an ioxidan s
A icle
Thio edoxin Down egula ion Enhances So a enib
E ec s in Hepa oca cinoma Cells
Ma ía JoséLópez-G ueso 1, Raúl González 2, Jo di Mun ané2,3,4 , JoséAn onio Bá cena 1,5,*
and C. Alicia Padilla 1,5
1Depa men o Biochemis y and Molecula Biology, Uni e si y o Có doba, 14071 Có doba, Spain;
[email p o ec ed] (M.J.L.-G.); [email p o ec ed] (C.A.P.)
2
Ins i u e o Biomedicine o Se ille (IBiS), Hospi al Uni e si y “Vi gen del Roc
í
o”/CSIC/Uni e si y o Se ille,
41013 Se illa, Spain; [email p o ec ed] (R.G.); [email p o ec ed] (J.M.)
3Depa amen o de Ci ugía Gene al, Hospi al Uni e si a io Vi gen del Rocío/Ins i u o de Biomedicina de
Se illa (IBiS)/CSIC/Uni e sidad de Se illa, 41013 Se illa, Spain
4Cen o de In es igación Biomédica en Red de En e medades Hepá icas y Diges i as (CIBERehd),
41013 Se illa, Spain
5Ins i u o Maimónides de In es igación Biomédica de Có doba (IMIBIC), 14004 Có doba, Spain
*Co espondence: bb1ba [email p o ec ed]
Recei ed: 11 Sep embe 2019; Accep ed: 21 Oc obe 2019; Published: 22 Oc obe 2019


Abs ac :
So a enib is he i s -line ecommended he apy o pa ien s wi h ad anced hepa oca cinoma
(HCC) in de-di e en ia ion s age (p esen ing epi helial–mesenchymal ansi ion, EMT). We s udied
he ole o he hio edoxin sys em (T x1/T xR1) in he sensi i i y o esis ance o HCC cells o he
ea men wi h So a enib. As a model, we used a se o h ee es ablished HCC cell lines wi h di e en
deg ees o de-di e en ia ion as occu s in me as asis. By quan i a i e p o eomics, we ound ha he
exp ession le els o T x1 and T xR1 ollowed he same end as canonical EMT ma ke s in hese cell
lines. T ea men wi h So a enib induced hiol edox educ i e changes in c i ical elemen s o oncogenic
pa hways in all h ee cell lines bu induced d as ic p o eome ep og aming only in HCC cell lines o
in e media e s age. T x1 down egula ion coun e ac ed he hiol educ i e e ec o So a enib on Signal
T ansduce and Ac i a o o T ansc ip ion 3 (STAT3) bu no on Mi ogen-Ac i a ed P o ein Kinase
(MAPK) o P o ein Kinase B (Ak ) and ans o med ad anced HCC cells in o So a enib-sensi i e cells.
Ten a ge s o he combined So a enib–siRNAT x1 ea men we e iden i ied ha showed a g adually
changing exp ession end in pa allel o changes in he exp ession o canonical EMT ma ke s, likely
as a esul o he ac i a ion o Hippo signaling. These indings suppo he idea ha a combina ion o
So a enib wi h hio edoxin inhibi o s should be aken in o accoun in he design o he apies agains
ad anced HCC.
Keywo ds: hepa oca cinoma; hio edoxin; so a enib; edox signaling; EMT
1. In oduc ion
Hepa oca cinoma (HCC) ep esen s 80% o he p ima y hepa ic neoplasms ha appea mainly in a
con ex o ch onic li e ci hosis. I is he six h mos equen neoplasm, he hi d cause o cance dea h,
and accoun s o 7% o egis e ed malignancies [
1
]. So a enib is he s anda d o ca e o ad anced-s age
HCC, as demons a ed in wo la ge-scale ials [2] and he Asia-Paci ic ial [3].
Epi helial–mesenchymal ansi ion (EMT) is an impo an p ocess ha happens in no mal
de elopmen in which epi helial cells lose many o hei p ope ies o become mesenchymal cells
by means o d as ic changes in a chi ec u e and beha io . A simila ansi ion also occu s du ing
umo p og ession and malignan ans o ma ion, leading o inc eased cell mo ili y and in asi eness.
An ioxidan s 2019,8, 501; doi:10.3390/an iox8100501 www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2019,8, 501 2 o 18
T ans o ming G ow h Fac o -ß (TGFB) is known as he main, al hough no exclusi e, induc o o
EMT, which akes place h ough a ious signaling pa hways [
4
]. A c i ical molecula e en o EMT is
he down egula ion o he cell adhesion molecule E-cadhe in and i s eplacemen by N-cadhe in [
5
].
Ac i a ion o Ak leads o a signi ican educ ion in E-cadhe in exp ession and o nuclea localiza ion o
SNAI1, sugges ing a ole o he PI3K/Ak signaling pa hway in he shi om E-cadhe in o N-cadhe in
exp ession and in EMT p og ession in cance [
6
,
7
]. The se ine/ h eonine kinase Ak is hough o be
esponsible o media ing he acqui ed esis ance o So a enib in HCC cells [
8
]. Ak can be ac i a ed by
insulin and su i al and g ow h ac o s h ough phospho yla ion media ed by mTOR2 and PDK1
a speci ic h eonine esidues. The p ocess can be e e ed by p o ein phospha ase 2 (PP2A) when
Ak is oxidized, sugges ing ha Ak is subjec ed o edox egula ion [
9
]. Mo eo e , i was shown ha
he in e ac ion be ween Ak and PP2A unde condi ions o oxida i e s ess could be impai ed by he
educ ion o a speci ic disul ide bond in Ak ca alyzed by glu a edoxin (G x). Ac i a ion o Ak a e
s imula ion wi h insulin and a ious g ow h and su i al ac o s, leading o he phospho yla ion a
Th 308 and Se 473 by PDK1 and mTOR2, espec i ely. Ak in i s oxidized o m can also be inac i a ed
by p o ein phospha ase 2 (PP2A) dephospho yla ion, which sugges s ha Ak is a edox- egula ed
p o ein [
9
]. I has been shown ha G x p e en ed Ak om o ming a speci ic disul ide bond be ween
Cys-297 and Cys-311 and supp essed i s associa ion wi h PP2A unde oxida i e s ess, [9–11].
STAT3 is a p o-su i al ansc ip ion ac o which is cons i u i ely ac i a ed in human cance cell
lines. Cons i u i e phospho yla ion o STAT3 causes impo an changes in apop osis, angiogenesis,
in asion, mig a ion, and p oli e a ion, esul ing in cell malignan ans o ma ion [
12
]. I has been
obse ed ha STAT3 ac i a ion is in ol ed in EMT, in asion, and gene a ion o me as asis in HCC [
13
].
Ty osine phospho yla ion o STAT3 is dependen on he hiol edox s a e modula ed by H
2
O
2
and P x2
le els and he hio edoxin sys em ac i i y [
14
]. T x1–STAT3 disul ide exchange in e media es we e
de ec ed, sugges ing ha T x1 may be a di ec media o o STAT3 disul ide educ ion.
O e exp ession o T x1 has been de ec ed in human ce ical neoplas ic squamous epi helial cells,
lung, colon, and HCC umo s [15–18]. Ta ge ing T x1 o cance he apy and as p ognos ic ma ke o
HCC has been conside ed because o he obse ed ela ionship be ween T x1 exp ession and umo
agg essi eness, al hough he mechanisms unde lying his associa ion a e s ill no well known [
19
].
O e exp ession o T x1 is one o he mechanisms o d ug esis ance in HCC ea men . A possible
s a egy ha is ga he ing s eng h is o combine T x1 gene he apy wi h chemo he apy o inc ease he
e ec i eness o ea men s in HCC [
20
]. Thio edoxin-in e ac ing p o ein (TXNIP) is a membe o he
alpha-a es in p o ein amily ha binds o he ac i e si e and coun e ac s he ac ion o T x1 unc ioning
as umo supp esso [
21
]. Down egula ion o TXNIP exace ba es cance p og ession, and i s exp ession
is ac ually educed in a ious human cance cells [
22
]. TXNIP de iciency enhances he induc ion o he
Zinc inge p o eins SNAI1 and SNAI2 by TGF-ß and p omo es TGF-ß-induced EMT [23].
A compa a i e genomic cha ac e iza ion o 19 cell lines de i ed om HCC allowed clus e ing
hem in o wo g oups (I and II) acco ding o hei gene exp ession pa e ns [24].
In his s udy, we selec ed h ee hepa oca cinoma cell lines, i.e., HepG2 belonging o g oup I and
SNU423 and SNU475 belonging o g oup II, o ca y ou a compa a i e s udy o hei basal p o eome
and o how i esponds o So a enib ea men and/o hio edoxin down egula ion. We also analyzed
edox modi ica ions and phospho yla ion o Ak , MAPK, and STAT3. The esul s ob ained suppo he
use o a combina ion o So a enib wi h T x1 inac i a ion in HCC he apy.
2. Ma e ials and Me hods
2.1. Ma e ials
HepG2 (HB-8065
™
, ATCC/LGC S anda ds, SLU, Ba celona, Spain), SNU423 (CRL-2238,
ATCC/LGC S anda ds) and SNU475 (CRL-2236, ATCC/LGC S anda ds) cell lines we e ob ained
om Ame ican Type Cul u e Collec ion (ATCC; LGC S anda ds, S.L.U., Ba celona, Spain). An i-T x1
was ob ained in-house om abbi immuniza ion wi h T x1. An ibodies agains STAT3 (#4904S),
An ioxidan s 2019,8, 501 3 o 18
MAPK (#9102), Th
202
/Ty
204
pMAPK (#4370P), Se
473
pAk (#4060S) we e ob ained om Cell Signaling
Technology. An ibodies agains Ty
705
pSTAT3 (#sc-7993), TXNIP (#sc-271237), Ak 1 (#sc-5298), and
ß-ac in (#sc-47778) we e om San a C uz Bio echnology, Inc. (Dallas, TX, USA). An i-T xR1 (#ab124954)
was p o ided by Abcam, Inc. Seconda y an ibodies we e om Sigma. ECL eagen was om GE
Heal hca e (Chicago, Illinois, USA); siRNA o T x1 was om Dha macon, Inc. (#L-006340-00; La aye e,
Colo ado, USA).
2.2. Cell G ow h Condi ions
Cell cul u es we e nega i e o mycoplasma con amina ion. Cells we e main ained in Eagle’s
minimum essen ial medium (EMEM), pH 7.4, supplemen ed wi h 10% FBS (#S181G-500, Biowes ),
sodium py u a e (1mM) (#L0642-100, Biowes ), and a penicillin–s ep omycin–ampho e icin solu ion
(100U/mL–100
µ
g/mL–0.25
µ
g/mL) (#L0010-100, Biowes ) a 37
◦
C in a humidi ied incuba o wi h 5%
CO
2
. The cells we e seeded a a densi y o 10
5
cells/cm
2
in 2D cul u e. When applied, So a enib
was added o he cell cul u e a a concen a ion o 10
µ
M, as se up in a p e ious s udy [
25
] and he
cells we e incuba ed o 24 h be o e cell lysis o measu e cell p oli e a ion and p o ein ac i i ies and
exp ession and o pe o m p o eomic analysis. Cells lysis was ca ied ou using 50 mM HEPES pH 7.5,
5 mM EDTA, 150 mM NaCl, 1% NP-40, a p o eases inhibi o cock ail (#P8340, Sigma-Ald ich), 1 mM
phenylme hylsul onyl luo ide (PMSF), 1 mM NaF, and 1mM Na
3
VO
4
. The lysa es we e incuba ed on
ice and o exed o 15 s in ou in e als o 5 min each. The samples we e cen i uged a 13,000 pm a
4◦C. The supe na an s we e collec ed o p o ein quan i ica ion and analysis.
2.3. De e mina ion o P oli e a ion and Caspase-3 Ac i i y Measu emen
Cell p oli e a ion was analyzed using a colo ime ic ELISA (Roche Applied Science, Penzbe g,
Ge many). In o al, 2
×
10
4
cells/cm
2
we e cul u ed in 96-well pla es a 37
◦
C and 5% CO
2
. A he end o
he expe imen , he cells we e incuba ed wi h a 10
µ
M B dU labelling solu ion o 6h a 37
◦
C ollowing
he p o ocol ecommended by he manu ac u e . Caspase-3-associa ed ac i i y was de e mined using
he pep ide-based subs a e Ac-DEVD-AFC (100 µM), as desc ibed elsewe e [10].
2.4. SDS-PAGE and Wes e n Blo ing
The p o ein exp ession le els o T x1, T xR1, TXNIP, STAT3, pSTAT3, Ak 1, pAk , MAPK, and
pMAPK we e de e mined by SDS-PAGE coupled o Wes e n blo ing analysis, ollowing he same
p ocedu e and using he same an ibody dilu ions as desc ibed be o e [10].
2.5. Silencing o T x1
Human T x1 was knocked down in HepG2, SNU423, and SNU475 cells using a pool o ou
speci ic siRNA in 6-well pla es (20,000 cells/cm
2
), acco ding o he manu ac u e ’s ecommenda ions
(Dha macon, GE Heal hca e Li e Sciences) and as desc ibed be o e o HepG2 cells [
10
]. Non- a ge ing
nega i e con ols we e un. Silencing o T x1 was always checked by Wes e n blo ing and ac i i y
assay o con i m ha i s le els we e educed by ≈80%.
2.6. Redox Mobili y Shi Assay
De ec ion o hiol edox changes in Ak , MAPK, and STAT3 p o eins was pe o med by di e en ially
labeling educed cys eines wi h 5 mM N-e hylmaleimide (NEM) and e e sibly oxidized cys eines by
educ ion wi h 5 mM is (2-ca boxye hyl) phosphine (TCEP), ollowed by labeling wi h 10 mM
4-ace amido-4’-maleimidyls ilbene-2,2’-disul onic acid (AMS) (The mo Scien i ic Pie ce), which
inc eases he molecula weigh o he na i e p o ein by 536.44 Da. The p o ocol was he same
as desc ibed be o e [10].
An ioxidan s 2019,8, 501 4 o 18
2.7. LC–MS/MS, Label-F ee MS P o ein Quan i ica ion, and Sys ems Analysis
P o eomic analyses we e pe o med a he P o eomics Facili y, Uni e si y o C
ó
doba (SCAI).
The p ocedu e s a ed wi h a cul u e o 2
×
10
4
cells/cm
2
, and he p o ocol was he same as
desc ibed p e iously [
26
]. Pep ides we e scanned and agmen ed wi h he The mo O bi ap Fusion
(Q-OT-qIT, The mo Scien i ic) equipped wi h a nano-UHLC Ul ima e 3000 (Dionex-The mo Scien i ics).
The analysis o MS aw da a was pe o med using he MaxQuan ( 1.5.7.0) so wa e [
27
] and he
label- ee quan i ica ion con igu a ion desc ibed be o e [
26
]. Th ee RAW da a iles pe sample om
h ee sepa a e expe imen s we e analyzed. Human UniP o KB/Swiss-P o p o ein da abase (Feb ua y
2018 e sion) was sea ched o p o ein iden i ica ion as desc ibed be o e [
26
]. The me hod used o he
impu a ion o missing alues was he mean impu a ion. Finally, only he condi ions wi h 3 alues pe
iden i ica ion we e conside ed and analyzed h ough ANOVA and Tukey’s pos -hoc es s. A p o ein
was conside ed di e en ially exp essed i iden i ied and quan i ied wi h a leas 2 unique pep ides and
had a old change o a leas 1.50 and p alue ≤0.05.
The di e en ially exp essed p o eins oge he wi h he old change alues we e analyzed wi h he
online IPA so wa e package (Qiagen, e sion 48207413) and he open so wa e DAVID [28]. Fo IPA,
each p o ein was mapped o i s co esponding gene objec in he Ingenui y Pa hways Knowledge.
The “co e analysis” unc ion was ca ied ou conside ing di ec and indi ec ela ionships expe imen ally
obse ed in all mammalian issues and species, as well as all node ypes, da a sou ces, and mu a ions.
The lis o signi ican ly en iched canonical pa hways, biological unc ions, and ups eam ac o s is
p esen ed oge he wi h he ac i a ion o inhibi ion z-sco e alues in a scale o colo s.
2.8. S a is ical Analysis
Resul s a e exp essed as mean
±
SD o da a om
≥
3 independen expe imen s. One-way ANOVA
wi h he leas signi ican di e ence Tukey’s es as pos -hoc mul iple compa ison analysis wi h a single
pooled a iance was used o compa isons; ou pu p alue anges we e handled in GP s yle: 0.0332 (*),
0.0021 (**), 0.0002 (***), <0.0001 (****). The h eshold o s a is ically signi ican di e ences was se a p
adjus ≤0.05 alue.
3. Resul s
3.1. T acing he P o eomic Signa u e o EMT in Human Hepa oca cinoma Cell Lines
A “label- ee” quan i a i e p o eomics analysis de ec ed 1170 p o eins wi h signi ican di e ences
be ween HepG2, SNU423, and SNU475 cell lines (Supplemen a y File S1). This is he i s compa a i e
p o eomic analysis ca ied ou wi h hese cell lines, and he esul s ob ained o canonical ma ke s
o EMT showed inc easing and dec easing g adien s, in ag eemen wi h he classi ica ion o hese
human HCC cells as epi helial o mesenchymal [
29
,
30
]. These esul s s ongly co ela e wi h p e ious
mic oa ay and Wes e n blo ing analyses o human HCC cell ypes [
29
,
30
] and cons i u e a de ini i e
p oo o concep o ou expe imen al app oach. E-cadhe in was no de ec ed, likely because ou
p o eomic p o ocol was no op imized o memb ane p o eins. The membe s o he T x sys em T x1
and T xR1 we e also p esen in inc easing le els om HepG2 o SNU475 cells (Figu e 1), which ag ees
wi h he ole desc ibed o T x1 as a p o-me as asis ac o [31].
An ioxidan s 2019,8, 501 5 o 18
An ioxidan s 2019, 8, x FOR PEER REVIEW 5 o 18
Figu e 1. Epi helial–mesenchymal ansi ion (EMT) ma ke s and hio edoxin sys em in h ee
hepa oca cinoma (HCC) cells lines. Da a o imen in, alpha- e op o ein, CD44 an igen, T x1, and
T xR1 we e e ie ed om he quan i a i e p o eomic analysis o HepG2, SNU423, and SNU475 cells
(Supplemen a y File S1). The scale in he e ical axis is he ela i e in ensi y om he LC–MS/MS
quan i a i e analysis and a ies be ween p o eins; he maximum alue o each p o ein anges om
3.25e + 007 o alpha- e op o ein in HepG2 cells o 8.97e + 008 o T x1 in SNU475 cells. (N = 3,
indi idual alues a e shown).
A sys em analysis o hese 1170 di e en ial p o eins yielded signi ican en ichmen s in se e al
canonical pa hways (Figu e 2A; he iden i ies o he p o eins a e shown in Supplemen a y File S1).
In eg in and ac in cy oskele on signaling, emodeling o epi helial adhe en junc ion, and PI3K/Ak
signaling, which ha e been desc ibed as being in ol ed in EMT, we e ac i a ed. On he e e se side,
he e was an o e all inac i a ion o amino acid me abolism, a y acid be a-oxida ion,
neu o ansmi e ca abolism, glu a hione me abolism, and oxido- educ ion p ocesses. SNU423 and
SNU275 cells showed simila ac i a ion and inac i a ion ends in many p ocesses, al hough hese
p ocesses we e a ec ed o a lesse ex en in SNU423 cells, in pa allel o hei deg ee o mesenchymal
p ope ies. Sys em analysis o 100 up egula ed and 156 down egula ed p o eins common o he i s
wo cell lines by Kyo o Encyclopedia o Genes and Genomes (KEGG) pa hways and Gene On ology
(GO) biological p ocesses using DAVID so wa e con i med and s essed hese simila i ies (Figu e
S1). A sea ch o ups eam egula o s b ough o ligh di e ences and commonali ies (Figu e 2B). To
name a ew, MYCN and MITF ansc ip ion ac o s appea ed o be ac i a ed in SNU423 bu
inac i a ed in SNU475 cells, whe eas HIF1A and he KDM5A his one H3 deme hylase beha ed in
he opposi e way.
Al oge he , hese esul s iden i ied a numbe o p o eins and egula o y elemen s ela ed o de-
di e en ia ion o HCC cells ha could be an in e es ing ocus o u he esea ch.
Figu e 1.
Epi helial–mesenchymal ansi ion (EMT) ma ke s and hio edoxin sys em in h ee
hepa oca cinoma (HCC) cells lines. Da a o imen in, alpha- e op o ein, CD44 an igen, T x1, and
T xR1 we e e ie ed om he quan i a i e p o eomic analysis o HepG2, SNU423, and SNU475 cells
(Supplemen a y File S1). The scale in he e ical axis is he ela i e in ensi y om he LC–MS/MS
quan i a i e analysis and a ies be ween p o eins; he maximum alue o each p o ein anges om
3.25e +007 o alpha- e op o ein in HepG2 cells o 8.97e +008 o T x1 in SNU475 cells. (N=3,
indi idual alues a e shown).
A sys em analysis o hese 1170 di e en ial p o eins yielded signi ican en ichmen s in se e al
canonical pa hways (Figu e 2A; he iden i ies o he p o eins a e shown in Supplemen a y File S1).
In eg in and ac in cy oskele on signaling, emodeling o epi helial adhe en junc ion, and PI3K/Ak
signaling, which ha e been desc ibed as being in ol ed in EMT, we e ac i a ed. On he e e se side,
he e was an o e all inac i a ion o amino acid me abolism, a y acid be a-oxida ion, neu o ansmi e
ca abolism, glu a hione me abolism, and oxido- educ ion p ocesses. SNU423 and SNU275 cells showed
simila ac i a ion and inac i a ion ends in many p ocesses, al hough hese p ocesses we e a ec ed o
a lesse ex en in SNU423 cells, in pa allel o hei deg ee o mesenchymal p ope ies. Sys em analysis
o 100 up egula ed and 156 down egula ed p o eins common o he i s wo cell lines by Kyo o
Encyclopedia o Genes and Genomes (KEGG) pa hways and Gene On ology (GO) biological p ocesses
using DAVID so wa e con i med and s essed hese simila i ies (Figu e S1). A sea ch o ups eam
egula o s b ough o ligh di e ences and commonali ies (Figu e 2B). To name a ew, MYCN and MITF
ansc ip ion ac o s appea ed o be ac i a ed in SNU423 bu inac i a ed in SNU475 cells, whe eas
HIF1A and he KDM5A his one H3 deme hylase beha ed in he opposi e way.

An ioxidan s 2019,8, 501 6 o 18
An ioxidan s 2019, 8, x FOR PEER REVIEW 6 o 18
Figu e 2. Sys ems Biology analysis o di e en ial p o eomic da a om h ee HCC cell lines.
Quan i a i e da a on p o ein le els in SNU423 and SNU475 cells ela i e o he le els in HepG2, he
mo e di e en ia ed cell line, we e analyzed. Up- and down- egula ed p o eins we e analyzed using
he IPA so wa e in e ms o (A) canonical pa hways based on he con en o he Ingenui y Knowledge
Base, and (B) Ups eam Regula o Analysis o iden i y he cascade o ups eam ansc ip ional
egula o s (see M&M sec ion 2.7 o mo e de ails). The deg ee o ac i a ion is ep esen ed in a colo
scale as indica ed. Red b acke s ha e been added o highligh signi ican da a commen ed on in he
main ex .
3.2. The T x/T xR/TXNIP Sys em is Highly Sensi i e o So a enib T ea men in HCC Cell Lines
The basal le els o T x1 and T xR1 in con ol cells we e ma kedly highe in mesenchymal
SNU475 cells (Figu e 1), bu So a enib induced a signi ican dec ease in hei le els in all HCC cell
lines (Figu e 3A,B). TXNIP beha ed in a ecip ocal manne ela i e o T x1 and T xR1. I was
up egula ed bo h by So a enib and by siRNAT x1, ei he independen ly o join ly, in all h ee cell
lines (Figu e 3C). These esul s indica e ha he down egula ion o he T x sys em is pa o he
an i umo al ac ion mechanism o So a enib and suppo ou hypo hesis ha co- ea men wi h T x1
inac i a ion adju an s would po en ia e So a enib-based an i umo al he apy.
Figu e 2.
Sys ems Biology analysis o di e en ial p o eomic da a om h ee HCC cell lines. Quan i a i e
da a on p o ein le els in SNU423 and SNU475 cells ela i e o he le els in HepG2, he mo e di e en ia ed
cell line, we e analyzed. Up- and down- egula ed p o eins we e analyzed using he IPA so wa e
in e ms o (
A
) canonical pa hways based on he con en o he Ingenui y Knowledge Base, and
(
B
) Ups eam Regula o Analysis o iden i y he cascade o ups eam ansc ip ional egula o s
(see M&M Sec ion 2.7 o mo e de ails). The deg ee o ac i a ion is ep esen ed in a colo scale as
indica ed. Red b acke s ha e been added o highligh signi ican da a commen ed on in he main ex .
Al oge he , hese esul s iden i ied a numbe o p o eins and egula o y elemen s ela ed o
de-di e en ia ion o HCC cells ha could be an in e es ing ocus o u he esea ch.
3.2. The T x/T xR/TXNIP Sys em is Highly Sensi i e o So a enib T ea men in HCC Cell Lines
The basal le els o T x1 and T xR1 in con ol cells we e ma kedly highe in mesenchymal SNU475
cells (Figu e 1), bu So a enib induced a signi ican dec ease in hei le els in all HCC cell lines
(Figu e 3A,B). TXNIP beha ed in a ecip ocal manne ela i e o T x1 and T xR1. I was up egula ed
bo h by So a enib and by siRNAT x1, ei he independen ly o join ly, in all h ee cell lines (Figu e 3C).
These esul s indica e ha he down egula ion o he T x sys em is pa o he an i umo al ac ion
mechanism o So a enib and suppo ou hypo hesis ha co- ea men wi h T x1 inac i a ion adju an s
would po en ia e So a enib-based an i umo al he apy.
An ioxidan s 2019,8, 501 7 o 18
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Figu e 3. Response o he hio edoxin sys em o So a enib and siRNAT x1 ea men s. Changes in he
le els o h ee componen s o he hio edoxin sys em, (A) T x1, (B) T xR1 and (C) TXNIP we e
de e mined in HepG2, SNU423 and SNU475 cells by Wes e n blo ing wi h hei speci ic an ibodies
on ea men wi h So a enib o siRNAT x1 independen ly o combined unde he condi ions
desc ibed in M&M. Densi ome ic da a no malized o ß-ac in a e shown; samples om 3 di e en
expe imen s (N = 3) o each cell line we e un in he same gel; p o ein le els be ween cells canno be
compa ed since each se o 3 eplicas o each cell line was de eloped on a di e en WB memb ane;
o a compa ison o T x1, T xR1 be ween cell lines, e e o quan i a i e p o eomic da a in Figu e 1; a
composi ion o ep esen a i e blo s is shown below each g aph. p alues 0.0332 (*), 0.0021 (**),
0.0002(***), <0.0001 (****), see Ma e ials & Me hods o s a is ical de ails.
3.3. The E ec o So a enib on he Global P o eome Depends on he Cell De-Di e en ia ion S age
So a enib alone was e y e ec i e in SNU423 cells, inducing signi ican changes in 465 p o eins,
whe eas in HepG2 and SNU475 cells, he numbe o p o eins a ec ed was es ic ed o only 23 and
83, espec i ely (Figu e 4A). The e ec o siRNAT x1 alone was a he low in all h ee cell ypes, as
should be expec ed since he e ec s o his an ioxidan p o ein a he ansc ip ional egula o y le el
a e limi ed. The cellula en i onmen de e mined which p o eins we e a ec ed by T x1
down egula ion, since he e we e no common siRNAT x1 a ge s among he h ee cell lines, al hough
mos o he p o eins a ec ed did also change when siRNAT x1 ea men was combined wi h
So a enib (Supplemen a y File S1). In e es ingly, a e y s ong syne gis ic e ec was obse ed when
So a enib was applied o T x1-down egula ed SNU475 cells (Figu e 4A). This syne gis ic e ec was
also obse ed in HepG2 cells, al hough o a lesse ex en , bu was absen in SNU423 cells.
Figu e 3.
Response o he hio edoxin sys em o So a enib and siRNAT x1 ea men s. Changes in
he le els o h ee componen s o he hio edoxin sys em, (
A
) T x1, (
B
) T xR1 and (
C
) TXNIP we e
de e mined in HepG2, SNU423 and SNU475 cells by Wes e n blo ing wi h hei speci ic an ibodies on
ea men wi h So a enib o siRNAT x1 independen ly o combined unde he condi ions desc ibed in
M&M. Densi ome ic da a no malized o ß-ac in a e shown; samples om 3 di e en expe imen s
(
N=3
) o each cell line we e un in he same gel; p o ein le els be ween cells canno be compa ed since
each se o 3 eplicas o each cell line was de eloped on a di e en WB memb ane; o a compa ison
o T x1, T xR1 be ween cell lines, e e o quan i a i e p o eomic da a in Figu e 1; a composi ion o
ep esen a i e blo s is shown below each g aph. p alues 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****),
see Ma e ials & Me hods o s a is ical de ails.
3.3. The E ec o So a enib on he Global P o eome Depends on he Cell De-Di e en ia ion S age
So a enib alone was e y e ec i e in SNU423 cells, inducing signi ican changes in 465 p o eins,
whe eas in HepG2 and SNU475 cells, he numbe o p o eins a ec ed was es ic ed o only 23 and 83,
espec i ely (Figu e 4A). The e ec o siRNAT x1 alone was a he low in all h ee cell ypes, as should
be expec ed since he e ec s o his an ioxidan p o ein a he ansc ip ional egula o y le el a e limi ed.
The cellula en i onmen de e mined which p o eins we e a ec ed by T x1 down egula ion, since
he e we e no common siRNAT x1 a ge s among he h ee cell lines, al hough mos o he p o eins
a ec ed did also change when siRNAT x1 ea men was combined wi h So a enib (Supplemen a y
File S1). In e es ingly, a e y s ong syne gis ic e ec was obse ed when So a enib was applied o
T x1-down egula ed SNU475 cells (Figu e 4A). This syne gis ic e ec was also obse ed in HepG2
cells, al hough o a lesse ex en , bu was absen in SNU423 cells.
An ioxidan s 2019,8, 501 8 o 18
An ioxidan s 2019, 8, x FOR PEER REVIEW 8 o 18
Figu e 4. Summa y o he quan i a i e p o eomic analysis o HCC cell lines subjec ed o So a enib
and/o siRNAT x1 ea men s. (A) The o al numbe o signi ican ly (adjus ed p ≤ 0.05) di e en
p o eins ( old change ≥1.5 and ≤0.6) quan i ied upon each ea men ela i e o hei con ols; he
numbe s abo e he b acke s and hin lines a e he “syne gy ac o ” eached by siRNAT x1 and
So a enib calcula ed by he a io be ween he numbe o p o eins a ec ed by he combined ea men
and he numbe o p o eins a ec ed by indi idual So a enib and siRNAT x1 ea men s. (B) Venn
diag ams o up egula ed and down egula ed p o eins by each ea men , by cell line; he colo code
indica ed in he legend applies o (A) and (B). (C) Venn diag ams o p o eins a ec ed in each cell line
by ea men .
Al oge he , hese p o eomic da a indica e ha So a enib is highly ac i e in mode a ely
di e en ia ed umo cells bu i has a limi ed e ec in T x1- ich and poo ly di e en ia ed cells, unless
he an ioxidan hio edoxin- ela ed signaling is weakened. I s ac ion is mino in epi helial HepG2
cells e en in combina ion wi h T x1 down egula ion.
A compa ison o he So a enib and T x1 silencing a ge s o each cell line (Figu e 4C) highligh ed
he ac ha he e we e no common a ge s be ween he se cell lines, sugges ing ha he e ec s o
each ea men a e dependen on he de-di e en ia ion s age o HCC cell lines. Howe e , he e we e
12 common a ge s o he combined ea men in he h ee cell lines (Figu e 4C and Figu e 5). These
common p o eins a e ela ed o lipids biosyn hesis, p o ein nuclea anspo , and ac in o ganiza ion.
The inc easing deg ee o down egula ion o mos o hese common p o eins uns pa allel o hei
deg ee o de-di e en ia ion.
Figu e 4.
Summa y o he quan i a i e p o eomic analysis o HCC cell lines subjec ed o So a enib
and/o siRNAT x1 ea men s. (
A
) The o al numbe o signi ican ly (adjus ed p
≤
0.05) di e en p o eins
( old change
≥
1.5 and
≤
0.6) quan i ied upon each ea men ela i e o hei con ols; he numbe s abo e
he b acke s and hin lines a e he “syne gy ac o ” eached by siRNAT x1 and So a enib calcula ed
by he a io be ween he numbe o p o eins a ec ed by he combined ea men and he numbe o
p o eins a ec ed by indi idual So a enib and siRNAT x1 ea men s. (
B)
Venn diag ams o up egula ed
and down egula ed p o eins by each ea men , by cell line; he colo code indica ed in he legend
applies o (A) and (B). (C) Venn diag ams o p o eins a ec ed in each cell line by ea men .
Al oge he , hese p o eomic da a indica e ha So a enib is highly ac i e in mode a ely di e en ia ed
umo cells bu i has a limi ed e ec in T x1- ich and poo ly di e en ia ed cells, unless he an ioxidan
hio edoxin- ela ed signaling is weakened. I s ac ion is mino in epi helial HepG2 cells e en in
combina ion wi h T x1 down egula ion.
A compa ison o he So a enib and T x1 silencing a ge s o each cell line (Figu e 4C) highligh ed
he ac ha he e we e no common a ge s be ween he se cell lines, sugges ing ha he e ec s o each
ea men a e dependen on he de-di e en ia ion s age o HCC cell lines. Howe e , he e we e 12
common a ge s o he combined ea men in he h ee cell lines (Figu es 4C and 5). These common
p o eins a e ela ed o lipids biosyn hesis, p o ein nuclea anspo , and ac in o ganiza ion. The
inc easing deg ee o down egula ion o mos o hese common p o eins uns pa allel o hei deg ee
o de-di e en ia ion.
An ioxidan s 2019,8, 501 9 o 18
An ioxidan s 2019, 8, x FOR PEER REVIEW 9 o 18
Figu e 5. Common a ge p o eins o he So a enib + siRNAT x1 ea men . Twel e p o eins ha
changed signi ican ly in HepG2, SNU423, and SNU475 cells a e shown. A b ie desc ip ion o hei
unc ion was ex ac ed om UniP o ; old changes ela i e o hei espec i e con ols ea ed wi h
sol en + Non- a ge siRNA a e indica ed in a colo scale. Quan i a i e p o eomic analysis as
desc ibed in Ma e ials and Me hods (N = 3). Ten old-change alues co espond o p o eins ha we e
p esen in he con ols bu we e no de ec ed in he ea ed samples.
Sys em analysis o he p o eomic da a p o ided some clues abou he di e en ial e ec o
So a enib in each cell ype (Figu e 6). The only pa hways al e ed by So a enib in SNU475 cells we e
hose o IGF-1 and enin–angio ensin signaling, which appea ed inac i a ed wi h he concomi an
di e en ial inac i a ion o se e al ups eam egula o s, i.e., NFE2L2, PDGF-B, a po en mi ogen o
cells o mesenchymal o igin, XBP1, in ol ed in he un olded p o ein esponse (UPR), and Ige (Figu e
6). None o hese pa hways and ups eam egula o s we e a ec ed in SNU423 cells.
Figu e 5.
Common a ge p o eins o he So a enib +siRNAT x1 ea men . Twel e p o eins ha
changed signi ican ly in HepG2, SNU423, and SNU475 cells a e shown. A b ie desc ip ion o hei
unc ion was ex ac ed om UniP o ; old changes ela i e o hei espec i e con ols ea ed wi h
sol en +Non- a ge siRNA a e indica ed in a colo scale. Quan i a i e p o eomic analysis as desc ibed
in Ma e ials and Me hods (N=3). Ten old-change alues co espond o p o eins ha we e p esen in
he con ols bu we e no de ec ed in he ea ed samples.
Sys em analysis o he p o eomic da a p o ided some clues abou he di e en ial e ec o So a enib
in each cell ype (Figu e 6). The only pa hways al e ed by So a enib in SNU475 cells we e hose o
IGF-1 and enin–angio ensin signaling, which appea ed inac i a ed wi h he concomi an di e en ial
inac i a ion o se e al ups eam egula o s, i.e., NFE2L2, PDGF-B, a po en mi ogen o cells o
mesenchymal o igin, XBP1, in ol ed in he un olded p o ein esponse (UPR), and Ige (Figu e 6). None
o hese pa hways and ups eam egula o s we e a ec ed in SNU423 cells.
An ioxidan s 2019,8, 501 16 o 18
siRNAT x1 combined ea men (“ST”); columns P–U show he abundance old change (“FC”) ela i e o he
con ol and hei espec i e s a is ical signi icance (p- alues, a alue o “1” means no signi ican FC). In he 4
h
shee , (“Cell lines”), columns A–D show p o ein ID, p o ein name, gene name, and numbe o unique pep ides;
columns E–M show he no malized abundance o each o h ee eplica es o un ea ed HepG2 cells (“HepG2”),
SNU423 cells (“S3”), and SNU475 cells (“S5”); columns N–Q show he p o ein abundance old change o SNU423
and SNU475 cells (“FCS3_HepG2” and “FCS5_HepG2”) ela i e o HepG2 oge he wi h he s a is ical signi icance
index (p alue). A alue o “1” means no signi ican FC.
Au ho Con ibu ions:
Concep ualiza ion, J.M., J.A.B., and C.A.P.; Fo mal analysis, M.J.L.-G. and R.G.; Funding
acquisi ion, J.M., J.A.B., and C.A.P.; In es iga ion, M.J.L.-G. and R.G.; Me hodology, M.J.L.-G. and R.G.; So wa e,
M.J.L.-G.; W i ing—o iginal d a , J.A.B., and C.A.P.; W i ing— e iew & edi ing, J.M.
Funding:
This esea ch has been inanced by g an s om he Spanish Minis y o Economy and Compe i i eness
(BFU2016-80006-P), he Ins i u e o Heal h Ca los III (ISCIII) (PI13/00021 and PI16/00090), and he Andalusian
Go e nmen (Conseje
í
a de Econom
í
a, Inno aci
ó
n, Ciencia y Empleo, BIO-0216 and CTS-6264; Conseje
í
a de
Igualdad, Salud y Pol
í
icas Sociales, PI-00025-2013, and PI-0198-2016). We hank he Biomedical Resea ch
Ne wo k Cen e o Li e and Diges i e Diseases (CIBERehd) ounded by he ISCIII and co- inanced by Eu opean
De elopmen Regional Fund “A way o achie e Eu ope” ERDF o hei inancial suppo .
Acknowledgmen s:
Technical suppo by he s a o he P o eomics acili y, Cen al Se ice o Resea ch Suppo
(SCAI) a he Uni e si y o Co doba, and he compu e esou ces, echnical expe ise and assis ance p o ided by
he PAB (Andalusian Bioin o ma ics Pla o m) cen e loca ed a he Uni e si y o Málaga a e acknowledged.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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