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The Haptoglobin b chain as a supportive biomarker for human lung
cancersw
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Published on 21 January 2011 on http://pubs.rsc.org | doi:10.1039/C0MB00242A
Sung-Min Kang,a Hye-Jin Sung,a Jung-Mo Ahn,a Jae-Yong Park,b Soo-Youn Lee,c
Choon-Sik Parkd and Je-Yoel Cho*a
Received 21st October 2010, Accepted 13th December 2010
DOI: 10.1039/c0mb00242a
Haptoglobin (Hp) is produced as an acute phase reactant during inflammation, infection, malignant
diseases, and several cancers. In proteomics analysis using human blood samples, the Hp peptide
levels were about 3-fold higher in lung cancer patients versus normal individuals. This study is
aimed at analyzing the elevation of which chain of Hp is closely related to lung cancers and can be
a serum biomarker for lung cancers. In Western blot (WB) analysis, we found that the Hp b chain
can be a better diagnostic biomarker for lung cancers. In the result of the Hp b chain ELISA
developed by us, the concentrations of the Hp b chain in the sera increased about 4-fold in 190
lung adenocarcinoma patients versus 190 healthy controls (8.0 3.8 mg ml 1 vs. 1.9 1.2 mg ml 1).
ELISA data showed that the serum levels of the Hp b chain in breast cancer (1.5 0.5 mg ml 1)
and hepatocellular carcinoma (HCC) (1.4 1.0 mg ml 1) patients remained similar to those of
healthy controls. Compared to lung adenocarcinoma, the Hp b chain levels in the plasma of
patients with other respiratory diseases such as tuberculosis (TBC), idiopathic pulmonary fibrosis
(IPF) and bronchial asthma (BA) were closer to those of healthy controls. Our data suggest
that an increase of the Hp b chain can be a potential serum biomarker for lung cancers.
Introduction
Lung cancer is one of the leading causes of cancer-related death
worldwide.1 According to a study of 5-year survival rates
between 2001 and 2005 by the Ministry for Health, Welfare
and Family Affairs, lung cancer (15.5%) has a very low survival
rate in Korea compared to other types of cancers, including
stomach cancer (56.4%), colon cancer (64.8%), breast cancer
(87.3%) and thyroid cancer (98.1%). Although several screening
techniques, such as X-rays and computed tomography (CT)
scans, are frequently used for lung cancer diagnosis, their high
cost, poor specificity, and high risk of radiation exposure are still
problematic for lung cancer diagnosis.2
Lung cancer has four major histological types that can be
grouped into two large categories: small-cell lung cancer
a
Department of Biochemistry, School of Dentistry and Brain Korea 21,
Kyungpook National University, and ProtAnBio, Dongin-dong 2 Ga 101,
Daegu, 700-422, South Korea. E-mail: jeycho@knu.ac.kr;
Fax: +82 53-421-1418; Tel: +82 53-420-4997
b
Internal Medicine, School of Medicine, Kyungpook National
University Hospital, Daegu, South Korea
c
Department of Laboratory & Genetics, Samsung Medical Center,
Sungkyunkwan University of Medicine, Seoul, South Korea
d
Division of Allergy and Respiratory Diseases, Department of Internal
Medicine, Soonchunhyang University Hospital, Seoul, South Korea
w Electronic supplementary information (ESI) available: Supplementary
Table 1: Unique peptides detected in each band by proteomic analyses.
Supplementary Table 2: Unique peptides detected in each chain by
proteomic analyses. See DOI: 10.1039/c0mb00242a
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(SCLC) and non-small-cell lung cancer (NSCLC). NSCLC,
which accounts for 80% of lung cancers, consists of squamouscell carcinoma representing 20–25% of lung cancers, largecell carcinomas constituting 15–20% of lung cancers, and
adenocarcinoma accounting for 30–40% of lung cancers.
Adenocarcinoma has been reported to be the most prevalent
histological subtype of lung carcinomas.3,4
Biomarkers, which are quantifiable measurements that
distinguish between normal and abnormal conditions, include
many different forms, such as physiological conditions, visual
characteristics, genes, and proteins.5 Recently, there have been
various attempts to improve the monitoring of lung cancer
therapies using genomic and proteomic techniques. Among the
many kinds of biomarkers, protein biomarkers are currently
being researched in conjunction with the development of
techniques in proteomics.6 Blood samples are good sources of
biomarkers because proteins in blood have significant physiological functions and secreted proteins are records of physiological
conditions. However, due to the existence of abundant proteins
in blood, the discovery of cancer biomarkers in serum is
challenging.7 Despite these difficulties, lung cancer biomarkers
have been characterized, and several genes and proteins have been
proposed as lung cancer-biomarker candidates: cytokeratin-19
fragment (CYFRA 21-1), carcinoembryonic antigen (CEA),
cancer antigen-125 (CA-125) and neuron-specific enolase (NSE)
etc. However, many of these biomarkers are not clinically used,
and most still lack sensitivity and specificity.8–10
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The acute-phase response is a remarkable systemic reaction
to disturbances in host homeostasis caused by infection,
trauma, surgery and immunological disorder. The acute-phase
response stimulates the liver to secrete acute-phase proteins,
which include the positive acute proteins, C-reactive protein
(CRP), serum amyloid A (SAA) and tumor necrosis factor
alpha (TNF-a), and the negative acute proteins, retinol binding
protein (RBP), transthyretin (TTR) and transferrin. Hp is a
positive acute-phase protein that is induced by the immediate
host inflammatory response to stimuli primarily in hepatocytes.11
The Hp molecule is a tetrameric protein of two a and b chain
dimers that are connected by disulfide bridges. These two
chains originate from a common precursor protein that is
proteolytically cleaved during protein synthesis. The main
function of Hp is to bind free plasma hemoglobin for protection
from oxidative stress and to assist in hemoglobin uptake by
the hemoglobin scavenger receptor CD163;12 however, under
conditions of inflammation, Hp is elevated through the activity of
IL-6. Several studies have reported Hp elevation in breast
cancer,13 ovarian cancer,14 pancreatic cancer,15,16 malignant
lymphoma,17 urogenital tumor18 and bladder cancer.19 Another
study reported that the fucosylation of Hp is increased in
pancreatic cancer and ovarian cancer.
In this study, we identified Hp using a LC-ESI-MS/MS
analysis of crude serum samples and then verified and quantified
Hp levels by WB analysis and ELISA. This analysis was done
on lung adenocarcinoma sera; samples from patients with
other types of solid tumors, including breast cancer and
HCC; and samples from patients with respiratory diseases.
Our data suggest that Hp b chain levels are increased in lung
adenocarcinoma compared with other types of cancers and
healthy controls. This increase of Hp b chain levels in lung
cancer may be a supportive biomarker for lung cancer.
Results
Identification of Hp by LC-ESI-MS/MS analysis
Serum samples were obtained from consenting individuals
with or without lung adenocarcinoma according to the serum
collection protocol described in Experimental procedures.
Clinical information about the samples used in this study is
summarized in Table 1. Using one-dimensional gel electrophoresis, 100 mg of protein from the pooled sera of five
individuals from the healthy control and lung adenocarcinoma
groups were separated. The gel was stained with Coomassie
Brilliant Blue (CBB) staining solution, which showed three
bands at 45, 15 and 10 kDa that had different intensities
between the normal and lung adenocarcinoma samples
(Fig. 1A). To identify the proteins that were elevated in the
lung adenocarcinoma sample, six bands of interest were
excised and in-gel digested; the tryptic peptides were then
analyzed by LC-ESI-MS/MS analysis. In each band,
20–30 proteins were identified and the 1 > false positive rate
(FPR) determined using a decoy database (Table 2). Hp
peptides were found in the bands at 45 kDa (band no. 1),
15 kDa (band no. 2) and 10 kDa (band no. 3; Fig. 1A). Hp
with 119, 91 and 42 spectral counts were detected in each band
of the lung adenocarcinoma samples, whereas 32, 28 and 19
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Table 1 Clinical data of the patients who provided plasma and serum
samples
Source
Sample type
Sex
Age
Serum
Healthy
Male
Female
Male
Female
Female
Male
Female
Male
Female
Male
Female
Male
Male
Male
Male
Male
56.2
52.1
60.3
62.4
47.8
53.7
61.0
69.1
58.5
64.4
53.0
54.4
60.4
55.8
60.5
54.9
AdenoCA
BreastCA
HCC
SQLC
SCLC
Plasma
Healthy
AdenoCA
TBC
IPF
BA
Total
9.3
10.4
10.3
9.6
6.4
6.0
6.7
6.4
12.4
9.0
0.0
3.3
5.2
4.2
12.3
3.0
118
83
164
75
22
16
4
36
4
39
1
50
30
50
50
50
Serum samples were obtained from the Samsung Medical Center and
the Kyungpook National University Hospital, and plasma samples
were obtained from the Soonchunhyang University Hospital.
Abbreviations used: AdenoCA, lung adenocarcinoma; BreastCA,
breast cancer; HCC, hepatocellular carcinoma; SQLC, squamous-cell
lung cancer; SCLC, small-cell lung cancer; TBC, tuberculosis; IPF,
idiopathic pulmonary fibrosis and BA, bronchial asthma. Data are
means SD.
were found in the healthy control sample bands. The average
ratio of the lung adenocarcinoma to the normal samples for
Hp precursor spectral counts detected by our mass analysis
was 3.1 (Table 3).
Hp is composed of a1, a2 and b chain polypeptides in
different combinations. In our MS/MS analysis results, the
major MS/MS spectrum of the Hp precursor of each band was
representative of the b, a2 and a1 chains, respectively. In band
no. 1, the peptide sequences SPVGVQPILNEHTFCAGMSK
and SCAVAEYGVYVK were detected with 21 and 20 peptide
hits, respectively, and these peptide sequences matched the
Hp b chain. For band no. 2 and band no. 3, the peptide fragments
LRTEGDGVYTLNDKK and AVGDKLPECEAVCGKPK
were primarily detected and each matched the Hp a2 and a1
chains with 28 and 6 peptide hits, respectively (Fig. 1B). The
unique sequences of Hp precursor in each band and in each
chain are summarized in ESIw Tables S1 and S2, respectively,
with the corresponding hit numbers. Hp proteins were
identified with the coverage of 15–38% range in each band
of the lung adenocarcinoma and normal samples. Fig. 1C
shows the full amino acid sequences of the Hp and the tryptic
peptide fragments detected in LC-MS/MS analysis are indicated
with boxes. In summary, considering that the spectral count of Hp
detected in the bands was much higher than that of the other
proteins (data not shown), each band indicated a different chain of
Hp and the key components of the elevated intensities of bands in
CBB staining are three different chains of Hp.
Identification of Hp in individual bands in immunoblotting
As previously described, Hp is composed of three different
chains, and each chain has different predicted molecular
weights: 9, 19 and 29 kDa. However, the immunoblotting
analysis with a polyclonal antibody (Ab) detected bands at 9,
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Fig. 1 Identification of Hp by LC-ESI-MS/MS analysis and MS/MS spectrum of Hp fragments. (A) Sera from a total of five healthy control and
five lung adenocarcinoma patients were pooled, and then 100 mg of protein of each pooled sample was separated by SDS-PAGE and stained with
Coomassie Brilliant Blue. Three bands at different molecular weights that showed different intensities were excised and in-gel digested. Lane 1,
pooled normal sera; Lane 2, pooled lung cancer patient sera. (B) Representative peptides of each band showing each chain of Hp. MS/MS
spectrum of the peptide fragment SPVGVQPILNEHTFCAGMSK (m/z = 2171.66, charge = 2+) from band 1, which is part of the Hp b chain,
with 21 peptide hits. In band 2, the Hp a2 chain fragment LRTEGDGVYTLNDKK (m/z = 1707.68, charge = 2+) was found with 28 peptide
hits. The band 3 peptide AVGDKLPECEAVCGKPK (m/z = 1857.46, charge = 2+) had 6 peptide hits and represented the Hp a1 chain. (C) The
figure shows the full amino acid sequence of Hp: blue letters, signal sequence; pink and green letters, a2 chain; green letters only, a1 chain; and
black letters, Hp b chain. Identified peptides are in black boxes, and expected modification sites are in red letters.
Table 2 The number of proteins identified by LC-MS/MS analysis
from individual bands
Table 3 Identification of Haptoglobin by LC-MS/MS analysis
Protein
Band 1
Band 2
Band 3
N
LC
N
LC
N
LC
Number of
proteins identifieda
Peptide
false-positive
rateb (%)
26
28
21
21
22
18
0.705
0.548
0.434
0.434
0.676
0.755
Description
Peptide numbers Coverage (%) Ratioa
N1
32
IPI00641737 Haptoglobin N2
precursor
28
N3
19
LC1
119
LC2
91
LC3
42
N1
26
N2
17
N3
15
LC1
38
LC2
31
LC3
33
3.71
3.25
2.21
a
Ratio is LC peptide quantitative value divided by N peptide
quantitative value.
a
Positive identification of a protein required a minimum of two
peptides (95% confidence) per protein. b Calculation of the FPR is
described in Experimental procedures.
19 and 45 kDa, which are different than the predicted
molecular weights of each band. We hypothesized that the b
chain might be N-glycosylated and thus detected at the higher
molecular weight of 45 kDa. To test this hypothesis, we first
searched for possible N-glycosylation motifs (NxS/T) in the
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sequences and found four possible sites in the b chain; which
are written in red characters in Fig. 1C. PNGase F treatment
was then performed because PNGase F cleaves asparaginelinked (N-linked) oligosaccharides from glycoproteins, which
deaminates asparagine to aspartic acid and leaves the
oligosaccharides intact. Following the PNGase F treatment,
the 45 kDa band shifted to 29 kDa, suggesting that the 45 kDa
band is a glycosylated form of the b chain (Fig. 2A).
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Fig. 2 Elevation of the Hp b chain compared with the a2 chain in sera of lung adenocarcinoma patients compared to healthy controls.
(A) Serum samples treated with PNGase F for the characterization of Hp bands were analyzed by immunoblotting with an anti-Hp
polyclonal antibody. PNGase F treatment resulted in a molecular mass reduction in the Hp b chain (29 kDa) but not in the a1 and a2 chains.
Lane 1, crude serum sample; Lane 2, PNGase F untreated sample; Lane 3, PNGase F treated sample. (B, C) Protein from the crude sera of
44 healthy controls, 78 lung adenocarcinoma patients, 22 breast cancer patients and 20 HCC patients was separated by SDS-PAGE, and
then Hp expression was detected by immunoblotting. Representative blots of the Hp a2 chain (B) and the b chain (C) expression in
each cancer patient and healthy control are shown. Each number indicates the patients’ number used for the immunoblotting. (D, E)
Densitometry plot of the respective bands for the a2 chain (D) and the b chain (E). Each blot contained the same standard sample; when
analyzing the results of immunoblotting by densitometry, the total pixels of each band (density area) analyzed by Scion image were normalized
to the total pixels of the standard. The boxes give the upper 75% and lower 25% quartiles of the measurements with respect to the median value
(horizontal line in each box) and each dot represents individual patient data, while the small open rectangle represents the mean value.
The expression levels of the Hp b chain showed significant differences between the sera of lung cancer patients and healthy controls, breast
cancer patients or hepatocellular carcinoma patients. Abbreviations used: AdenoCA; lung adenocarcinoma, BreastCA; breast cancer and HCC;
hepatocellular carcinoma (* indicates P o 0.05).
Higher increase level of the Hp b chain than the a2 chain in lung
adenocarcinoma
To validate the Hp expression levels, the sera of 44 healthy
controls and 78 lung adenocarcinoma patients were tested
to estimate the expression level of Hp by immunoblotting
analysis. The a2 and b peptide chains of lung cancer samples
showed higher expression levels than those of healthy
controls, and the differences were great enough to be detected
by WB analysis (representative data in Fig. 2B and C).
We analyzed 44 healthy and 78 lung adenocarcinoma samples
by WB analysis, and the densities of the a2 band and b
chain bands in the WB were photographically measured
by Scion image analysis. The relative protein levels were
measured by densitometry analysis of the Hp a2 and b chain
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bands using the equation: (pixel number of sample band / pixel
number of standard band) 100 individuals; Fig. 2D
and E. To analyze the applicability for differential diagnosis,
we also analyzed different types of cancers, including
breast cancer and HCC. The results showed that, although
the Hp a2 and b chains were both potential markers
for lung cancer diagnosis, b chain levels were better
indicators than a chain levels for the identification of
lung adenocarcinoma. In our study, the Hp a1 chain
was not clearly detectable in most of the healthy and
cancer samples. The expression levels of the Hp b chain
showed significant differences between lung adenocarcinomas
and healthy control samples (p = 0.04299) and also
between adenocarcinomas and other types of solid cancers,
including breast cancer and HCC (p = 0.01941 and 0.00457;
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Increased expression levels of the Hp b chain in lung
adenocarcinoma
To better quantify the levels of the Hp b chain in the sera, we
developed an ELISA using a b chain-specific Ab for detection.
The specificity of the Ab was tested by WB using lung cancer
serum. The Ab recognized a single specific band of the Hp b
chain (Fig. 3A). Then, the sera of 190 lung adenocarcinoma
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Fig. 2E); however, the a2 chain level was slightly increased
in lung adenocarcinomas compared to healthy controls
(p = 0.002), and there was not much difference between lung
cancer and other types of tumors (breast cancer, p = 0.20366;
HCC, p = 0.06402; Fig. 2D). Altogether, these data show that
the Hp b chain is a better marker for lung adenocarcinoma
diagnosis compared to the a chain.
Fig. 3 ELISA showed an increase of the Hp b chain in the sera of lung cancer patients. (A) To verify the specificity of the Hp b chain antibody
used for ELISA, immunoblotting was performed for crude serum of lung cancer patients, PNGase F-untreated serum of lung cancer patients and
serum of lung cancer patients after PNGase F treatment. Lane 1, crude serum sample; Lane 2, PNGase F untreated sample; Lane 3, PNGase F
treated sample. (B) Concentrations of the Hp b chain in the sera of 190 healthy controls and 190 lung adenocarcinoma patients were measured by
ELISA. On average, the Hp b chain levels in the lung adenocarcinoma samples (8.0 mg ml 1) were 4.2-fold higher than in healthy controls
(1.9 mg ml 1). Sera from patients with other histological types of lung cancers, including squamous-cell lung cancer (SQLC) and small-cell lung
cancer (SCLC), were also subjected to ELISA analysis. The average value for SQLC was 8.2 mg ml 1 and the average value for SCLC was
8.9 mg ml 1. (C) When the serum samples were grouped by lung cancer stages, the Hp concentration in serum was correlated with the stages,
with the exception of stage II, which had a lower number of samples. (D) The Receiver Operating Characteristic (ROC) curve of Hp b chain with
the sensitivity and specificity values from the cut-off range of 2 mg ml 1. The detection of cancer samples with respect to healthy controls
(* indicates P o 0.05).
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patients and 190 healthy controls were analyzed by ELISA
(Fig. 3B). The ELISA data showed that the average concentration of the Hp b chain was different between lung
adenocarcinoma (8.0 3.8 mg ml 1) patients and healthy
controls (1.9 1.2 mg ml 1). The ELISA results from lung
adenocarcinoma samples were analyzed by cancer stage
in order to evaluate the correlation between Hp b chain
concentration and cancer progression (Fig. 3C). The Hp b
chain concentration showed a gradual increase that
depended on the cancer stage. The average of stage I was
6.2 2.5 mg ml 1, stage III was 9.0 4.6 mg ml 1 and stage IV
was 11.8 4.9 mg ml 1; however, the data for stage II did not
show correlation between stage and Hp b chain concentration
unlike other stages. It seems that this is due to the
small number of samples. In the ELISA analysis for lung
adenocarcinoma and healthy control samples, when the cut-off
was set at 2 mg ml 1, the AUC value was 0.822 and the
specificity and sensitivity of the Hp b chain as a diagnostic
marker were estimated to be 63.2% and 82.6%, respectively
(Fig. 3D). These data suggest that the Hp b chain can be a
potential biomarker for both the detection and monitoring of
lung adenocarcinoma.
Similar pattern of the Hp b chain increase in other histological
types of lung cancer
To determine whether the increase of the Hp b chain was
specific for lung adenocarcinoma or common to other
histological types of lung cancers, we measured the Hp b
chain levels by ELISA in SQLC and SCLC samples. The
average concentrations of the Hp b chain were not different
from that of lung adenocarcinoma samples (8.2 3.4 mg ml 1
for SQLC; 8.9 3.6 mg ml 1 for SCLC) (Fig. 3B). Our ELISA
data showed that the increase of the Hp b chain in sera was not
specific to lung adenocarcinoma because the Hp b chain was
also increased in other histological types of lung cancers, such
as SQLC and SCLC.
Comparison of the Hp b chain levels between lung
adenocarcinoma patients and other cancer and respiratory
disease patients
ELISA analysis of the Hp b chain was also performed on sera
from patients with other types of solid tumors and respiratory
diseases, and these results were compared to the lung
adenocarcinoma samples to test the usefulness of the Hp b
chain for differential diagnosis. ELISA showed that the Hp b
chain levels of the serum in breast cancer and HCC were
1.5 0.5 and 1.4 1.0 mg ml 1, respectively (Fig. 4A).
The data showed that the breast cancer and HCC levels were
not different from that of healthy controls. Except for one
sample in HCC, all of the breast cancer and HCC samples had
the Hp b chain concentrations that were lower than our cut-off
value (5 mg ml 1). We also analyzed the Hp b chain by ELISA
in plasma samples of patients with TBC, IPF and BA and
compared the results to the plasma samples of healthy
controls and lung adenocarcinoma patients. The plasma
concentration of the Hp b chain was about 3-fold higher in
lung adenocarcinoma patients (11.4 4.3 mg ml 1) compared
to healthy controls (3.2 1.3 mg ml 1). The Hp b chain
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Fig. 4 Higher levels of the Hp b chain in the serum and plasma of
lung adenocarcinoma patients relative to patients with other types of
solid tumors or respiratory diseases. (A) Validation of the Hp b chain
expression in other types of tumors, including 20 breast cancer and
20 hepatocellular carcinoma (HCC) patients, was performed by
ELISA. When a cut-off value of 5 mg ml 1 was applied, only one
HCC sample was detected above the threshold. (B) Validation of the
Hp b chain expression level was also performed by ELISA in the
plasma samples from healthy controls, lung adenocarcinoma (AdenoCA)
patients and patients with other respiratory diseases, including
tuberculosis (TBC), idiopathic pulmonary fibrosis (IPF) and bronchial
asthma (BA). The concentration of the Hp b chain was significantly
higher in the lung adenocarcinoma group than in healthy controls or
patients with other respiratory diseases (* indicates P o 0.05).
concentrations in patients with other respiratory diseases were
all significantly lower than that in the lung adenocarcinoma
patients: TBC (4.6 2.0 mg ml 1), IPF (5.8 2.6 mg ml 1) and
BA (3.7 1.6 mg ml 1) (Fig. 4B). These results suggest that the
Hp b chain might be a potential biomarker for lung cancers
compared to other types of solid tumors, such as breast cancer
and HCC, and, to some extent, may also be a biomarker for
respiratory diseases.
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Discussion
In this study, we discovered that Hp expression was higher in
the sera of lung cancer patients compared to healthy controls
by LC-MS/MS analysis, and this result was validated and
quantified by immunoblotting and ELISA. Among the three
different chains of Hp, Hp a2 and b chains showed differences
compared to healthy controls, but only the Hp b chain showed
a significant difference between lung cancers and other tumors.
The Hp b chain was increased not only in the sera of lung
adenocarcinoma patients, but also in other histological types
of lung cancers; however, the level of increase was different
(4-fold higher on average) between lung cancer and other types
of tumors or respiratory diseases.
Hp is an acute phase-reactant tetrameric (a2b2) glycoprotein. The main function of Hp is to remove free plasma
hemoglobin.20,21 However, Hp has also been shown to
have angiogenic22 and antioxidant23 properties and has an
important role in cell migration.24 Although Hp is mainly
synthesized in the liver, the local differential expression of Hp
has also been demonstrated in cancer tissues.25–27
It has been reported that the Hp a subunit is a potential
biomarker in ovarian cancer and SCLC. Ye et al. identified Hp
by SELDI-MS/MS and then examined the Hp a subunit
expression level by WB and quantified the a chain by
ELISA.28 The Hp a chain levels in 13 SCLC samples were
also about 3-fold higher than in 4 healthy controls when
measured by densitometry of western blots.29 In our lung
cancer study, the data of the Hp a subunit showed a similar
increase compared to ovarian cancer and SCLC, indicating
that the Hp a chain may be similarly increased in other types
of cancers. Although Hp is reportedly a potential biomarker
for pancreatic and ovarian cancers,15,30 few studies have been
conducted investigating Hp in relation to lung cancers. In
previous reports of Hp in lung cancers, most studies have
measured the total Hp without discriminating between each
chain,31,32 and none of the researchers specifically measured
the b chain of Hp. In this study, however, we found that the
Hp b chain had a greater increase in the sera of lung cancer
patients compared to the other chains, a1 and a2, and could be
a better biomarker for the identification of lung cancers and
for differentiating between lung cancer and other cancers such
as breast cancer and hepatocellular cancers. The quantitative
measurement of the Hp b chain was possible because we
setup an ELISA method using a Hp b chain specific
monoclonal antibody. Our data suggest that the increased
level of the Hp b chain in the sera of lung cancer patients can
be a supportive serum biomarker for lung cancer diagnosis
and monitoring.
Although our data showed that the p-values for the expression
level of the Hp b chain in the respiratory diseases are not
significant compared to healthy controls, the statistical analysis
for the levels of the Hp b chain between lung cancer and other
respiratory diseases turned out to be significant (Fig. 4B).
However, there are still overlapping ranges of the Hp b chain
levels between lung adenocarcinoma and other respiratory
diseases. Thus, caution is needed when the Hp b chain is to
be used as a potential biomarker to differentiate lung cancer
patients from other respiratory disease patients.
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We determined that the Hp b chain is N-glycosylated using
a PNGase F treatment experiment (Fig. 2A). Our data are also
supported by previous studies showing that the Hp b chain has
four glycosylation sites and might exist as a glycosylated form
in colon cancers.33 Another report also showed that Hp
is a fucosylated protein and suggested that the increased
fucosylation of Hp in cancers may indicate that cancer can
induce the fucosylation of the molecule.34 An increase of
fucosylated Hp in pancreatic cancer has also been reported
and did not appear to be correlated with the total amount of
Haptoglobin.30 One of the possible reasons why we observed a
better diagnostic predicted value of the Hp b chain relative to
the a chains might be due to the glycosylation characteristics
of the b chain. Because the serum proteins in cancer patients
are known to be further glycosylated, it is possible that the b
chain is more glycosylated in lung cancer patients than in
normal controls. Because the monoclonal antibody we used
for the WB and ELISA can similarly detect the Hp b chain in
its glycosylated and unglycosylated forms (Fig. 3A), the
difference may not be caused by differences in the detection
ability of the antibody for the Hp b chain. It is quite possible
that the glycosylated Hp b chain in the sera of lung cancer
patients might be more stable and might have a longer half-life
in the serum. Thus, we would see a greater increase in the Hp b
chain than the Hp a chain in the sera of lung cancer patients.
For Hp to be applied to lung cancer diagnosis, the increase in
the total protein amount and the fucosylated status should be
combined.
Conclusion
We identified Hp in serum and verified the elevation of
the glycosylated b chain in the lung cancer patients. Although
the Hp a2 chain was slightly increased, a greater increase of the
Hp b chain was found. The marked increase of the Hp b chain
in lung cancers compared to other types of cancers or respiratory
diseases suggests that it can be a potential serum biomarker
for lung cancers.
Experimental procedures
Human serum samples
Serum and plasma samples were obtained from patients with
lung cancer, other types of solid cancers and respiratory
diseases from the Samsung Medical Center (IRB No.
2008-06-007-005), the Kyungpook National University Hospital
(IRB No. 74005-833) and the Soonchunhyang University
Hospital, a member of the National Biobank of Korea.
Informed consent was obtained from all donors. We used
serum samples from 239 lung adenocarcinoma patients, 201
healthy controls, and 40 patients with other histological types
of SQLC and SCLC lung cancers and other types of solid
cancers, including 22 breast cancer and 20 HCC samples. We
also used plasma samples from 50 healthy controls, 30 lung
adenocarcinoma patients and 50 patients with the respiratory
diseases TBC, IPF and BA. Serum and plasma were separated
from whole blood by centrifugation within 4 h after collection
and then stored at 70 1C until use. Additional information
Mol. BioSyst.
View Online
on the samples is described in Table 1. Among samples described
above, samples for WB analysis and ELISA were randomly
selected and some of them were used for both analyses.
with a protein that has a 95% probability, according to the
Protein Prophet algorithm,38 and a minimum of 2 peptides are
matched with the protein sequence, each with a 95%
probability, based on the Peptide Prophet algorithm.
Downloaded by Kyungpook National University on 24 January 2011
Published on 21 January 2011 on http://pubs.rsc.org | doi:10.1039/C0MB00242A
Identification by LC-ESI-MS/MS analysis
Separation by SDS-PAGE and Coomassie Blue staining.
Protein (100 mg) from the pooled sera of five lung adenocarcinoma
patients and healthy individuals was separated by SDS-PAGE
(sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
The gels were then washed three times with ddH2O for 5 min
and stained with Bio-Safe Coomassie Stain solution
(Coomassie G250 Stain; BioRad, Hercules, CA) for 1 h with
gentle shaking at room temperature (RT). The gels were
destained by incubation in ddH2O overnight.
In-gel digestion. After being stained with Coomassie, the
protein bands of interest were excised, sliced and in-gel
digested with trypsin as previously reported.35 Briefly, six
bands from lung cancer and healthy samples, which showed
different intensities, were excised and subjected to in-gel
digestion. The gel was sliced into six bands and destained by
incubation in 75 mM ammonium bicarbonate/40% ethanol
(1 : 1). The gel pieces were then shake incubated in a 5 mM
dithiothreitol solution with enough volume to cover the gel.
For the alkylation of proteins, the gel pieces were incubated in
300 ml 55 mM IAA solution and then dehydrated in 700 ml of
100% acetonitrile. The gel pieces were dried in a speed vacuum
and treated with trypsin (Roche Applied Science) overnight at
37 1C. Tryptic peptides were eluted with 0.1% formic acid.
LC-ESI-MS/MS analysis and data analysis. LC-MS/MS
analysis was performed using the Thermo Finnegan ProteomeX
workstation and LTQ linear IT MS (Thermo Electron, San
Jose, CA, USA) equipped with an NSI source (San Jose, CA).
The analysis conditions for mass spectrometry were the same
as previously reported.35 MS data analyzed as previously
reported. Briefly MS/MS data were searched based on the
IPI human protein database (version 3.29, 69 965 entries)
using the SEQUEST algorithm sorcerer 3.4 beta 2 (Sorcerer
software 3.10.4, sorcerer web interface 2.2.0 r334 and Trans
proteomic Pipeline 2.9.5). SEQUEST search parameters were
set to a fragment ion mass tolerance of 1.00 Da and a parent
ion tolerance of 1.5 Da. Oxidation of methionine and
iodoacetamide modification of cysteine were specified as fixed
modifications. Scaffold calculates the quantitative value
number by normalizing the spectral counts across our Scaffold
experiment. Protein identifications were accepted if they could
be established at greater than 95.0% probability as specified
by the peptide prophet algorithm36 and contained at least two
identified peptides. Protein probabilities were assigned by the
Protein Prophet algorithm.37 Proteins that contained similar
peptides and could not be differentiated based on MS/MS
analysis alone were grouped to satisfy the principles of
parsimony. The peptide false positive (FPR) rate was
calculated using the Scaffold software. For each charge state,
the incorrect assignments are tabulated to calculate the
FPRi = [(#Assigned Incorrect at 95% probability)/
(Total# Incorrect Assigned)] 100, with i being the charge
state. The assignment is considered correct if it is associated
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Deglycosylation with peptide-N-glycosidase F (PNGase F)
One microgram of glycoprotein was denatured in buffer at
100 1C for 10 min and then cooled to RT. For complete
deglycosylation, the protein was incubated in reaction buffer
with 1 ml PNGase F (New England BioLabs, UK) for 1 h at
37 1C. The deglycosylation of proteins was confirmed by
immunoblotting.
Measurement of Hp in serum samples
Western blot and analysis by densitometry. Western blots
(WBs) were performed as previously reported.39 Briefly, 0.5 mg
of crude serum protein was separated by 15% SDS-PAGE and
then transferred. The membrane was incubated with an
anti-Hp Ab at a 1 : 3000 dilution (polyclonal Anti-human
Hp Ab, DaKoCytomation, Denmark) for 1 h at RT, followed
by incubation with a horseradish peroxidase-conjugated
anti-Rabbit IgG secondary Ab at 1 : 2000 (Stressgen, BC,
Canada) for 30 min at RT. The ECL Western blot analysis
system (Amersham Biosciences, UK) was used to detect the
immunoreactive proteins. The densitometric analysis of the
thickness of bands was performed with Scion Image (Scion,
Frederick, MD). An image of a calibration grid slide was
measured using Scion Image and a distance of 250 mm was
calculated to be equivalent to 733 pixels.
Enzyme-linked immunosorbent assay (ELISA). Quantification
of the Hp b chain levels in serum and plasma samples was
performed using in-house Sandwich ELISA. The ELISA starter
kit (Koma Biotech Inc., Korea) was used, which included coating
solution, washing solution, blocking solution and color-reaction
solution. Three antibodies, anti-Hp b chain monoclonal Ab
(Ab frontier, Seoul, Korea), anti-Hp polyclonal Ab (Ab frontier)
and anti-rabbit secondary Ab (Stressgen), were used. The
anti-Hp b chain monoclonal Ab was diluted in coating buffer
to 1 mg ml 1, and then 100 ml of the prepared coating solution
was dispensed into each well. The plate was incubated overnight
at 4 1C. After blocking with 200 ml blocking solution for 1 h at
RT, serum or plasma samples were diluted 200-fold in PBS, and
then 100 ml was added to each well and the samples were
incubated for 1 h at RT. Purified human Hp protein (SigmaAldrich, MO, USA) was used as a standard at a concentration
range of 0 ng ml 1 to 600 ng ml 1. After blocking, 100 ml of
anti-Hp polyclonal Ab (1 : 20 000, Ab frontier) was added to the
96-well plates, which were then incubated for 1 h. For detection,
the plates were incubated with 100 ml of goat anti-rabbit Ab
(1 : 5000, Stressgen) for 1 h at RT followed by the addition of
PINK-ONE TMB solution (Koma Biotech Inc.). To determine
the concentration of the Hp b chain, the absorbance at 450 nm of
the plates were read using an ELISA reader (Shimadzu).
Statistical analysis
All data are presented as the means standard deviations
(SD). Statistical differences among the densitometry and
ELISA results of the groups were evaluated by two-sample
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c
The Royal Society of Chemistry 2011
View Online
t-test. A statistical significance of p o 0.05 is indicated by an
asterisk (*). The area under the curve (AUC) value was
determined using the Panel Composer Program, provided by
Yonsei Proteome Research Center (Seoul, Korea).
Downloaded by Kyungpook National University on 24 January 2011
Published on 21 January 2011 on http://pubs.rsc.org | doi:10.1039/C0MB00242A
Acknowledgements
This work was supported by a National Research Foundation
of Korea (NRF) grant funded by the Korean government
(MEST) (No. 2009-0093611), by grant FPR08A2-120 of the
21C Frontier Functional Proteomics Project from the Korean
Ministry of Education, Science and Technology, and by grant
No. RTI04-01-01 from the Regional Technology Innovation
Program of the Ministry of Knowledge Economy (MKE).
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