Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of Colorectal Cancer
Li SJ (2013) Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of
Colorectal Cancer. PLoS ONE 8(8): e70550. doi:10.1371/journal.pone.0070550
Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of Colorectal Cancer
Yifan Wang 0
Xingye Wu 0
Qiang Li 0
Shangrong Zhang 0
Shu Jie Li 0
Rakesh K. Srivastava, The University of Kansas Medical center, United States of America
0 1 Department of Biophysics, School of Physics Science, Nankai University , Tianjin , China , 2 Department of Pathology, Tonghua Center Hospital , Tonghua , China
Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn2+ respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.
Funding: This work was supported by National Natural Science Foundation of China (31271464). The funder had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The voltage-gated proton channel Hv1 was identified using
bioinformatics searches based on known cation channels, which is
mainly expressed in immune cells such as macrophages,
neutrophils, and eosinophils [1,2]. Hv1 in mammalian phagocytes was
proposed to be responsible for the proton-transporting pathway,
which regulates intracellular pH during oxygen consumption
associated with phagocytosis, called respiratory burst [3,4]. Hv1
is activated by depolarization and intracellular acidification, whose
activity maintains intracellular pH neutral to keep reactive oxygen
species (ROS) generation [5,6]. Hv1 not only regulates pH in
cytoplasm, but can also provide protons in the phagosome, a
closed membrane compartment for killing and digestion of a
pathogen . Hv1 is extremely selective for H+, with no detectable
permeability to other cations [7,8]. The voltage activation
relationship of Hv1 depends strongly on both the intracellular
pH (pHi) and extracellular pH (pHo). Increasing pHo or lowering
pHi promotes H+ channel opening by shifting the activation
threshold to more negative potentials . Furthermore, Hv1
current is inhibited by submillimolar concentrations of Zn2+ and
Cd2+ and other divalent cations .
Hv1 contains three predicted domains: N-terminal acid and
proline-rich domain, transmembrane voltage-sensor domain
(VSD), and C-terminal domain. Voltage-gated K+ channels are
comprised of four subunits, each of which has a pore domain and
a VSD. The four pore domains come together to form one single
central pore, and four peripheral VSDs control the gate of the
pore . In contrast to the voltage-gated K+ channels, the Hv1
contains a VSD but lacks the pore domain. Recent studies showed
that Hv1 functions as a dimer in which the intracellular
Cterminal domain is responsible for the dimeric architecture of the
protein, and each subunit contains its own proton-transporting
pathway . The intracellular C-terminal domain of Hv1
forms a dimer via a parallel a-helical coiled-coil and is essential for
the protein localization .
Tumor cells often exist in a hypoxic microenvironment, and
possess high-glycolytic activity and produce acidic metabolites
[15,16]. To avoid the acidosis resulting from reducing in cytosolic
pH, tumor cells must extrude excessing cytosolic protons to
maintain cytosolic pH, which results in acidic tumor
microenvironment. The hypoxic and acidic tumor microenvironment plays
a key role in cancer development, progression, and metastasis .
Our previous work showed that Hv1 is specifically expressed in
highly metastatic human breast tumor tissues and cell lines, and
promotes breast cancer cell progression and metastasis, through
regulating breast cancer cell intracelular pH [17,18]. In the
present study, we investigated the expression of Hv1 in colorectal
Age (yr) median 62.4, range 3676
tumor tissues and cell lines and its potential association with
clinicopathological features and post-resectional survival.
Inhibitions of Hv1 expression and activity in the highly metastatic
colorectal cancer cells markedly decrease the cell invasion and
migration, restraint proton extrusion. Our results suggest that Hv1
over-expression may be used as an independent biomarker for the
prognosis and diagnosis of patients with colorectal cancer.
Materials and Methods
All of the procedures were done in accordance with the
Declaration of Helsinki and relevant policies in China. We
obtained the written informed consent from all participants
involved in our study. The study obtained ethics approval for
our study from the ethics committee of Tonghua Center Hospital.
Patients and samples
Colorectal cancer tissue samples were obtained from patients
who underwent routine curative surgery at the Department of
Surgery, Tonghua Center Hospital between 2001 and 2007. The
patients were not pretreated with radiotherapy or chemotherapy
prior to surgery. 139 colorectal cancer tissues and paired adjacent
non-tumor colorectal tissues were fixed in 10% formalin and
embedded in paraffin for immunohistochemical analysis. The
clinicopathological features of these patients were shown in
Table 1. In addition, to verify the expression of Hv1 in
premalignant dysplastic lesions, 10 normal colorectal, 18
hyperplastic polyp and 20 adenoma tissues were also examined using
immunohistochemistry. Each patients clinical status was classified
according to the pathologic tumor grade, tumor size, lymph node
status. Tumor differentiation was graded by the Edmondson
grading system. This study was approved by the Ethics Committee
of Tonghua Center Hospital, and informed consent was obtained
from each patient.
Generation of an anti-Hv1 polyclonal antibody
An anti-Hv1 polyclonal antibody was generated against the
carboxyl terminal domain of Hv1 (residues 221273 of Hv1,
QEIERLNKLLRQHGLLGEVN). The protein was purified to
homogeneity after expression in Escherichia coli . The purified
protein was injected into mice and the anti-Hv1 polyclonal
antibody was purified by rProtein A Sepharose (GE, Healthcare)
column. HRP-conjugated goat anti-mouse IgGs were purchased
from Jackson ImmunoResearch Laboratories, Inc. (West Grove,
Expression vector and transfection
Hv1 cDNA was cloned into pEGFP-N1 (Clontech) to create
Hv1 expression plasmid pHv1-EGFP, fused with the enhanced
green fluorescent protein (EGFP) moiety attached to the
Cterminus of Hv1 . 293 T cells were cultured in DMEM
(Dulbeccos modified Eagles medium; GIBCO) with 10% fetal
bovine serum plus antibiotics (100 units/ml penicillin and 100 g/
ml streptomycin, GIBCO) in a 5% CO2 incubator at 37uC. Cells
grown on glass coverslips at 5070% confluence in a six-well plate
were transiently transfected with pHv1-EGFP plasmid by using
lipofectamine 2000 (Invitrogen) following the manufacturers
protocol. Cells were used for experiments 3648 h after
Lymph node status
Western blotting was performed with an antiHv1 polyclonal
antibody (1 mg/ml) as described above with a final dilution of
1 1000. The denatured proteins were separated by 12.5%
SDSPAGE and then transferred to a PVDF membrane (GE,
Healthcare) by wetting electroblotting devices. Nonspecific protein
absorption was prevented using 5% (w/v) skim milk in
phosphatebuffered saline containing 0.1% Tween 20 (PBS-T) for 1 h.
Primary antibody incubation in PBS-T was performed for 1 h at
room temperature. The HRP-coupled anti-mouse secondary
antibody was used at a final dilution of 1 15,000 in PBS-T, and
HRP was revealed with a chemiluminescent detection system
Histological diagnoses of tumourous and non-tumourous
formalin-fixed and paraffin-embedded tissues were confirmed in
haematoxylin and eosin-stained sections. Immunohistochemistry
was performed with an anti-Hv1 polyclonal antibody. The
antiHv1 antibody (1.0 mg/ml) was diluted 100-fold with PBST
(phosphate-buffered saline containing 0.1% tween-20) containing
1% (w/v) BSA. The paraffin-embedded sections filled with 10 mM
ethylenediaminetetracetic acid (EDTA) buffer (pH 8.0) were
heated in a microwave oven for 12 min. After cooling, the
sections were treated with 0.5% Triton X-100 in PBS for 10 min,
exposed to 3% (v/v) hydrogen peroxide (H2O2) for 10 min to
inhibit endogenous peroxidase activity. Subsequently, the sections
were incubated in PBST containing 5% fetal bovine serum and
2% BSA for 30 min to reduce nonspecific binding. Incubation
with primary antibody was performed overnight at 4uC in a
humidified chamber. HRP-coupled anti-mouse secondary
antibody was used at a final dilution of 1 400 for 1 h. Finally, the
visualization signal was developed with diaminobenzidine (DAB)
and the slides were counterstained in hematoxylin. Negative
control was performed to treat with non-immune mouse serum as
the primary antibody instead of anti-Hv1 antibody.
Stained sections were evaluated in a blinded manner without
prior knowledge of the clinical information using the German
immunoreactive score, Immuno-Reactive-Score (IRS). Briefly, the
IRS assigns sub-scores for immunoreactive distribution (04) and
intensity (0-3), then multiplies them to yield the IRS score. The
percent positivity was scored as 0 (,5%), 1 (525%), 2 (25
50%), 3 (5075%), 4 (.75%). The staining intensity was
score according to the area of Hv1-positive staining cells as 0, 2
(negative, ,5%), 1, + (weakly positive, 525%), 2, ++
(positive, 2550%), and 3, +++ (strongly positive, .50%). The
final Hv1 expression score was calculated from the values of
percent positivity score and staining intensity score, which was
ranged from 0 to 12. We estimated IRS by averaging the values in
eight fields at 6500 magnification for each specimen. Hv1
expression levels were defined as follows: low expression (score
#3) and high expression (score .3). Immunohistochemical
analysis and scoring were performed by two independent
The human colorectal cancer cell lines SW620, HT29, LS174T,
Colo205 and SW480 were cultivated at 37uC in an atmosphere of
95% air and 5% CO2 with DMEM supplemented with 10% fetal
bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml
streptomycin, and 20 mM L-glutamine.
SW620, HT29, LS174T, Colo205 and SW480 cells grown on
glass coverslips at confluence in six-well tissue culture plates were
fixed with 4% (w/v) paraformaldehyde in PBS at room
temperature for 30 min, washed in PBS, treated with 0.5% Triton
X-100 in PBS for 20 min, exposed to 3% (v/v) hydrogen peroxide
(H2O2) for 15 min, and blocked with 5% fetal bovine serum and
2% BSA in PBST for 20 min at room temperature. The blocked
coverslips were incubated with the anti-Hv1 antibody (1.0 mg/ml)
at a dilution of 1 200 with PBST containing 1% (w/v) BSA at
37uC for 1 h. After washing in PBS for three times, the coverslips
were further incubated with HRP-coupled anti-mouse secondary
antibody at a final dilution of 1 400 for 1 h. Signals were
visualized by the HRP/DAB system. The cell nuclei were stained
Quantitative real-time PCR
The mRNA expression levels of Hv1 in SW620, HT29,
LS174T, Colo205 and SW480 cells, were evaluated by
quantitative real-time PCR using ABI PRISM 7000 Sequence Detection
System (Applied Biosystems, Foster City, CA). Total RNAs were
extracted using RNAiso Reagent (Takara), and reverse-transcribed
by MMLV super transcriptase (Takara). Real-time quantitative
reverse transcription PCR was performed with SYBR
PrimeScriptTM RT-PCR Kit (Takara) according to the manufacturers
instructions. The primers were as follows: Hv1,
59-CAGGTCATCATCATCTGCTTG-39 (forward) and
59CCGTTCTGAACGTGTCTTAAC-39 (Reverse); GAPDH,
59CCAAGGTCATCCATGACAAC-39 (forward) and
59-AGAGGCAGGGATGATGTTCT-39 (reverse). PCR thermal condition
used was: 94uC for 30 s; annealing, 52uC for 30 s; extension, 72uC
for 30 s. Relative mRNA expression levels of proteins were
calculated according to the equation: 22DDCT, in which
DDCT = (CTHv1 - CTGAPDH) - (CTHv1 - CTGAPDH)SW620. All
experiments were performed in triplicate.
SW620 and SW480 cells grown on glass coverslips were fixed
with 4% (v/v) paraformaldehyde in phosphate-buffered saline
(PBS) at room temperature for 30 min, washed in PBS, treated
with 0.5% Triton X-100 in PBS for 20 min, and blocked with 5%
fetal bovine serum and 2% BSA in PBST for 1 h. The blocked
coverslips were incubated with anti-Hv1 antibody (1.0 mg/ml) at a
dilution of 1 200 in 2% BSA at 4uC overnight. After washing with
PBS for 5 min for four times, the coverslips were further incubated
for 1 h at room temperature with a FITC-conjugated goat
antimouse IgG at a dilution of 1 400 in 2% BSA, followed by another
washing as described above. Confocal images of FITC
fluorescence of SW620 and SW480 cells were recorded on a Leica TCS
SP5 confocal microscopy (LEICA, Germany) with the FITC-filter
set for Hv1 and the DAPI filter set for the nuclear DAPI dye. The
images were later processed by Adobe Photoshop software.
Suppressing Hv1 mRNA expression
The sequence of the siRNA targeting the Hv1 gene was
59CTACAAGAAATGGGAGAAT-39, and the random sense
sequence was 59-TTCTCCGAACGTGTCACGT-39, both of
which were obtained from Ribobio (Guangzhou, China) .
SW620 cells grown at confluence were passed in 6-well plates at
30% confluence and incubated overnight, then transfected with
the siRNA and the negative control, respectively, using
lipofectamine 2000 (Invitrogen), according to the manufacturers protocol.
The final concentration of siRNA was 100 nM. Silencing was
examined 48 h after transfection. The efficiency of siRNA in
suppressing Hv1 expression was determined by quantitative
realtime PCR, immunocytochemistry and western blotting using
antiHv1 antibody as described above.
To examine the effect of Hv1 on the migratory ability of
colorectal cancer cells, migrations of SW620, SW480, and SW620
cells down-regulated Hv1 expression and inhibited Hv1 activity by
siRNA and Zn2+ respectively, were assessed in wounded
monolayer model. To down-regulate Hv1 expression, SW620 cells were
grown to confluence and transfected with siRNA and negative
control for 24 h, respectively. To inhibit Hv1 activity, 1 M ZnCl
solution was added into the DMEM medium to a final
concentration of 100 mM [1,2]. The SW620 and SW480 cells
were planted in a 24-well plate (1.56105 per well) and cultured for
24vh to confluence and subsequently wounded with a tip. Cell
movement was observed under phase-contrast microscopy, and
were captured with a digital camera every 12 h.
Invasion and migration assays
In vitro invasion and migration assays were performed to assess
the effects of Hv1 on invasive and migratory abilities of SW620
and SW480 cells. SW620 and SW480 cells were cultured in
sixwell plate in DMEM medium with 10% FBS at confluence,
transfected with the siRNA and negative control respectively for
24 h, and then trypsinized, washed and counted. For cell invasion,
transwells with 8 mm pore size filters (Millipore) were covered with
matrigel (Becton Dickinson) and inserted into 24-well plates. And
for cell migration, the transwells were not coated with matrigel.
DMEM medium (500 ml) containing 10% FBS was added to the
lower chamber, and 200 ml of a cell suspension (56104 cells) was
placed in the upper chamber. The plates were incubated at 37uC
in humidified atmosphere containing 5% CO2 for 24 h. To inhibit
Hv1 activity, 1 M ZnCl solution was added into the DMEM
medium to a final concentration of 100 mM [1,2]. The penetrated
cells were fixed by paraformaldehyde, stained with crystal violet
solution, and photographed. Each experiment was conducted four
times. The migration and invasion rates were calculated as
[migration cell No. of test/migration cell No. of controlSW620]
6100% and [invasion cell No. of test/invasion cell No. of control
SW620] 6100%, respectively.
Activity of Hv1 channel
The activity of Hv1 in colorectal cancer cells was assessed as a
change in intracellular pH (pHi) in response to membrane
depolarization by BCECF fluorescence . The cells were
incubated with 3.0 mM of BCECF-AM (Molecular Probe) in
serum-free DMEM medium for 30 min, respectively, and washed
with PSS solution (140 mM NaCl, 5 mM KCl, 5 mM glucose,
1 mM CaCl2, 1 mM MgCl2, 20 mM Tris, pH 7.5) for 3 times.
Cells were incubated with NH4Cl/NMDG (N-methyl
D-glucamine) solution (100 mM NMDG, 40 mM NH4Cl, 5 mM KCl,
5 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 20 mM Tris, pH 7.3)
for 20 min and washed by ammonium free solution (140 mM
NMDG, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgCl2,
20 mM Tris, pH 7.4), rapidly inducing intracellular acidification
. Membrane depolarization was achieved by loading high K+
solution (145 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM
MgCl2, 20 mM Tris, pH 7.5). Intracellular pH changes
acidloaded cells were detected by fluorescent probe BCECF at
excitation wavelengths of 490 nm and 440 nm and an emission
wavelength of 525 nm under membrane depolarizing condition
using RF-5301PC Spectrofluorophotometer (Shimadzu, Japan).
Measurements of intracellular pH
Intracellular pH was measured using the pH sensitive
fluorescent probe BCECF-AM. Cells cultured in the monolayers were
incubated with 3.0 mM of BCECF-AM (Molecular Probe) in
bicarbonate-free DMEM medium at 37uC for 30 min. After
loading, the cells were washed three times with HEPES buffer to
remove the extracellular dye, and made to remain in the identical
buffer. The HEPES buffer contained 140 mM NaCl, 5 mM KCl,
1 mM MgSO4, 1 mM CaCl2, 1 mM NaH2PO4, 5.5 mM glucose,
and 20 mM HEPES, pH 7.4. The fluorescence at excitation
wavelengths of 490 nm and 440 nm was recorded at an emission
wavelength of 525 nm using RF-5301PC
Spectrofluorophotometer (Shimadzu, Japan). Calibration of fluorescence vs pH
was performed by equilibration of external and internal pH with
nigericin (10 mM) in a high K+ buffer with a range of pH from 5.5
to 8.0. The high K+ buffer contained 145 mM KCl, 5 mM
glucose, 1 mM CaCl2, 1 mM MgCl2, and 20 mM HEPES (or
MES). The relative fluorescence ratio values were plotted against
corresponding pHi values, which allowed determination of the
All statistics were performed using SPSS16.0 software.
Measurement data was represented as mean 6 SD. Comparison of the
mean between groups was performed by t test. P values ,0.05
were considered significant. Survival analysis was assessed using
Kaplan-Meier method and survival rate was compared by
Clinicopathologic profiles from the 139 cases selected for this
study were reviewed in Table 1. The average age of the patients
was 62.4 years (range, 3676), including 68 males and 71 females.
The locations of their cancers were 29 left-sided and 43 right-sided
colon, and 47 rectum cases, respectively. Among the 139 resected
cases, the primary tumor size varied as follows: ,5 cm in 72 cases,
and $5 cm in 67 cases. Twenty of 139 tumors showed poor
cytological differentiation. The tumor extent was limited (T1 or
T2) in 45 cases and advanced (T3 or T4) in 94 cases. Tumor
metastasis to the lymph nodes was observed in 59 of 139 cases.
Increased expression of Hv1 in colorectal cancer
Hv1 expressed in immune cells is associated with respiratory
burst [3,4], but its function in tumorigenesis has not been
identified. To investigate Hv1 for use as a potential biomarker and
therapeutic target for colorectal cancer, Hv1 expression in 139
colorectal cancer tissues and paired normal tissues, 10 normal
colorectal, 20 colorectal adenoma and 18 hyperplastic polyp
tissues, was detected using immunohistochemistry with an
antiHv1 polyclonal antibody that was generated in house. To examine
the specificity of the antibody, 293 T cells were transfected with
pHv1-EGFP expression plasmid. And the expression of
Hv1EGFP was detected by immunocytochemistry and western blotting
with the antibody, and EGFP fluorescence. The results showed
that the antibody specifically recognizes Hv1 and EGFP is a
marker for Hv1 expression (Fig. 1A and B).
As shown in Fig. 1C and Table 2, Hv1 staining was mainly
moderate or strong positive in colorectal cancer tissues, but not in
normal colorectal and hyperplastic polyp tissues. Hv1 was mainly
observed in the plasma membrane of tumor cells in colorectal
tissues, as shown in Fig. 1C (h, j and l) (as indicated by
arrowheads). In colorectal adenoma tissues, the staining was
negative or weakly positive (Fig. 1C, e and f; Table 2). Hv1 in
colorectal cancer tissues was significantly expressed compared with
that in normal colorectal, hyperplastic polyps and adenoma
tissues, suggesting that Hv1 may be involved in colorectal
tumorigenesis. Overall, 106 of the 139 (76.3%) cases showed high
expression Hv1 in the tumor tissues (IRS over 3), while 33 (23.7%)
of the cases showed low expression (IRS 03). Generally, Hv1
density was significantly higher in cancer tissues than in adenoma
tissues (7.2063.25 versus 2.2062.12) (Table 2).
High Hv1 expression is associated with a poor prognosis
The correlations between Hv1 expression and clinicopathologic
characteristics are summarized in Table 3. There were significant
associations with the depth of tumor classification (P = 0.007), age
(P = 0.021), tumor size (P = 0.000), lymph node status (P = 0.000),
clinical stage (P = 0.000) and p53 status (P = 0.014) in patients who
had high Hv1 expression compared with patients who had low
Hv1 expression. There was no significant association between Hv1
expression and the other clinical features, such as gender,
differentiation and Ki-67 expression. In addition, the correlations
between the expression levels of p53, Ki-67, TopoII, GST-p and
P-gp and clinicopathologic characteristics were showed in Table 4.
p53 status related to tumor classification (P = 0.011), Ki-67 to
differentiation (P = 0.010), TopoII to differentiation (P = 0.010),
and GST-p to tumor size (P = 0.007) and differentiation
(P = 0.000). However, P-gp did not show statistical significance
with clinicopathologic parameters.
Kaplan-Meier survival curves showed that patients who had
high Hv1 expression were more likely to have a shorter overall
Mean Standard deviation Score range
Type of tissues
Normal colorectal 0.00
Hyperplastic polyps 1.15
Colorectal cancer 7.20
*, 0.001; **, 0.001; and ***, 0.001 were compared with normal colorectal.
expression of Hv1 were prone to have an early recurrence
compared with patients who had low expression of Hv1 (37.463.0
vs 47.2610.7, P,0.008) (Table 5).
(n = 33, 23.7%)
survival (P = 0.008, Fig. 2A) and recurrence-free survival
(P = 0.008, Fig. 2B) compared with patients who had low Hv1
expression, suggesting that Hv1 over-expression may be associated
with a poor clinical prognosis. Patients who had high Hv1
expression had a poor recurrence-free survival (P = 0.008)
compared with patients who had low Hv1 expression (univariate
analysis) (Table 5). Overall survival examined by Cox univariate
analysis also indicated that high expression of Hv1 was
significantly associated with shorter survival (P = 0.008). Univariate Cox
regression analyses showed that Hv1 expression level was
significantly associated with recurrence-free and overall survival,
whereas other clinical characteristics lost their predictive
significance. Furthermore, multivariate Cox regression analyses revealed
that high expression of Hv1 was independent risk factor for overall
survival (relative risk [RR] = 0.443, P = 0.015) and recurrence-free
survival (RR = 0.427, P = 0.026) (Table 6). Patients who had high
Tumor classification T1/T2
Lymph node status Negative
Distance metastasis M0
Left colon 15(51.7) 14(48.3) 0.415 13(44.8) 16(55.2) 0.768 6(20.7)
0.720 17(58.6) 12(41.4) 0.491
Tumor size (cm) ,5
30(41.7) 42(58.3) 0.076 32(44.4) 40(55.6) 0.274 15(20.8) 57(79.2)
0.007 50(69.4) 22(30.6) 0.510
15(33.3) 30(66.7) 0.011 21(46.7) 24(53.3) 0.547 11(24.4) 34(75.6)
0.241 29(64.4) 16(35.6) 0.670
Right colon24(55.8) 19(44.2)
30(44.1) 38(55.9) 0.268 36(52.9) 32(47.1) 0.551 12(17.6) 56(82.4)
12(17.6) 0.106 44(64.7) 24(35.3) 0.589
42(53.2) 37(46.8) 0.251 43(54.4) 36(45.6) 0.271 18(22.8) 61(77.2)
0.530 53(67.1) 26(32.9) 0.958
Table 4. Correlation of Hv1 expression levels in colon cancer with clinicopathological parameters, p53, Ki-67, TopoII, GST-p and
Pgp expression levels.
Moderately 32(55.2) 26(44.8)
For p53 and Ki-67, #25%, as a low expression; .25%, as a high expression. For TopoII, GST-p and P-gp, 2/+, as a low expression; .+, as a high expression.
38(47.5) 42(52.5) 0.696 39(48.8) 41(51.2) 0.658 19(23.8) 61(76.2)
10(12.5) 0.910 51(63.8) 29(36.2) 0.357
65(49.2) 67(50.8) 0.742 66(50.0) 66(50.0) 0.713 30(22.7) 102(77.3) 0.720 116(87.9) 16(12.1) 0.207 89(67.4) 43(32.6) 0.573
38(48.7) 40(51.3) 0.957 38(48.7) 40(51.3) 0.662 19(24.4) 59(75.6)
10(12.8) 0.959 50(64.1) 28(35.9) 0.427
27(44.3) 34(55.7) 0.458 24(39.3) 37(60.7) 0.010 9(14.8)
Distribution of Hv1 in human colorectal cell lines
To examine whether Hv1 is also expressed in human colorectal
cancer cell lines, the expression of Hv1 in human colorectal cancer
cell lines, SW620, HT29, LS174T, Colo205 and SW480, was
detected by immunocytochemistry and real time RT-PCR. As
shown in Fig. 3, the expression levels of Hv1 among these
colorectal cancer cell lines have significant difference. Hv1 is
expressed at a high level in SW620, HT29, LS174T, and Colo205
cells, but not in SW480 cells. The localization of Hv1 in SW620
cells was determined by a confocal microscopy. As shown in
Fig. 3C, Hv1 is highly expressed in SW620 cells, which is localized
0.000 36(59.0) 25(41.0) 0.212
in both intracellular sites and plasma membrane (as indicated by
arrowheads), whereas Hv1 is hardly expressed in SW480 cells. The
result that Hv1 expression is higher in SW620 cells than that in
SW480 cells is identical with the results from
immunocytochemistry (Fig. 3A) and real time RT-PCR (Fig. 3B).
Inhibition of Hv1 activity decreases invasion and
migration in highly metastatic colorectal cancer cells
Invasion and migration are two prominent hallmarks of tumor
malignancy. To evaluate the contribution of Hv1 to invasive and
migratory potential in colorectal cancer cells, we performed
invasion and migration assays. First, we examined the kinetics of
migratory ability of SW620, SW480 and HT29 cells. Fig. 4A, B
and C showed the migration kinetics of SW620 (Fig. 4A), SW480
(Fig. 4B) and HT29 (Fig. 4C) cells. A wounded monolayer of
SW620 cells allowed for wound closure after 48 h (Fig. 4A, a, b, c,
d and e). SW620 and HT29 (Fig. 4C, a, b, c, d and e) cells closed
the wound dramatically faster than SW480 cells (Fig. 4B, a, b, c, d
and e). In order to down-regulation of Hv1 expression and
inhibition of Hv1 activity, the siRNA targeting Hv1 and a final
concentration of 100 mM ZnCl2 [1,2] were used, respectively. The
efficiencies of Hv1 knock-down with siRNA in SW620, SW480
and HT29 cells were assessed by western blotting, which showed
the reduction in Hv1 protein levels upon siRNA knockdown in
SW620 and HT29 cells (Fig. 3D). Suppressions of Hv1 expression
by siRNA (f, g, h, i and j in Fig. 4A, B and C) and Hv1 activity by
100 mM ZnCl2 (k, l, m, n and o in Fig. 4A, B and C) clearly
No. of Patients
Median (95% CIa)
No. of Patients
aCI indicates confidence interval.
decreased the migration in SW620 and HT29 cells, but almost
without affecting on SW480 cells (Fig. 4B, f, g, h, i and j for
siRNA; k, l, m, n and o for 100 mM ZnCl2). The time-dependent
wound distances of SW620 (Fig. 4A), SW480 (Fig. 4B) and HT29
(Fig. 4C) were shown in right panels.
We then studied invasion and migration of SW620, SW480 and
HT29 cells using transwell inserts. As shown in Fig. 4D and E,
SW620 cells have markedly higher invasive and migratory abilities
than SW480 cells, which is consistent with the results from
migration kinetics study above. Inhibitions of Hv1 expression by
siRNA and Hv1 activity by ZnCl2 remarkably decreased the
invasion and migration of SW620 cells, but almost did not
influence SW480 cells (Fig. 4D and E). The data indicated that
suppression of Hv1 expression and activity could inhibit the
invasion and migration of the highly metastatic colorectal cancer
cells in vitro, which suggested that Hv1 is involved in the invasion
and migration of the metastatic human colorectal cancer cells.
Table 6. Multivariate Cox proportional hazards analysis for recurrence-free survival and overall survival according to Hv1
No. of Patients
RRa (95% CIa)
No. of Patients
Median (95% CIa)
Median (95% CIa)
aRR and CI indicate relative risk and confidence interval, respectively.
bP values were obtained by Cox proportional hazards analysis modeled for high and low/negative levels of Hv1 expression.
Hv1 involved in regulating intracellular pH
The H+ channel activity of Hv1 in SW620 and SW480 cells was
measured with a pH-sensitive probe BCECF. BCECF is a widely
used pH indicator for estimating intracellular pH (pHi) [20,21]. Its
fluorescence intensity at maximum emission wavelength is
pHdependent: a fall in pH with a decrease in fluorescence intensity,
and to a rise in pH with an increase in fluorescence intensity. We
acid-preloaded and exposed SW620 and SW480 cells to an
outward-acting proton force (in high-K+ medium) that will drive
an efflux of H+ ions. As shown in Fig. 5A the sharp increase on the
fluorescence intensity of BCECF at membrane depolarization was
observed for SW620 cells (Fig. 5A, a (nc)), indicating that an
increase in pHi occurred. In contrast to SW620 cells, the
florescence intensity of BCECF at membrane depolarization
almost did not change for SW480 cells (Fig. 5A, b (nc)).
Suppression of Hv1 expression obviously decreased the florescence
intensity of BCECF at membrane depolarization in SW620 cells
(Fig. 5A, a (si)), whereas did not affect on SW480 cells (Fig. 5A, b
(si)). Inhibition of Hv1 activity by 100 mM ZnCl2 also inhibited
outward proton extrusion in SW620 cells (Fig. 5A, a (Zn2+)). These
results revealed that the pHi recovery was due to active Hv1.
To examine the effect of Hv1 on intracellular pH (pHi), we
measured the pHi in SW620 and SW480 cells using BCECF.
Down-regulation of Hv1 expression by siRNA and inhibition of
Hv1 activity by 100 mM ZnCl2 induced a decrease in intracellular
pH in the highly metastatic colorectal cancer SW620 cells, but not
the poorly metastatic colorectal cancer SW480 cells (Fig. 5B). As
shown in Fig. 5B, down-regulation of Hv1 expression in SW620
cells significantly increased acidity of intracellular pH from 7.5 to
7.0, while inhibition of Hv1 activity by 100 mM ZnCl2 more
remarkably induced acidity of intracellular pH from 7.5 to 6.9 in
SW620 cells. The finding showed that the inhibitions of Hv1
expression and activity in SW620 cells notably suppressed proton
In the present study, our data revealed that Hv1 expression was
markedly higher in colorectal cancer tissues than in normal
colorectal tissues, colorectal adenoma tissues and colorectal
hyperplastic polyp tissues. We observed that Hv1 expression in
colorectal cancer was significantly associated with tumor
recurrence and metastasis. Patients who had high expression of Hv1
were remarkably poor recurrence-free and overall survival
compared with patients who had low expression of Hv1.
Multivariate analysis demonstrated that Hv1 expression level
was an independent prognostic factor for recurrence-free and
overall survival in patients with colorectal cancer. Our results
clearly demonstrated that high Hv1 expression is associated with
poor prognosis and unfavorable clinical outcome of colorectal
To elucidate the mechanism that the effect of Hv1 on colorectal
cancer development and metastasis, the expression of Hv1 in
colorectal cancer cell lines was also detected, and the role of Hv1
in migration and invasion of colorectal cancer cells has been
assessed. We found that Hv1 is highly expressed in highly
metastatic colorectal cell lines, but lowly in poorly metastatic
colorectal cell lines. Down-regulation of Hv1 expression or
inhibition of Hv1 activity notably decreases the migratory and
invasive abilities of the highly metastatic colorectal cancer cells.
Suppression of Hv1 activity in the highly metastatic colorectal
cancer cells restrains the extrusion of intracellular protons and
induces the reduction of intracellular pH (pHi). Therefore, we
conclude that Hv1 regulates intracellular pH (pHi) in the highly
metastatic colorectal cancer cells.
Maintenance of cytosolic pH is vital for all biological processes
in cells. Solid tumor cells often exist in a hypoxic
microenvironment with an acidic extracellular pH (pHo) value than that of
surrounding normal cells . The high glycolytic activity and
acidic metabolites in cancer cells result in the excessive production
of intracellular acidity. To overcome the hypoxic
microenvironment and prevent the intracellular accumulation of the increased
acidic metabolites, tumor cells must be enhanced by the ability to
dispose of the increased intracellular protons. Several pHi
regulatory mechanisms in tumor cells have been described, such
as proton pumps, Na+/H+ exchangers, bicarbonate (HCO32)
transporters, and proton-lactate symporters, which have been
shown to be involved in cancer progression and as promisingly
therapeutic targets for future anticancer therapy [15,2531].
Recent researches have highlighted the fundamental role of the
tumours extracellular metabolic microenvironment in malignant
invasion. This tumour cell microenvironment is acidified primarily
by vacuolar H+-ATPases (V-ATPases) , Na+/H+ exchanger
NHE1 , carbonic anhydrase  and H+/lactate
cotransporter , which are activated in some cancer cells.
The importance of V-ATPases in cancer malignancy has been
repeatedly demonstrated in several human cancer tumors and cell
lines such as hepatocellular carcinoma [15,32]. Inhibition of
VATPase function via knockdown of the protein subunit ATP6L
expression using RNA interfering technology can effectively retard
the growth and metastasis of human hepatocellular carcinoma
xenografts [15,32]. Proton pump inhibitors (PPI), associated with
the inhibition of V-ATPase activity and increasing in both
extracellular pH and the pH of lysosomal organelles, trigger a
rapid cancer cell apotosis in human melanomas,
adenocarcinomas, lymphomas and B cells, as a result of intracellular
acidification, caspase activation and early accumulation of reactive
oxygen species in tumour cells . Research groups in all
over the world have recently started an International Society of
Proton Dynamics in Cancer (ispdc) in January 2010 to investigate
various aspects of proton dynamics in cancer cells . The newly
formed society contributes to stimulate interdisciplinary
collaboration for the development of more specific and less toxic
therapeutic strategies based on proton dynamics in tumor cell
In our previous work, we showed that Hv1 function relates to
breast tumor growth and metastasis through proton extrusion
. Hv1 is also expressed in airway epithelia, which mediates
pH-regulated acid extrusion and acidify an alkaline airway surface
liquid . In the present work, the close relationship between
Hv1 expression and clinicopathological features in colorectal
cancer also predicted that Hv1 might boost carcinogenesis and
tumor progression through regulating intracellular pH. Therefore,
the voltage-gated proton channel Hv1 is a new candidate for some
tumor cell intracellular and extracellular pH regulation.
The glycolysis and proton secretion in tumor cells are proposed
to contribute to the proliferation and invasion of cancer cells
during the process of tumorigenesis and metastasis [16,38]. The
cytosolic pH value is extremely important for tumor cells,
inasmuch as a decrease of cytosolic pH possibly stops tumor cell
metabolism and induces cell death [15,33]. An alkaline cytosolic
pH and an acidic extracellular pH resulting in high glycolytic
activity and acidic metabolites are characteristics of tumor cells.
The aberrant pH gradient between the alkaline cytosol and the
acidic extracellular environment is involved in tumor progression
and malignancy, which might be maintained by up-regulated
activity of Hv1 that extrude protons outside the cell and acidify
intracellular vesicles in colorectal cancer cells.
In conclusion, we demonstrated here that Hv1 is over-expressed
in patients with colorectal cancer and high Hv1 expression
correlated with the disease progression and poor clinical outcome
in colorectal cancer. Furthermore, Hv1 was proved to be a risk
factor for tumor recurrence and an independent molecular marker
of prognosis for colorectal cancer and may become a novel
molecular target in the strategies for the prediction of tumor
recurrence and prognosis or treatment of colorectal cancer.
Conceived and designed the experiments: YFW XYW SJL. Performed the
experiments: YFW XYW QL SRZ. Analyzed the data: YFW XYW.
Contributed reagents/materials/analysis tools: YFW XYW SJL. Wrote the
paper: YFW XYW.
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