
29
)
(Supplemen a y Fig. S1). Impo an ly, all hese h ee genes ha e been
p e iously linked o human malignancy
30–32
. Con e sely, among
SFB-down egula ed genes ela ed o me abolism, he mi ochond ial
enzyme py u a e dehyd ogenase (lipoamide) alpha 1 (pdha1) ca a-
lyzes he i e e sible con e sion o py u a e o ace yl-CoA, linking
he glycolysis wi h he ica boxylic acid (TCA) cycle and espi a-
ion
33
. Thus, o e all, his gene signa u e poin s o a glycoly ic adap -
i e esponse o mi ochond ial ailu e, as i is also consis en wi h
e idence o enhanced glucose consump ion and lac a e p oduc ion
in SFB- ea ed LCSC-2 cells (Fig. 3D).
Glycolysis p o ides, beside ATP and anabolic in e media es, also
educing equi alen s ( h ough he Pen ose Phospha e Pa hway, PPP)
ha main ain he GSH/GSSG edox bu e , and inc eased glycolysis
may coun e ac SFB ac ion, in pa , by imp o ing he cell an ioxidan
capaci y
34
. In suppo o his iew, LCSC-2 cells wi h cons i u i ely
low le els o ROS a e in insically esis an , unlike cells wi h high
ROS, o he d ug ac ion (Fig. 6 b and c). In e es ingly, a simila Low-
ROS p o ile iden i ies pu a i e s em cells om no mal and umo
b eas cance cell popula ions
6
, sugges ing he possibili y ha esis -
ance o SFB and a low oxidan con en cha ac e izes a subse LCSC-2
o cells wi h he p ope ies o Cance S em Cells. By ex ension, es-
is ance o CSC o SFB may explain he limi ed cu a i e capaci y o
Figu e 3
|
So a enib modula es ene gy me abolism. (a). ATP measu emen in LCSC-2 cells ea ed wi h SFB. Values a e in ligh uni s no malized o
p o ein con en . Ba s a e Mean 6SD o duplica e sample. S a is ics a e by wo- ailed - es . His og am ep esen a i e o wo independen expe imen s.
(b). Immunode ec ion o phospho-(Th 172) AMPK phospho yla ion unde he indica ed s imuli; an i AMPK ( o al) immunoblo ing was used as
loading con ol. (c). An i p-AMPK immunoblo showing no inhibi o y e ec o Ebselen on AMPK phospho yla ion by SFB o SFB 12DG. To al AMPK
was used as loading con ol. Rele an bands a e indica ed by a ows. Pic u e ep esen a i e o a leas wo independen expe imen s. Black colums a e
Mean 6SD o densi ome ic alues om 2–3 independen expe imen s, no malized o a e age band indesi y. (d). Glycolysis- ela ed genes modula ed by
So a enib in LCSC-2 cells. Fold change e e s o he un ea ed (Dmso) sample. Up egula ed genes a e highligh ed in ed, down egula ed genes in g een.
(e). Real Time PCR alida ion o h ee selec ed genes om panel C, a. Fold change (SFB/Dmso) o each gene was calcula ed by he DC me hod
(see Supplemen a y Me hods); ba s a e mean 6SD o h ee independen eac ions. S a is ics a e by wo- ailed - es . ( ), (g) and (h). Ba g aphs displaying
enhanced 6NBDG up ake and inc eased glucose me abolism in LCSC-2 cells a e 48 hou s exposu e o SFB. Ba s a e Mean SD o 2–3 independen
samples. *p,0.05 (ANOVA); *** p,0.0001 ( - es ).
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 5

his d ug
10
.Wi h his espec i is no ewo hy ha glycolysis blockade
o e comes SFB esis ance in ashion ha is ROS-independen (Fig. 6
a and Supplemen a y Fig. 6) and he e o e ci cum en s, unlike SFB
alone, po en ial an ioxidan s-based su i al s a egies implemen ed
by cance (s em) cells.
LCSC-2 esis ance o So a enib also in ol es AMP kinase. Dose
dependen induc ion o AMPK phospho yla ion by SFB is mos
likely a consequence o cell de-ene giza ion, and appea s o be inde-
penden o ROS ( ig. 3 c) bu equi es ac i e mi ochond ia, as shown
by lack o esponse in HEK-293 cells g own in high glucose o de oid
o mi ochond ial DNA by a POLG1 dominan nega i e mu an ( ig. 5
e and Supplemen a y Fig. S5). Mo eo e , a p o ec i e ole o he
kinase agains SFB-induced cell dea h is s ongly sugges ed by expe i-
men s o gene ic and pha macologic inhibi ion o he enzyme ( ig. 6
d– ) and by co ela i e e idence o cons i u i e AMPK phospho yla-
ion and esis ance o SFB in HEK-293T POLG1 D890N cells ( igs. 4
d, 5e and Supplemn a y Fig. S5). Mul iple p o ec i e mechanisms,
including down egula ion o mTOR signaling wi h educed ATP
consump ion
35
, induc ion o au ophagy ( ig. 5 b and c), and gene ic
ep og amming (Supplemen a y Fig. S1) may accoun o his e ec .
While his aspec needs o be u he cla i ied, he abo e obse a ions
a e ansla ionally ele an o he possibili y o a ge ing AMPK in
combina ion wi h SFB o inc ease cell esponse o he d ug. Mo eo e ,
simila o o he mi ochond ia- a ge ing d ugs like Biguanides, umo
sensi i i y o SFB may be p edic ed by loss o unc ion o he umo
supp esso and AMPK ac i a ing kinase LKB1
36
, and boos ed by lim-
i ed glucose a ailabili y in he umo mass
37
. O no e, while his manu-
sc ip was in p epa a ion, i was epo ed ha SFB an i umo ac ion is
media ed by AMPK/mTOR in b eas cance cells
38
, indica ing ha
oles o AMPK downs eam o his d ug may be umo - and con ex -
dependen .
In conclusion, we ha e he e p o ided no el e idence o impo -
an me abolic e ec s o he mul ikinase inhibi o So a enib on li e
cance cells. Ou obse a ions, al hough wi h he in insic limi a ions
Figu e 4
|
So a enib oxici y is inc eased by he glycoly ic inhibi o 2-deoxy-glucose. (a). Cy o oxic e ec o SFB (5 mM) and 2DG (20 mM), applied as
single agen s o in combina ion, in LCSC-2 cells. Pe cen age o dead (P opidium Iodide-pe mean ) cells was de e mined by low cy ome y a e 24 hou s
exposu e o he d ugs. The dashed line indica es he expec ed alue o simple addi i i y, clea ed o backg ound cell dea h (Dmso), calcula ed by he
Bliss Independence Model (see Supplemen a y Me hods). A whi e ci cle in he SFB 12DG ba indica es he amoun o cell dea h en i ely a ibu able o
he d ug combina ion, clea ed o he backg ound. ** indica es p ,0.01 compa ed o ehicle; in e ac ion deno es a signi ican ‘‘cell ( ow 3column)
e ec ’’ in he wo-way ANOVA es . His og am ep esen a i e o se e al independen expe imen s (b). MTT Assay unde he same condi ions as in a. Ba s
a e mean 6SD o h ee independen expe imen s, each in duplica e. Line and s a is ics a e as in panel a. (c). E alua ion o SFB oxici y and SFB 12DG
in e ac ion in B16 mouse melanoma cells. Cells we e analysed o PI exclusion as in a, a e 24 hou s exposu e o he d ugs. Ro enone (Ro , 5 mM), mimics
he e ec o SFB (2.5 mM), and syne gizes wi h 2DG. Ba s a e mean 6SD o duplica e samples. *5p,0.05 and ** 5p,0.01 compa ed o Dmso.
S a is ics as in panel a. Panel ep esen a i e o wo independen expe imen s. (d) MTT assay showing lack o esponse o SFB (5 mM) o HEK293 –POLG1
(D890N) cells g own in doxycycline 50 ng/ml o 10 days o deple e m DNA (Dox1); Dox- a e non-induced cells ha e ain m DNA. Pic u e
ep esen a i e o wo independen expe imen s. S a is ics as in panel a.
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 6
o s udies in i o, indica e in mi ochond ia an impo an a ge o
he d ug’s ac ion, in ol e ROS in SFB-dependen cy o oxici y, and
iden i y mul iple (AMPK, me abolic ep og amming, an ioxidan
capaci y) po en ial mechanisms o d ug esis ance, o be ci cum-
en ed by no el and mo e e ec i e combina ion he apies.
Me hods
Reagen s and an ibodies.Mos o he Chemicals we e ob ained om Sigma-Ald ich
(Milan, I aly). So a enib (Nexa a , BAY 43-9006) was kindly p o ided by Baye
Pha maceu icals. An ibodies and siRNA a e lis ed in SI.
Plasmids.p LC3 (mRFP-GFP andem luo escen - agged LC3) was a gi om
Tamo su Yoshimo i (Addgene plasmid # 21074).
Cell lines and ans ec ions.The a hepa ocholangioca cinoma cell line LCSC-2 was
kindly p o ided by D . Thomas D. Shupe and D . B yon E. Pe e sen (Dep . o
Pa hology, Uni e si y o Flo ida, Gaines ille, FL). Cells we e main ained in
Dulbecco’s modi ied Eagle medium (DMEM 4.5 g/L d-glucose)/Ham’s F12 medium
50550, supplemen ed wi h 8% FBS and 1% an ibio ics (LCSC-medium). Flp-In
TM
T-
REx
TM
HEK-293 cells s ably exp essing he D890N dominan nega i e mu an o he
mi ochond ial Polyme ase gamma (POLG1) unde a Te acycline-inducible
p omo e ha e been p e iously desc ibed
21
. Cell we e ou inely main ained in
s anda d DMEM (4.5 g/L d-glucose). Induc ion o he ecombinan POLG1-myc
p o ein was eadily de ec able a e 24 hou s ea men wi h 50 ng/ml Doxycycline
(Sigma), and was main ained o 10 days in o de o se e ely deple e mi ochond ial
DNA. Addi ional cell lines a e desc ibed in SI.
T ans ec ion o cDNA and siRNA in o LCSC-2 cells was pe o med wi h
Lipo ec amineH2000 (Li e Technologies) and HiPe Fec (QIAGEN), espec i ely,
acco ding o he manu ac u e ’s ecommenda ions.
Cy o oxici y assays.Fo MTT assay, cells we e seeded in 24-well pla es (2 310
5
/well)
o 96 well pla es (5 310
4
/well) and challenged wi h SFB o he s imuli in se um- ee
medium.
A e 24 hou s o incuba ion MTT solu ion (1510 dilu ion o he 5 mg/ml s ock)
was added o cells and incuba ed a 37uC o h ee addi ional hou s. The MTT-
Figu e 5
|
AMPK links me abolic damage by SFB o he mTOR/au ophagy cascade. (a). Wes e n blo analysis o p o ein lysa es om LCSC-2 s imula ed
o 4–6 hou s wi h he indica ed combina ions o SFB and 2DG. Phospho yla ion o AMP (Se 172) and o he mTOR e ec o S6 (Se 235/236) we e
moni o ed by phospho-speci ic an ibodies. To al AMPK is also displayed. In he lowes wo panels, appea ance o a as mig a ing LC3B band (LC3B II)
and dec eased an i-p62 signal in SFB and SFB 12DG ea ed samples deno e enhanced au ophagy. Pic u e ep esen a i e o h ee independen
expe imen s wi h compa able esul s. (b). Con ocal analysis o LCSC-2 cells ansien ly ans ec ed wi h a andem ed-g een lu escen agged LC3 and
exposed o SFB o 6 hou s. Red punc a (au olysosomes) and yellow punc a (au ophagosomes) we e coun ed and he a io calcula ed o each cell. a. Ba s
a e mean 6SD o n 551 (Dmso) and n 532 (SFB); s a is ics by - es . (c). ep esen a i e luo escen mic opho og aphs displaying enhanced au ophagic
lux (p e alence o ed o e yellow do s) in he sample exposed o SFB. (d). Exp ession o myc- agged POLG1 (D890N) in HEK293 cells en days a e
cul i a ion in he p esence o 50 ng/ml doxycyclin. (e). Induced (Dox1) and non induced (Dox2) HEK-293 POLG1 (D890N) cells we e s imula ed o
4 hou s as indica ed (SFB 155mM) and phospho yla ion o AMPK and S6 e alua ed by immunoblo ing as in a. To al AMPK is also depic ed.
In he la e panel, appea ance o a ain slow mig a ing band abo e he main one con i ms AMPK hype phospho yla ion. SFB has li le e ec on AMPK in
his highly glycoly ic cell line, unless glycolysis is inhibi ed (2DG and No Glucose). Mi ochond ia deple ion cancels SFB s imula o y e ec on AMPK.
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 7
con aining medium was hen emo ed, and he con e ed dye solubilized wi h 500 ml
(24 well pla e) o 100 ml (96 well pla e) o acidic isop opanol (0.04 M HCl in absolu e
isop opanol o Isop opanol/Dmso 151 / . Abso bance was ead a 570 nm.
Fo P opidium Iodide exclusion assay, medium con aining loa ing cells was
emo ed and a ached cells we e ypsinized and pooled wi h he loa ing ones in low
cy ome y es ubes. Few seconds a e addi ion o P opidium Iodide (1 mg/ml inal
concen a ion) cells we e analysed o ed luo escence by low cy ome y (Exc.
488 nm, ed luo escence channel FL-3). Pe cen age o PI posi i e (dead) cells was
de e mined, a e exclusion o cell deb is.
Colony o ma ion assay
Fo he de e mina ion o he colony o ma ion capabili y, cells we e seeded in 6-well
pla es (500/well) in comple e cul u e medium. A e wo weeks cells we e ixed wi h
3,7% Fo malin o i e hou s and s ained wi h 0.02% Giemsa.
In acellula ATP measu emen .Fo in acellula ATP de ec ion, cells we e seeded in 12
well pla es (4 310
5
/well/ml) in p esence o in absence o ele an s imuli. A e 12 h cells
we e p ocessed and ATP was de ec ed by using P omega ki , ENLITENHATP Assay
Sys em Bioluminescence De ec ion, acco ding o he p o ide ’s ecommenda ions
39
.
High esolu ion espi ome y on in ac cells.A suspension o LCSC-2 in DMEM/
F12, p ocessed espec i ely wi h DMSO (c l) and So a enib 2.5 mM, was added o
each Oxyg aph Chambe (Chambe A and Chambe B), a a densi y o 0.5 3
10
6
cells/ml.
The expe imen s began wi h he measu emen o he Rou ine espi a ion
(i.e. he espi a ion o d ug- ea ed cells esuspended in he medium wi hou
he addic ion o subs a es), ollowed o abou 10 minu es (STATE R). Then,
2ml o 4 mg/ml Oligomycin, an ATP syn hase blocke , we e added in each
chambe (STATE L) and espi a ion was eco ded o 6 minu es.
Subsequen ly, 5 mg/ml o FCCP (a p o onopho e - H
1
ionopho e - and
uncouple o oxida i e phospho yla ion) we e added (STATE E) and es-
pi a ion was eco ded o 3 minu es; highe FCCP concen a ions o wide /
b oade ime in e als exe an inhibi o y e ec on cellula espi a ion
depending on mi ochond ia.
All inhibi o s and uncouple s used in he p o ocol a e able o c oss he cell mem-
b ane and do no equi e a p io cells pe meabiliza ion
40
.
Con ocal analysis o au ophagic lux in LCSC-2 cells.Quan i a i e assessmen o
au ophagosome o-au olysosome ma u a ion dynamic he was pe o med acco ding
o
24
using a luo escen agged LC3 p obe.
Figu e 6
|
LCSC-2 cell killing by SFB in ol es ROS and is coun e ac ed by AMPK. (a) PI exclusion assay depic ing he e ec o Ebselen (2 mM, g ay ba s)
on LCSC-2 cell g ow h inhibi ion by SFB and SFB 12DG. Ba s a e mean 6SD o duplica e o iplica e samples. S a is ics a e by wo-way ANOVA.
** p,0.01 compa ed o ehicle. Signi icance o he in e ac ion SFBxEBS is also indica ed. Rep esen a i e o wo independen expe imen s.
(b). Iden i ica ion and so ing o LCSC-2 cell popula ions wi h low and high con en o ROS, based on H2-DCFDA luo escence dis ibu ion on g een
luo escence FL-1 his og am. (c). g ow h inhibi ion e alua ed by MTT assay. Values a e pe cen ages o Dmso/PBS. Ba s a e mean 6SD o duplica e
samples. S a is ics o ele an compa isons by wo-way ANOVA a e indica ed. Signi icance o he in e ac ion e ec (ROS x SFB) is indica ed. Pic u e
ep esen a i e o wo independen expe imen s. (d). Knock-down o AMPKaby siRNA sensi izes LCSC-2 cells o SFB. (e). PI exclusion assay on
mock (siC l) and AMPK-silenced (siAMPKa) LCSC-2 cells exposed o SFB alone o in combina ion wi h 2DG o 24 hou s. Signi ican di e ences
(AMPK e sus C l) a e indica ed by as e isks. *p,0.05; **p,0.01 by wo-way ANOVA. Ba s a e mean 6SD o duplica e samples. Pic u e
ep esen a i e o wo independen expe imen s. (e). wes e n blo analysis con i ming AMPK down egula ion and impai ed pS6 inhibi ion/LC3B
lipida ion in siAMPK- ea ed cells exposed o SFB. Rep esen a i e o wo independen expe imen s. ( ). PI exclusion assay illus a ing LCSC-2 sensi i i y
o 5 mM SFB upon pha macological modula ion o AMPKa; inhibi o s we e added o cells 2 hou s p io o SFB, and iabili y was assessed 22 hou la e .
*p,0.05 ** p,0.01 by one-way ANOVA. Values a e mean 6SD o duplica e samples. Pic u e ep esen a i e o wo independen expe imen s.
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SCIENTIFIC REPORTS | 5 : 9149 | DOI: 10.1038/s ep09149 8
Isola ion o mi ochond ia om a li e .Mi ochond ia we e isola ed om he li e
o an adul emale Wis a a , using he ki MITOISO1 om Sigma. De e mina ion o
o ganelle ansmemb ane po en ial wi h JC-1 was pe o med acco ding o he ki
ins uc ions, as desc ibed elsewhe e
41
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Acknowledgmen s
We wish o hank D . Thomas D. Shupe and P o . B yon Pe e sen (Depa men o
Pa hology, Immunology and Labo a o y Medicine, College o Medicine, Uni e si y o
Flo ida, Gaines ille, Flo ida, USA), o ha ing kindly p o ided he LCSC-2 cell line. This
wo k was pa ially suppo ed by Baye Pha maceu ical ( o A.G.), by he I alian associa ion
o Cance Resea ch (AIRC, g an IG8634/2009 o G.P.) and by he I alian Minis y o
Uni e si y and Resea ch (MIUR, PRIN 2008, g an nu2008T7WRTM_003 o G.P.). JNS is
suppo ed by he Academy o Finland [CoE unding] and he Ne he lands O ganiza ion o
Scien i ic Resea ch [NWO: VICI g an 865.10.004].
Au ho con ibu ions
G.P. and A.G. concei ed and supe ised he s udy; G.P. and V.T. ca ied ou he
expe imen s and analyzed da a; D.S. pa icipa ed in expe imen al p ocedu es; G.M.
pe o med con ocal analysis o au ophagic lux; A.C.P. and G.M. analyzed da a; M.B., L.C.
and C.B. ca ied ou he Mic oa ay Analysis; R.S. and A.P. pe o med he oxyme y
expe imen s, J.N.S. con ibu ed undamen al eagen s and M.A.P. con ibu ed o da a
analysis. G.P., V.T. and A.C.P. we e in ol ed in w i ing he pape . All he au ho s had inal
app o al o he submi ed e sion.
Addi ional in o ma ion
Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/
scien i ic epo s
Compe ing inancial in e es s: V.T.’s ellowship was pa ially unded by Baye
Pha maceu ical. The au ho s decla e no o he po en ial compe ing inancial in e es s.
How o ci e his a icle: Teso i, V. e al. The mul ikinase inhibi o So a enib enhances
glycolysis and syne gizes wi h glycolysis blockade o cance cell killing. Sci. Rep. 5, 9149;
DOI:10.1038/s ep09149 (2015).
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