Sodium/p o on exchange iso o m 1 egula es in acellula pH and cell
p oli e a ion in human o a ian cance
Ca los Sanhueza
a,
⁎, Joaquín A aos
a
, Luciano Na anjo
a
, Fe nando Toledo
a,b
, Ana R Bel án
c,d
,
Ma co A Ramí ez
a,d
, Jaime Gu ié ez
a,e
,FabiánPa do
a,
,And eaLei a
a
, Luis Sob e ia
a,g,h,
⁎⁎
a
Cellula and Molecula Physiology Labo a o y (CMPL), Di ision o Obs e ics and Gynaecology, School o Medicine, Facul y o Medicine, Pon ificia Uni e sidad Ca ólica de Chile,
San iago 8330024, Chile
b
Depa men o Basic Sciences, Facul y o Sciences, Uni e sidad del Bío-Bío, Chillán 3780000, Chile
c
Depa men o Educa ion, Facul y o Educa ion, Uni e sidad de An o agas a, An o agas a 1270300, Chile
d
Biomedical Depa men , Facul y o Heal h Sciences, Uni e sidad de An o agas a, An o agas a 1270300, Chile
e
Cellula Signalling Di e en ia ion and Regene a ion Labo a o y (CSDRL), Heal h Sciences Facul y, Uni e sidad San Sebas ian, San iago 7510157,Chile
Me abolic Diseases Resea ch Labo a o y, Cen e o Resea ch, De elopmen and Inno a ion in Heal h - Aconcagua Valley, San Felipe Campus, School o Medicine, Facul y o Medicine,
Uni e sidad de Valpa aíso, San Felipe 2172972, Chile
g
Depa men o Physiology, Facul y o Pha macy, Uni e sidad de Se illa, Se ille E-41012, Spain
h
Uni e si y o Queensland Cen e o Clinical Resea ch (UQCCR), Facul y o Medicine and Biomedical Sciences, Uni e si y o Queensland, He s on, QLD 4029, Queensland, Aus alia
abs ac a icle in o
A icle his o y:
Recei ed 16 Augus 2016
Recei ed in e ised o m 5 Oc obe 2016
Accep ed 18 Oc obe 2016
A ailable online 21 Oc obe 2016
Cance cells gene a e p o ons (H
+
) ha a e ex uded o he ex acellula medium mainly ia he Na
+
/H
+
exchange 1 (NHE1), which egula es in acellula pH (pHi) and cell p oli e a ion. In p ima y cul u es o
human asci es-de i ed o a ian cance cells (haOC) we assayed whe he NHE1 was equi ed o pHi modula ion
and cell p oli e a ion. Human o a y exp esses NHE1, which is highe in haOC and A2780 (o a ian cance cells)
compa ed wi h HOSE cells (no mal o a ian cells). Basal pHi and pHi eco e y ( ollowing a NH
4
Cl pulse)
was highe in haOC and A2780, compa ed wi h HOSE cells. Zonipo ide (NHE1 inhibi o ) caused in acellula
acidifica ion and pHi eco e y was independen o in acellula bu e capaci y, bu educed in NHE1 knockdown
A2780 cells. Zonipo ide educed he maximal p oli e a ion capaci y, cell numbe , hymidine inco po a ion, and
ki67 (ma ke o p oli e a ion) fluo escence in haOC cells. SLC9A1 ( o NHE1) amplifica ion associa ed wi h
lowe o e all pa ien su i al. In conclusion, NHE1 is exp essed in human o a ian cance whe e i has a
p o-p oli e a i e ole. Inc eased NHE1 exp ession and ac i i y cons i u e an un a ou able p ognos ic ac o in
hese pa ien s.
© 2016 Else ie B.V. All igh s ese ed.
Keywo ds:
NHE1
pHi
Human
O a ian cance
Cell p oli e a ion
1. In oduc ion
O a ian cance is equen ly diagnosed a ad anced s ages [1,2].
Cance cells gene a e p o ons (H
+
)[3], which a e eleased by
memb ane anspo sys ems esul ing in low ex acellula pH (pHo),
i.e., acidi y, bu high in acellula pH (pHi), i.e., alkaliza ion [4].The
memb ane anspo sys ems in ol ed in pHi con ol include he
sodium (Na
+
)/H
+
exchange s (NHEs) [5–8]. NHE iso o m 1 (NHE1)
p edomina es and plays c i ical oles in cance [4,8–10]. NHE1 ac i i y
causes in acellula alkaliza ion and ex acellula acidifica ion [4,11].
Since NHE1 inac i a ion educes p oli e a ion o human umou
gas ic [12] and small lung cance [13] cells, NHE1 may ac as a
p o-p oli e a i e ac o in human umou cells.
Mechanisms o pHi con ol in he human o a y o asci es-cance
cells a e unknown [8]. Since non-human o a y cells exhibi NHEs ac i -
i y [14–18], NHE1 may con ol he pHi in he human o a y. Howe e ,
he e a e no epo s add essing exp ession o ac i i y o NHE1 in
human o a ian cance cells [8]. In his s udy, we show ha unc ional
NHE1 is exp essed in human o a ian cance cells playing a c i ical ole
in pHi eco e y and cell p oli e a ion. Thus, NHE1 exp ession and
ac i i y a e ac o s ha could esul in pHi-dependen modula ion o
human o a ian cance cells p oli e a ion. The la e could be ele an
o he educed su i al o pa ien s su e ing om his disease.
Biochimica e Biophysica Ac a 1863 (2017) 81–91
Abb e ia ions: NHEs, Na
+
/H
+
exchange s; NHE1, NHE iso o m 1; haOC, human
asci es-de i ed o a ian cance cells; K, cell g ow h a e; D
, doubling ime; K/D
, maximal
p oli e a ion capaci y; HMA, 5-N,N-hexame hylene amilo ide; V-ATPases, V ype ATPases;
1/
pHi
E, pHi modula ion e ficiency; TCGA, The Cance Genome A las; CNA, DNA copy-
numbe al e a ions.
⁎Co esponding au ho .
⁎⁎ Co espondence o: L. Sob e ia Cellula and Molecula Physiology Labo a o y (CMPL),
Di ision o Obs e ics and Gynaecology, School o Medicine, Facul y o Medicine, Pon ificia
Uni e sidad Ca ólica de Chile, P.O. Box 114-D, San iago 8330024, Chile.
E-mail add esses: [email protected] (C. Sanhueza), sob e [email protected]
(L. Sob e ia).
h p://dx.doi.o g/10.1016/j.bbadis.2016.10.013
0925-4439/© 2016 Else ie B.V. All igh s ese ed.
Con en s lis s a ailable a ScienceDi ec
Biochimica e Biophysica Ac a
jou nal homepage: www.else ie .com/loca e/bbadis
2. Ma e ials and me hods
2.1. S udy g oups
Pa ien s included in his s udy (3 pa ien s) cou sed wi h o a ian
cance (high deg ee o o a ian se ous papilla y ca cinoma s age IIIC o
IV, and endome ioid mix ype ca cinoma wi h high deg ee se ous
di e en ia ion) and we e all subjec ed o p ima y su ge y. O a ian
asci es samples we e ob ained om he Hospi al Clínico UC-CHRISTUS
in San iago de Chile (collec ed by D Mau icio Cuello om Di ision o
Obs e ics and Gynaecology, School o Medicine, Pon ificia Uni e sidad
Ca ólica de Chile). E hnici y o pa ien s included in his s udy was
A
B
0.0
0.5
1.0
1.5
2.0
2.5
Immunosignal o NHE1
(CTCF x106)
Tumou epi helium S oma
*
C
NHE1 CK7
S
Te
NHE1
Fig. 1. NHE1 exp ession in human o a y. A. NHE1 and cy oke a in-7 (CK7) p o eins (a ows) de ec ed by immunofluo escence in human o a ian cance issue sec ions. B. NHE1 p o ein a he epi helial
o igin (Te) and s oma non-epi helial (S) egions (do ed line limi s Te and S). C. NHE1 immunosignal in Te and S om B. Ba s: 50 μm in A and 200 μminB.*Pb0.05. Mean ± S.E.M. (n = 3).
82 C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
Hispanic. The in es iga ion con o ms o he p inciples ou lined in he
Decla a ion o Helsinki. E hics Commi ee app o al om he Facul y o
Medicine o he Pon ificia Uni e sidad Ca ólica de Chile and in o med
consen o pa ien s we e ob ained.
2.2. Cell cul u e
P ima y cul u es o human asci es-de i ed o a ian cance cells
(haOC) we e es ablished by mixing equal amoun s o eshly isola ed
asci es wi h MCDB 105 (Sigma-Ald ich, S Louis, MO, USA)/medium
199 (M199) (Gibco, G and Island, NY, USA) (1:1 / ) cul u e
media con aining 10% e al bo ine se um (FBS), 12.5 mmol/L
NaHCO
3
, 4.5 mmol/L D-glucose, 12.5 mmol/L (4-(2-hyd oxye hyl)-
1-pipe azinee hanesul onic acid (HEPES), 100 IU/mL penicillin, and
100 μg/mL s ep omycin (37°C, pH 7.4) (MCDB 105/M199 cul u e medi-
um). Cells a 70% confluence we e ha es ed wi h ypsin/e hylenediamine
e aace ic acid (EDTA) (0.05/0.2%, 3 minu es, 37°C) and g ew up o passage
2[19]. The human o a y cell lines HOSE (no mal o a ian su ace
epi helium) and A2780 (o a y cance cells) (kindly p o ided by
D Ca men Rome o om he Hospi al Clínico Uni e sidad de Chile,
San iago, Chile) we e main ained in Dulbecco’smodified Eagle’s
F12 medium (DMEM-F12) (Gibco) con aining high (5 mmol/L)
D-glucose and supplemen ed wi h 10% FBS, 16 mmol/L NaHCO
3
,
15 mmol/L HEPES, 100 IU/mL penicillin and 100 μg/mL s ep omycin
(DMEM-F12 cul u e medium).
2.3. Cell p oli e a ion assay
HOSE (6.5 x10
3
cells/cm
2
), A2780 (2.0 x10
4
cells/cm
2
), and p ima y
cul u es o haOC (1.0 x10
4
cells/cm
2
) we e seeded in 24 well pla es and
cul u ed o 0-48 hou s in MCDB 105/M199 o haOC o DMEM-F12
cul u e media o HOSE and A2780 in he absence (Con ol) o p esence
o 100 nmol/L zonipo ide (NHE1 inhibi o ) [20]. Cells we e esuspended
ollowing ypsin/EDTA (0.05/0.2%, 3 minu es, 37°C) diges ion and
coun ed in a haemocy ome e as desc ibed [21]. Cell g ow h a es (K)
we e de i ed om he exponen ial g ow h equa ion:
K¼ln C
ðÞ−ln Ci
ðÞ
•ln eðÞ
whe e is ime in cul u e, C
is numbe o cells a a gi en ime in cul u e,
C
i
is numbe o cells a he beginning o he expe imen (i.e., = 0 hou ),
and eis 2.7182. The K alues we e exp essed as numbe o cells x10
3
pe
cm
2
o cell cul u e su ace pe hou [21].Doubling ime(D
) o cell
g ow h was de i ed om 0.6932/Kand exp essed in hou s. Co ec ed
g ow h a es by he co esponding doubling imes a any gi en ime
(i.e., maximal p oli e a ion capaci y) in cul u e was es ima ed om K/D
.
2.4. Thymidine inco po a ion
Cells in he loga i hmic phase o cell g ow h (i.e., 40-45% conflu-
ence) we e incuba ed wi h [
3
H] hymidine (2 μCi/mL, 6-[
3
H] hymidine,
6.7 Ci/mmol (NEN, D eieich, Ge many), 48 hou s, 37°C) in
MCDB105/M199 o haOC o DMEM-F12 cul u e media o HOSE
and A2780 cells in he absence o p esence o zonipo ide. A e
his incuba ion pe iod he cells we e insed wi h phospha e-bu e ed
sal (PBS) solu ion ((mmol/L) NaCl 130, KCl 2.7, Na
2
HPO
4
0.8, KH
2
PO
4
1.4 (pH 7.4, 4°C)) and exposed o 5% ichlo oace ic acid (TCA, 200
mL, 10 minu es). TCA was emo ed and cells insed wi h 99% me hanol
(200 mL) and diges ed wi h 25 mmol/L o mic acid o adioac i i y
de e mina ion as desc ibed [21].
A
B
haOC
HOSE A2780
0
5
10
15
20
NHE1
(a bi a y uni s)
HOSE A2780 haOC
*
*
HOSE A2780 haOC
NHE1
-Ac in
-ac in/
Fig. 2. NHE1 p o ein abundance in human o a ian umou cells. A. Wes e n blo o NHE1 and β-ac in (in e nal e e ence) p o ein abundance in HOSE, A2780, and haOC cells. Lowe panel:
NHE1/β-ac in a io densi ome ies no malized o 1 in HOSE. B. Immunofluo escence o NHE1 (a ows show NHE1). Ba s: 10 μm. *Pb0.01. Mean ± S.E.M. (n = 3-7).
83C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
2.5. Measu emen o pHi
Cell monolaye s in 96 well pla es (70% confluence) we e incuba ed
o 10 minu es a 37°C wi h he fluo escen pH sensi i e p obe 2,7-
bica boxye hyl-5,6-ca boxyfluo escein ace oxyme hyl es e (BCECF-
AM, 12 μmol/L) (Sigma-Ald ich), as desc ibed [22]. Some expe imen s
we e also pe o med in cells incuba ed o 1 minu e wi h BCECF-AM,
and he esul s o pHi we e no significan ly di e en om 10 minu es
incuba ion (no shown). The excess o p obe was emo ed by inse
h ee imes wi h con ol solu ion (CS) ((mmol/L) NaCl 145, KCl 5,
NaH
2
PO
4
1Na
2
SO
4
1, CaCl
2
1.8, MgCl
2
1, HEPES 30, D-glucose 5
(pH 7.4, 37°C). The fluo escence a ios was egis e ed in a fluo ime e
Tecan M200P o (Un e sbe gs , Aus ia) a e al e na e cell exci a ion
a 440 and 490 nm, and emission a 530 nm egis e ed e e y 2 seconds
in e al o a pe iod o 150 seconds. pHi was es ima ed by in e pola ion
o emission a ios using s anda d calib a ion cu es in cells incuba ed
wi h 10 μmol/L nige icin in a calib a ing solu ion ((mmol/L) KCl 130,
NaCl 20, CaCl
2
1, MgCl
2
1, HEPES 5 (pH 6.2, 7.2, o 8.2)) as desc ibed
[21,22]. Cells we e incuba ed wi hou o wi h 5 μmol/L 5-N,N-
hexame hylene amilo ide (HMA, NHEs gene al inhibi o ) [23],100
nmol/L zonipo ide [20],0.1μmol/L concanamycin A (V ype ATPases
(V-ATPases) inhibi o ) [24],and10μmol/L Sche ing 28080 (H
+
/K
+
ATPase inhibi o ) [25].
2.6. Reco e y o pH
i
The pH
i
eco e y was examined by applying he NH
4
Cl pulse
echnique [21,22]. In b ie , BCECF-AM loaded cells we e incuba ed in
CS un il he basal pH
i
was s abilized (~3 minu es). Cells we e exposed
(2 minu es) o CS supplemen ed wi h NH
4
Cl (NH
4
Cl/CS solu ion)
((mmol/L) NaCl 121, KCl 5.4, CaCl
2
1, KH
2
PO
4
0.4, MgCl
2
0.5, MgSO
4
0.4, Na
2
HPO
4
0.3, HEPES 10, D-glucose 0.6, NH
4
Cl 20 (pH 7.4, 37°C)).
A e his incuba ion pe iod he NH
4
Cl/CS solu ion was eplaced
by insing he cells wi h CS ee o NH
4
Cl (
0
Na
+
/CS) ((mmol/L)
N-me hyl-D-glucamine (NMDG) 120, KCl 5, CaCl
2
1.8, MgCl
2
1, HEPES
30, D-glucose 5 (pH 7.4, 37°C)), wi hou o wi h HMA, zonipo ide,
concanamycin A and/o Sche ing 28080 as abo e.
Reco e y a es o pH
i
(dpHi/d ) we e calcula ed om da a collec ed
o he fi s 60 seconds o he eco e y (i.e., a e emo ing he NH
4
Cl
load) and fi ed by a fi s o de lineal eg ession as desc ibed [21,22].
The esul s we e exp essed in pH
i
uni s/minu e. The NHEs-media ed
componen o dpHi/d (
NHEs
) was es ima ed om:
NHEs ¼
HMAα−δ
HMAþCAþSchα−δ•100
whe e
HMA
αis dpHi/d inhibi ed by HMA,
HMA+CA+Sch
αis dpHi/d
inhibi ed by HMA + concanamycin A + Sche ing, and δ ep esen s
he co esponding emaining dpHi/d (backg ound) on each condi ion.
The in ol emen o NHE1 (
NHE1
)in he
NHEs
componen was
es ima ed by
NHE1
=
NHEs
–
Z
α,whe e
Z
αis dpHi/d inhibi ed
by zonipo ide. The in ol emen o V-ATPase (
V
)andH
+
/K
+
ATPase (
H/K
) componen s we e es ima ed as o
NHEs
conside ing
CA
α(dpHi/d inhibi ed by concanamycin A) o
Sch
α(dpHi/d inhibi ed by
Sche ing) and
HMA+Z+Sch
α(dpHi/d inhibi ed by HMA + zonipo ide +
Sche ing) o
HMA+Z+CA
α(dpHi/d inhibi ed by HMA + zonipo ide +
concanamycin A), espec i ely.
Ini ial eloci y o pHi eco e y was de i ed om slope o linea
phase o dpHi/d adjus ed o he one phase exponen ial associa ion
equa ion conside ing he leas squa es fi [26]:
i¼dpHi
d 1−e−k ðÞ
whe e
i
is ini ial eloci y, dpHi/d is mayo eco e y a e a a gi en ime
( ) (in his s udy = 0.03 seconds) and eand ka e cons an s. The
capaci y o cells o change he pHi alue in ce ain uni s o pHi as a esul
in he dpHi/d modifica ions (i.e., e ficiency o pHi modula ion (1/
pHi
E))
was es ima ed om:
1=pHiE¼ΔdpHi=d ðÞ
ΔpHi •α
whe e ΔdpHi/d is he change in dpHi/d om con ol, ΔpHi is he change
in pHi om con ol, and αis a gi en pHi uni .
2.7. In insic bu e ing capaci y
The abili y o in insic cellula componen s o bu e changes in pHi,
i.e., in acellula bu e capaci y (βi), was measu ed as desc ibed [21].
A e de e mining he basal pHi he cells we e incuba ed in a 0.5
mmol/L KCl-con aining
0
Na
+
/CS plus Sche ing 28080 + concanamycin
A (pH 7.4, 37°C) un il he pHi was s abilized unde his expe imen al
condi ion (~3 minu es). Cells we e hen incuba ed in he la e solu ion
con aining dec easing concen a ions o NH
4
Cl (50, 20, 10, 5, 2.5 o 1
mmol/L). To assay he e ec o each o he concen a ions o NH
4
Cl he
cells we e insed h ee imes wi h he co esponding lowe NH
4
Cl con-
cen a ion used in his s udy. The βiwas calcula ed om he exp ession:
βi¼ΔNHþ
4
i
ΔpHi
whe e he in acellula NH
4
+
concen a ion ([NH
4
+
]
i
) was ob ained om
he Hende son-Hasselbalch equa ion on he assump ion ha [NH
3
]
i
Table 1
pHi and pHi eco e y a es in human o a y cells.
HOSE A2780 haOC
pHi
Con ol 7.391 ± 0.034 7.621 ± 0.044 * 7.840 ± 0.053 *
HMA 7.262 ± 0.112 †7.574 ± 0.145 *†7.648 ± 0.095 *†
Zonipo ide (Z) 7.270 ± 0.079 †7.383 ± 0.161 †7.656 ± 0.069 *†
Concanamycin A (CA) 7.601 ± 0.183 7.546 ± 0.031 7.858 ± 0.196
Sche ing 28080 (Sch) 7.692 ± 0.132 7.607 ± 0.043 7.782 ± 0.163
dpHi/d (pHi uni s/minu e)
Con ol 0.114 ± 0.081 0.152 ± 0.025 * 0.194 ± 0.050 *
HMA 0.018 ± 0.007 * 0.026 ± 0.017 * 0.004 ± 0.001 *
Z 0.051 ± 0.006 *†0.026 ± 0.008 * 0.049 ± 0.006 *†
CA 0.110 ± 0.010 ‡0.062 ± 0.018 *‡0.196 ± 0.086 ‡
Sch 0.106 ± 0.013 ‡0.080 ± 0.011 *‡0.157 ± 0.082 ‡
HMA + Z 0.025 ± 0.002 *§ 0.025 ± 0.017 *§ 0.001 ± 0.001 *§
HMA + CA 0.012 ± 0.015 *§ 0.034 ± 0.008 *§ 0.001 ± 0.005 *§
HMA + Sch 0.014 ± 0.009 *§ 0.037 ± 0.011 *§ 0.001 ± 0.030 *§
HMA + Z + CA 0.012 ± 0.029 *§ 0.039 ± 0.016 *§ 0.009 ± 0.001 *§
HMA + Z + Sch 0.025 ± 0.004 *§ 0.033 ± 0.018 *§ 0.005 ± 0.014 *§
HMA + CA + Sch 0.011 ± 0.002 *§ 0.012 ± 0.007 *§ 0.011 ± 0.002 *§
HMA + Z + CA + Sch 0.026 ± 0.016 *§ 0.006 ± 0.011 *§ 0.001 ± 0.023 *§
Z + CA 0.053 ± 0.032 * 0.031 ± 0.010 *# 0.047 ± 0.011 *
Z + Sch 0.061 ± 0.057 * 0.022 ± 0.015 *# 0.037 ± 0.009 *
Z + CA + Sch 0.051 ± 0.055 * 0.011 ± 0.015 *# 0.037 ± 0.009 *
CA + Sch 0.112 ± 0.013 $ 0.033 ± 0.025 *£ 0.120 ± 0.051 $
The in acellula pH (pHi) was measu ed in BCECF-AM–p eloaded HOSE, A2780 and
human asci es o a ian cance cells (haOC) in he absence (Con ol) o p esence o 5
μmol/L 5-N,N-hexame hylene amilo ide (HMA) (NHEs gene al inhibi o ), 100 nmol/L
zonipo ide (Z) (NHE1 inhibi o ), 0.1 μmol/L concanamycin A (CA) (V ype ATPases
inhibi o ), o 10 μmol/L Sche ing 28080 (Sch) (H
+
/K
+
ATPase inhibi o ) as desc ibed in
Ma e ials and Me hods. Ini ial a es o pH
i
eco e y (dpHi/d ) we e de e mined in cells
subjec ed o an acid pulse (NH
4
Cl assay) as abo e. In pHi sec ion: *Pb0.05 e sus Con ol
in HOSE. †Pb0.05 e sus co esponding Con ol alues. In dpHi/d sec ion: *Pb0.05 e sus
Con ol in HOSE o in he co esponding cell ype. †Pb0.05 e sus all o he co esponding
alues excep Z + CA, Z + Sch, and Z + Ca + Sch. ‡Pb0.05 e sus co esponding HMA and
Z. §Pb0.05 e sus co esponding alues in Z, CA, o Sch. ¶Pb0.05 e sus co esponding
alues in HMA (alone o in mix wi h o he inhibi o s), CA, and Sch. #Pb0.05 e sus
co esponding alues in CA o Sch. $Pb0.05 e sus co esponding alues in HMA and Z
(alone o in mix wi h o he inhibi o s), CA o Sch. £Pb0.05 e sus all o he co esponding
alues excep CA and Sch. Values a e mean ± S.E.M. (n = 3-22).
84 C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
(in acellula NH
3
) was equi alen o [NH
3
]
o
(ex acellula NH
3
), and Δ
pHi is he ac ion o change in uni s o pHi alue.
Knowing he dpHi/d and he βi alues (a simila pHi alues in
each cell ype), he a e o o e all ansmemb ane H
+
flux (J
H+
) was
calcula ed om he ollowing exp ession:
JHþ¼dpHi
d •βi
2.8. Wes e n blo ing
To al p o ein was ob ained om confluen cells insed (x2) wi h
ice-cold PBS and ha es ed in 100 μL o lysis bu e (100 mmol/L NaCl,
10 mmol/L Na
4
P2O
7
x10-H
2
O, 10 mmol/L NaF, 1% T i on X100,
1 mmol/L sodium o ho anada e, 50 mg/mL leupep in, 20 mmol/L
HEPES (pH 7.4, 4°C) as desc ibed [22]. Cells we e sonica ed (6 cycles,
5 seconds, 100 W, 4°C) and o al p o ein was sepa a ed by
cen i uga ion (13500 g, 15 minu es, 4°C). P o eins (70 μg) we e
sepa a ed by polyac ylamide gel (8%) elec opho esis, ans e ed o
Immobilon-P poly inylidene difluo ide memb anes (BioRad Labo a o ies,
He o dshi e, UK) and p obed wi h p ima y monoclonal abbi an i-
NHE1 (1:500 dilu ion, 12 hou s, 4°C) o monoclonal mouse an i-β-ac in
(1:2500 dilu ion, in e nal e e ence) (San a C uz Bio echnology,
San a C uz, CA, USA) an ibodies. The memb anes we e insed in
PBS/0.1% Tween 20 (PBS-T) and u he incuba ed (1 hou ) in
PBS-T/0.2% bo ine se um albumin (BSA) con aining seconda y
ho se adish pe oxidase-conjuga ed goa an i- abbi o an i-mouse
an ibodies (The mo Scien ific, Rock o d, IL, USA). P o eins we e
de ec ed by enhanced chemiluminescence in a ChemiDoc-I 510
Imagen Sys em (UVP, LCC Upland, CA, USA) and quan ified by
densi ome y [22].
2.9. Immunofluo escence mic oscopy
Pa a o maldehyde-fixed, pa a fin-embedded o a ian cance se ial
sec ions (4 μm) we e dewaxed and subsequen ly ehyd a ed wi h se ial
incuba ions using 100, 95, o 70% e hanol solu ions. His ological
slices we e pe meabilized (3% Tween 20, 10 minu es) and blocked
wi h de a ed-cow milk (5% in PBS, 1 hou , 22°C). NHE1, cy oke a in-7
(CK-7) (ma ke o epi helial cells) and (ma ke o p oli e a ion)
we e immunolocalized ollowing incuba ion (o e nigh a 4°C) wi h
p ima y policlonal abbi an i-NHE1 (1:200 dilu ion) (San a C uz
Bio echnology), monoclonal mouse an i-CK-7 (1:200 dilu ion) (Dako,
Glos up, Denma k) o an i-ki67 (1:50 dilu ion) (The mo Scien ific,
Rock o d, IL, USA) in PBS con aining 2% BSA (PBS/2% BSA) and ollowed
by incuba ion (1 hou , 22°C) wi h he seconda y an ibody Alexa Fluo
568 goa an i- abbi o an i-mouse IgG (H+L, λexc/λem: 578/603 nm,
1:750) (Li e Technologies).
024681012
6.5
7.0
7.5
8.0
8.5
Time (minu es)
pHi
haOC
HOSE
A2780
+ NH
4
Cl - NH
4
Cl
0.0 0.5 1.0 1.5 2.0
0.0
0.1
0.2
0.3
0.4
Time (seconds)
pHi uni s
haOC
A2780
HOSE
6.0 6.5 7.0 7.5 8.0
0
10
20
30
40
50
pHi
i
((mmol/L) /pHi uni s)
HOSE
A2780
haOC
0.0
0.5
1.0
1.5
2.0
2.5
JH+
((mmol/L)/second)
HOSE A2780 haOC
*
*
0
2
4
6
8
10
12
i
((mmol /L)/pHi uni s)
HOSE A2780 haOC
A
ED
C
B
Fig. 3. pHi eco e y. A. HOSE, A2780, and haOC cells we e exposed o 20 mmol/L NH
4
Cl (+NH
4
Cl). NH
4
Cl was emo ed (–NH
4
Cl) and ini ial a es o pHi eco e y we e calcula ed (see
Me hods). B. Ini ial eloci y o pHi eco e y as in A. C. In acellula bu e ing capaci y (βi) de e mined as desc ibed in Me hods. D.βi o co esponding pHi om da a in C. E.H
+
flux
a es (J
H+
) (see Me hods). *Pb0.05 e sus all o he alues. Values a e mean ± S.E.M. (n = 3-10).
85C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
Cells we e g own on mic oscope co e glasses (6 x10
4
cells pe slide)
(Sail B and, Shangai, China) in co esponding cul u e media o 70% con-
fluence as desc ibed [27].Inb ie ,cellswe efixed (4% pa a o maldehyde,
15 minu es), pe meabilized (0.1% T i on X-100, 10 minu es) and blocked
(PBS/2% BSA, 1 hou , 22°C). NHE1 and ki67 we e immunolocalized
ollowing incuba ion (o e nigh a 4°C) wi h p ima y policlonal abbi
an i-NHE1 (1:200 dilu ion) o monoclonal mouse an i-ki67 (1:50
dilu ion) in PBS/2% BSA and ollowed by incuba ion (1 hou , 22°C) wi h
he seconda y an ibody Alexa Fluo 488 goa an i-mouse IgG (H+L,
λexc/λem: 488/519 nm, 1:750 dilu ion) o Alexa Fluo 568 goa
an i- abbi IgG (H+L, λexc/λem: 578/603 nm, 1:750 dilu ion) (Li e
Technologies). Cell nuclei we e s ained wi h Hoesch 33342 (4 μmol/L,
10 minu es, 22°C) (The mo Scien ific). Images we e ob ained unde an
EVOS FL Imaging Sys em (AMF 4300) (Li e Technologies). Fluo escence
quan ifica ion was pe o med by he me hod o Co ec ed To al
Fluo escence In ensi y (CTCF) as desc ibed [28] in egions o op ical
in e es (ROI) in se ial slices. ROI was defined as a sec ion o he issue
ha was posi i e (epi helial) o nega i e (s oma) o he epi helial
ma ke CK-7 whe e NHE1 and ki67 s aining was de e mined. Images
we e p ocessed wi h Image J e sion 1.48 (Wayne Rusband NIH, USA).
2.10. NHE1 supp ession
Supp ession o NHE1 exp ession was done using he comme cially
a ailable sho in e e ence RNAs (siRNA) NHE-1 siRNA (h) o
con ol sequence (siC) (San a C uz Bio echnology) ollowing
manu ac u e ’s ins uc ions (h p://da ashee s.scb .com/siRNA_
p o ocol.pd ). HOSE and A2780 cells knockdown o NHE1
(
KD-NHE1
HOSE and
KD-NHE1
A2780, espec i ely) we e gene a ed.
haOC cells we e no iable in i o unde his app oach, hus
KD-NHE1
haOC
cells was no possible.
2.11. TCGA da a analysis
Genomic, mRNA exp ession and clinical in o ma ion o high-g ade
se ous cance we e ob ained in silico om The Cance Genome A las
O a ian Cance (h ps://confluence.b oadins i u e.o g/display/GDAC/
Home). Analyses o pa ien ’s su i al and NHE1 signalling pa hway
we e pe o med in cBioPo al pla o m o Cance Genomics (h p://
www.cbiopo al.o g/s udy.do?cance _s udy_id=o _ cga) acco ding o
p e iously published p o ocols [29,30].
2.12. S a is ical analysis
The alues a e mean ± S.E.M., whe e nindica es he numbe o di -
e en biological samples (3 asci es om pa ien s wi h o a ian cance )
and co esponding cell cul u es wi h 3-4 eplica es pe expe imen .
Compa isons be ween wo g oups we e pe o med by means o
S uden ’s unpai ed - es . The di e ence be weenmo e han wo g oups
was pe o med by ANOVA (one o wo ways). I he ANOVA demon-
s a ed a significan in e ac ion be ween a iables, pos hoc analyses
we e pe o med by he mul iple-compa ison Bon e oni es . To
es ima e he co ela ion be ween NHE1 and ki67 exp ession in issue
samples, he co ela ion analysis was pe o med using Pea son’s
co ela ion coe ficien ( ). Pa ien su i al analyses we e add essed by
Kaplan Meie me hodology using log- ank, Wilcoxon pa ame e s.
AC
BD
Fig. 4. NHE1 in ol emen in pHi eco e y. A. pHi eco e y a es (dpHi/d ) in HOSE, A2780, and haOC cells wi hou (Con ol, 100%) o wi h 5 μmol/L 5-N,N-hexame hylene
amilo ide (HMA), 100 nmol/L zonipo ide (Z), 0.1 μmol/L concanamycin A (CA), and/o 10 μmol/L Sche ing 28080 (Sch). B.dpHi/d ac ion media ed by NHEs (
NHEs
), V ype
ATPases (
V
), o H
+
/K
+
ATPase (
H/K
) componen s. A ows indica e he dpHi/d ac ion media ed by NHE1 (
NHE1
)o
NHEs
componen (see Me hods). ‘All’is all componen s
o dpH/d .C.Wes e nblo o NHE1andβ-ac in (in e nal e e ence) p o ein abundance in HOSE and A2780 exp essing NHE1 (
NHE1
HOSE,
NHE1
A2780) o NHE1 knockdown
(
KD-NHE1
HOSE,
KD-NHE1
A2780) cells. Lowe panel:NHE1/β-ac in a io densi ome ies no malized o 1 in
NHE1
HOSE cells. D.dpH/d in cells as in C. In A: *Pb0.04 e sus Con ol,
†Pb0.05 e sus all o he co esponding alues, ‡Pb0.05 e sus all o he alues excep o Z in HOSE cells. In B: *Pb0.05 e sus all o he alues, †Pb0.04 and ‡Pb0.05 e sus
co esponding alues in HOSE and haOC cells. In C and D: *Pb0.05 e sus co esponding alues in
NHE1
HOSE o
NHE1
A2780. In D: †Pb0.05 e sus all o he alues. Values a e mean ±
S.E.M. (n = 3-10).
86 C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
Pb0.05 alue was conside ed s a is ically di e en . The s a is ical
so wa e G aphPad InS a 3.0b and G aphPad P ism 7.0b (G aphPad
So wa e Inc., San Diego, CA, USA) we e used o da a analysis. Pb0.05
was conside ed s a is ically significan .
3. Resul s
3.1. NHE1 exp ession in human o a y
The fi s goal o his s udy was o de e mine whe he NHE1 was
exp essed in human o a ian cance issue. O a ian cance issue was
posi i e o NHE1 and CK7 (Fig. 1A). NHE1 was de ec ed in umou
epi helium and s oma (Fig. 1B), wi h highe fluo escence a he epi he-
lium, compa ed wi h s oma (Fig. 1C). We hen assayed whe he NHE1
was exp essed in cul u ed human o a ian cance cells. NHE1 p o ein
was de ec ed in all cell ypes (Fig. 2A) and was highe in haOC and
A2780 compa ed wi h HOSE cells. NHE1 p o ein was de ec ed in he
cells and in he cell pe iphe y (Fig. 2B).
3.2. Basal pHi and dpHi/d
Since NHE1 exp ession associa es wi h pHi modula ion in se e al
umou cells [4–10], we de e mined basal pHi and he po en ial
unc ional in ol emen o his and o he memb ane anspo
mechanisms o egula e in acellula H
+
con en . Basal pHi in
haOC and A2780 was highe han in HOSE cells (Table 1). Zonipo ide
ABC
D
Fig. 5. NHE1 in ol emen in cell p oli e a ion. A. HOSE, A2780, and haOC cells cul u ed in he absence (Con ol) o p esence o 100 nmol/L zonipo ide. Cell numbe was de e mined in a
haemocy ome e (see Me hods). B. Cell numbe , hymidine inco po a ion, and ki67 posi i e cells in he absence (Con ol) o p esence o zonipo ide (+Zonipo ide) as in A. C.
Immunofluo escence o NHE1 and ki67 p o ein in human o a ian cance issue sec ions (ba s: 50 μm). The g aph ep esen s NHE1 e sus ki67 immunosignal adjus ed o a fi s -o de
equa ion (Pea son’s co ela ion coe ficien ( ) = 0.83, goodness-o -fi R
2
=0.672,Pb0.0001, n = 32 ROIs in 3 pa ien s). (D) Cell numbe in HOSE and A2780 exp essing NHE1
(
NHE1
HOSE,
NHE1
A2780) o NHE1 knockdown cells (
KD-NHE1
HOSE,
KD-NHE1
A2780). Values a e no malized o 1 in
NHE1
HOSE o
NHE1
A2780 cells. In B: *Pb0.05 e sus Con ol. In D, *Pb0.04
e sus co esponding
NHE1
HOSE o
NHE1
A2780. Values a e mean ± S.E.M. (n = 3-10).
Table 2
E ec o zonipo ide on he pa ame e s o human o a y cells p oli e a ion.
K(1/hou ) D
(hou ) K/D
(1/(hou )
2
)
HOSE
Con ol 0.01702 ± 0.00477 41 ± 11 0.00042 ± 0.00011
Zonipo ide 0.01131 ± 0.00402 61 ± 22 0.00019 ± 0.00007 *
A2780
Con ol 0.03949 ± 0.00304 †17 ± 1 †0.00232 ± 0.00016 †
Zonipo ide 0.03328 ± 0.00401 †21 ± 3 †0.00158 ± 0.00023 *†
haOC
Con ol 0.01357 ± 0.00131 51 ± 1 0.00027 ± 0.00003
Zonipo ide 0.00791 ± 0.00074 * 88 ± 1 * 0.00009 ± 0.00001 *
HOSE, A2780, and human asci es o a ian cance cells (haOC) cells we e seeded in 24-well
pla es (2.05 cm
2
su ace) and cul u ed o 48 hou s in p ima y cul u e medium wi hou
(Con ol) o wi h 100 nmol/L zonipo ide. Cell numbe was es ima ed by coun ing a
di e en pe iods o ime and cell g ow h a es (K) and doubling ime (D
) o cell g ow h
was defined as desc ibed in Ma e ials and Me hods. K/D
ep esen s co ec ed cell g ow h
a e by doubling imes (maximal p oli e a ion capaci y) o cells in cul u e. *Pb0.05 e sus
co esponding Con ol alues. †Pb0.05 e sus co esponding alues in HOSE and haOC
cells. Values a e mean ± S.E.M. (n = 3-22).
87C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
and HMA, bu no concanamycin A o Sche ing 28080 caused
in acellula acidifica ioninallcell ypes.Toes ima e he
capaci y o cells o eco e om an acidic pHi cells we e subjec ed
o he NH
4
Cl pulse assay [21–23]. The acidic pHi caused by he
NH
4
Cl pulse was ully es o ed a e ~3 minu es (Fig. 3A). Basal
dpHi/d (Table 1)and
i
o dpHi/d (Fig. 3B) in haOC (
i
= 10.2 ±
0.001 pHi uni s/minu e) and A2780 cells (
i
= 9.8 ± 0.002 pHi
uni s/minu e) we e highe compa ed wi h HOSE cells (
i
=7.8±
0.001 pHi uni s/minu e).
3.3. ßi and J
H+
Since a change in basal pHi and dpHi/d depends on he ßi capaci y
o cells [21], his pa ame e was es ima ed and used o de e mine J
H+
.
The esul s show ha ßi inc eased as pHi alue dec eased (Fig. 3C)
and ßi a equal pHi a e a 20 mmol/L NH
4
Cl pulse (pHi HOSE = 6.95,
pHi haOC = 6.93, pHi A2780 = 6.95) we e simila in all cell ypes
(Fig. 3D). In addi ion, J
H
+
was highe in haOC and A2780 compa ed
wi h HOSE cells (Fig. 3E).
3.4. NHE1, V-ATPase, and H
+
/K
+
ATPase in ol emen on dpHi/d
To know he ela i e con ibu ion o he memb ane anspo
mechanisms in ol ed in he egula ion o pHi, he dpH/d was
measu ed in he absence o p esence o specific inhibi o s o
NHE1, V-ATPase, and H
+
/K
+
ATPase. The esul s show ha HMA
educed dpHi/d in all cell ypes (Table 1). Pa ial inhibi ion was
de ec ed in HOSE and A2780 cells; howe e , HMA abolished dpHi/d
in haOC cells (Fig. 4A). Zonipo ide caused a pa ial educ ion o
dpHi/d in HOSE and haOC cells, which was simila o HMA e ec in
A2780 cells. Concanamycin A and Sche ing 28080 caused pa ial e-
duc ion (47-59%) o dpHi/d only in A2780 cells. The
NHEs
componen
accoun ed o a ac ion o dpHi/d simila in HOSE and haOC cells
(Fig. 4B). Howe e ,
NHEs
accoun ed o almos all dpHi/d in A2780
cells. In ol emen o
NHE1
componen in
NHEs
esul ed in a ac ion
o dpHi/d ha was simila in HOSE and haOC cells; howe e , in
A2780 cells accoun ed o all he
NHEs
componen . The
V
and
H/K
componen s we e de ec ed only in A2780 cells.
Wi h he aim o co obo a e ha NHE1 exp ession is equi ed o
a pHi eco e y, cells knockdown o his p o ein we e used o
0
1×10
7
2×10
7
3×10
7
SLC9A1, mRNA exp ession
(RNA Seq V2 RSEM) (log2)
Homologous
dele ion
Shallow
dele ion
Diploidism Gain Ampli ica ion
050100150200
0
25
50
75
100
Mon hs
Pe cen su i al
Ampli ica ion
Homologous dele ion
Diploid
Gain
Shallow dele ion
A
B
CD
Fig. 6. NHE1 exp ession and su i al in pa ien s wi h o a ian cance . A. Analysis o he DNA copy-numbe al e a ions (CNA) o SLC9A1 in human o a ian cance (The Cance Genome A las
O a ian Cance (TCGA) da abase) ( he numbe o cases wi h he indica ed gene ic al e a ions (Gene ic Al e a ion) is gi en). B. Rela ion be ween mRNA numbe o copies and al e a ions in
CNA o SLC9A1 in pa ien s wi h high-g ade se ous o a ian cance . C. Rep esen a ion o NHE1 pa hway as in C. G een a ows depic s di ec -modula o y pa hway o SLC9A1 by endo helin-1
(END1)/endo helin ecep o A (ENDRA) and epide mal g ow h ac o (EGF)/EGF ecep o (EGFR). D. O e all su i al o pa ien s wi h o a ian cance acco ding o he indica ed CNA
al e a ions (by cBioPo al pla o m o Cance Genomics).
88 C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
es ima e dpHi/d . The NHE1 p o ein abundance was lowe in
KD-NHE1
HOSE
and
KD-NHE1
A2780 cells compa ed wi h cells exp essing NHE1 (Fig. 4C).
The
KD-NHE1
HOSE and
KD-NHE1
A2780 cells also show lowe dpHi/d
compa ed wi h cells exp essing NHE1 (Fig. 4D).
3.5. Cell p oli e a ion and NHE1 and ki67 co ela ion
Since an acidic pHo due o inc eased ac i i y o NHE1 is associa ed
wi h inc eased umou cells p oli e a ion [12,13], cell p oli e a ion was
assayed in he p esence o a NHE1 inhibi o . The esul s show ha
zonipo ide educed p oli e a ion (Fig. 5A) and he K/D
in all cell ypes
(Table 2). The educ ion in he K/D
was less p onounced in A2780
compa ed wi h HOSE o haOC cells. Cell g ow h a e was highe , bu
doubling imes we e lowe in A2780 cells compa ed wi h HOSE o
haOC cells. Zonipo ide dec eased g ow h a e and inc eased he
doubling ime only in haOC cells. Resul s in he kine ics o cell
g ow h we e complemen ed wi h an es ima ion o he changes in
he ac ual cell numbe changes and nucleic acid u no e . The cell num-
be and [
3
H] hymidine inco po a ion we e educed by zonipo ide
(Fig. 5B). Addi ionally, he numbe o ki67 posi i e cells (ma ke o
cell p oli e a ion) in he p esence o zonipo ide was lowe in A2780
and haOC, bu unal e ed in HOSE cells.
To confi m a pa allel inc ease in NHE1 and ki67 p o ein exp ession
in human o a ian issue, a po en ial co ela ion be ween hese p o eins
exp ession was analysed. The esul s show ha NHE1 and ki67 p o eins
we e de ec ed in he same egions in o a ian cance issues, wi h a pos-
i i e co ela ion o NHE1 and ki67 p o ein abundances (Fig. 5C). Addi-
ionally, he cell numbe coun ed in
KD-NHE1
HOSE and
KD-NHE1
A2780
cells was lowe compa ed wi h hese cells exp essing NHE1 (Fig. 5D).
3.6. SLC9A1 and o e all su i al
In he aim o knowing whe he NHE1 o e exp ession associa es
wi h ad e se p ognos ic in pa ien s wi h high-g ade se ous o a ian can-
ce , we analysed in silico he DNA copy-numbe al e a ions (CAN) o
SLC9A1. Analysis o he CNA o his gene in human o a ian cance e-
eals ha 4% o cases will p esen wi h gene ic changes including ampli-
fica ion (nine pa ien s), deep dele ions (one pa ien s), and up egula ion
(six pa ien s) o down egula ion ( wo pa ien s) o mRNA exp ession
(Fig. 6A). SLC9A1 mRNA exp ession inc eases acco ding o he ype o
CNA al e a ion in pa ien s wi h high-se ous o a ian cance (Fig. 6B).
The analysis in silico e eals ha SLC9A1 is in di ec -pa hway wi h
endo helin-1 (END1)/endo helin ecep o A (ENDRA) and wi h epide -
mal g ow h ac o (EGF)/EGF ecep o (EGFR) in pa ien s wi h o a ian
cance (Fig. 6C). SLC9A1 amplifica ion associa ed wi h educed o e all
su i al (~50%) compa ed wi h pa ien s wi hou changes in SLC9A1
(diploid (98 pa ien s) (41.4 e sus 20.5 mon hs in nine pa ien s wi h
SLC9A1 amplifica ion, espec i ely) (Log- ank = 0.0005, Wilcoxon =
0.0013) o wi h mild CNA al e a ion (shallow dele ion (108 pa ien s)
o gain (63 pa ien s)) (Fig. 6D). The e was no a di e ence be ween am-
plifica ion and homologous dele ions (5 pa ien s, Log- ank = 0.36,
Wilcoxon = 0.68), o homologous dele ion and o e all su i al when
compa ed wi h unal e ed o mild CNA al e a ion.
4. Discussion
This s udy shows ha NHE1 is exp essed in he human o a ian can-
ce and p ima y cul u es o human asci es o a ian cance cells (haOC).
Tumou cells show in acellula alkaliza ion compa ed wi h no mal
issues [4,31,32]. This phenomenon depends on he exp ession o mem-
b ane anspo sys ems o H
+
emo al [8,33]. haOC cells show pHi
alues highe (~0.35 pHi uni s) han in non- umou cells, sugges ing
ha pHi egula ion is al e ed by his pa hology. This condi ion may be
esponsible o cell malignancy since an inc ease in 0.2-0.4 pHi uni s
associa es wi h highe cance cell p oli e a ion [4,21,33]. NHE1, 2, 3,
and 4 a e c i ical egula o s o pHi in umou issues [8,34]. Ou esul s
show ha NHE1 p o ein is exp essed in CK7 posi i e umou epi helium
in he human o a y. Addi ionally, haOC cells show inc eased NHE1 ex-
p ession (~12 old) compa ed wi h non- umou cells, sugges ing i s po-
en ial ole in his ype o cance . haOC cells also show inc eased dpHi/d
and pHi ha was compa able o ha in non- umou cells, sugges ing
ha in acellula alkaliza ion may esul om a highe dpHi/d .Indeed,
inc eased
i
o dpHi/d in haOC ag ees wi h o he cance cells [4,8,31,
32]. Since haOC and non- umou cells in insic bu e capaci y (ßi)
we e simila , inc eased dpHi/d and J
H
+
a e no due o abno mal cell
capaci y o bu e a change in pHi. In e es ingly, he ßi alues de e -
mined in HOSE, haOC, and A2780 cells a e in he same ange as o
o he umou cells such as T
84
cells (~31 (mmol/L)/pHi uni s) [22,35],
and lowe han in non- umou MDCK cells (~110 (mmol/L)/pHi uni s)
[21]. The di e ences in he βi alues could be due o specie di e ences
(human e sus dog) since he expe imen al condi ions o de e mine
his pa ame e we e simila in hese s udies.
HMA educed he dpHi/d and pHi, sugges ing a ole o NHEs in
hese phenomena. Since HMA- educed pHi was highe (~1.5 old) in
haOC han non- umou cells, haOC cells a e mo e sensi i e o NHEs
ac i i y-dependen pHi egula ion. Same esul s we e ound in cells
ea ed wi h zonipo ide. Thus, haOC cells equi e NHE1 ac i i y by
ala ge ac ion(
NHE1
/
NHEs
~0.8), in his phenomenon.
NHE1
in ol emen o inc ease pHi is suppo ed by he lowe dpHi/d de ec ed
in NHE1-knockdown o a ian umou cells.
The maximal p oli e a i e capaci y (K/D
) o haOC was simila o
non- umou cells, sugges ing ha o a ian cance does no al e o a ian
cells p oli e a ion. NHE1’s ac i i y plays a p e e en ial ole in haOC cells
p oli e a ion since zonipo ide- educed K/D
was ~1.3 old highe han
non- umou cells. Since a posi i e co ela ion be ween NHE1 and ki67
p o ein abundance was ound (i.e., NHE1-exp ession dependen p oli -
e a ion), and zonipo ide educes K/D
(i.e., NHE1-ac i i y dependen
p oli e a ion), NHE1 exp ession and ac i i y a e equi ed o haOC
cells p oli e a ion. The la e is suppo ed by esul s showing
zonipo ide- educed haOC cell numbe and DNA u no e .
haOC cells show pHi modula ion e ficiency (1/
pHi
E~4.74 pHi
uni s/minu e) lowe han in non- umou cells. Thus, haOC cells
may coun wi h a less e ficien mechanism p o ec ing o in acellu-
la acidifica ion han in non- umou cells. Zonipo ide- educed K/D
in haOC cells may depend on a lowe 1/
pHi
Ein haOC (1/
pHi
E~0.17
pHi uni s/minu e in he p esence o zonipo ide) compa ed wi h
non- umou cells (1/
pHi
E~0.11 pHi uni s/minu e in he p esence o
zonipo ide) [(K/D
)/1/
pHi
E~0.31 in haOC and ~0.11 in HOSE cells].
This mechanism will a ou umou cells su i al since in acellula
acidifica ion causes cell dea h [36–38]. In e es ingly, a high pHi
a ou s he ansi ion om phase S o G2/M in he cell cycle and
DNA/RNA syn hesis [6,39]. Ou esul s show ha zonipo ide educed
[
3
H] hymidine inco po a ion. Thus, blocking NHE1 ac i i y leads o
acidic pHi-dependen educed p oli e a ion likely due o educed cell
cycle p og ession o haOC cells. These findings a e suppo ed by he e-
duced dpHi/d de ec ed in A2780 and non- umou NHE knockdown
cells. Thus, NHE1 exp ession and ac i i y a e p o-p oli e a i e ac o s
in human o a ian cells.
Pa allel analysis o CNA in SLC9A1 ( o NHE1) e ealed ha a ac ion
o pa ien s showed wi h CNA including highe amplifica ion. Since
SLC9A1 mRNA exp ession is inc eased depending on CNA al e a ions,
his change in SLC9A1 could de e mine human o a ian cance s age.
CNA al e a ions may modula e he SLC9A1 exp ession by endo helin-1
and epide mal g ow h ac o , complemen ing epo s in ol ing
endo helin-1 and endo helin ecep o A in his ype o cance [40]
and endo helin-1 in combina ion wi h β-a es in-1 ac i a ion inc eas-
ing chemo esis ance [41]. Equally, ac i a ion o epide mal g ow h
ac o /epide mal g ow h ac o ecep o signalling is in ol ed in highe
angiogenesis [42] and esis ance o pacli axel [43] in human o a ian
cance . Highe se um epide mal g ow h ac o [44] and endo helin-1
in he pe i oneal fluid in pa ien s wi h malignan asci es [45,46] a e
epo ed in human o a ian cance . Thus, epide mal g ow h ac o and
89C. Sanhueza e al. / Biochimica e Biophysica Ac a 1863 (2017) 81–91
