1
Unconjugated bilirubin is a novel prognostic biomarker for
nasopharyngeal carcinoma and inhibits its metastasis via
anti-oxidation activity
Cheng-Cheng Deng1,2, Miao Xu1,2, Jing Li3, Xiao-Lin Luo1,4, Yu-Jia Zhu1,5, Rou
Jiang1,6, Meng-Xia Zhang1,6, Jin-Ju Lei1,2, Yi-Fan Lian1,2, Xiong Zou1,6, Rui
You1,6, Li-Zhen Chen1,2, Qi-Sheng Feng1,2, Jin-Xin Bei1,2, Ming-Yuan Chen1,6,
Yi-Xin Zeng1,2
1. Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in
South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou
510060, China.
2. Department of Experimental Research, Sun Yat-sen University Cancer Center,
Guangzhou 510060, China.
3. Department of Oncology, the First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, China.
4. Department of Gynecologic Oncology, Sun Yat-sen University Cancer Center,
Guangzhou 510060, China.
5. Department of Radiation Oncology, Sun Yat-sen University Cancer Center,
Guangzhou 510060, China.
6. Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer
Center, Guangzhou 510060, China.
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Cheng-Cheng Deng, Miao Xu and Jing Li contributed equally to this work.
Running title: Unconjugated bilirubin inhibits metastasis in NPC
Key words: unconjugated bilirubin; metastasis; matrix metalloproteinase-2;
anti-oxidation; nasopharyngeal carcinoma
Financial support:
National Basic Research Program of China (973 Plan 2011CB504302; to Y.-X. Zeng),
National Natural Science Foundation of China (NSFC 81302370;to M. Xu), the
New Century Excellent Talents in University (NCET-12-0562; to M.-Y. Chen), Sun
Yat-Sen University Clinical Research 5010 Program (201310; to M.-Y. Chen),
Guangdong Provincial Natural Science Foundation of China (S2013020012726; to
M.-Y. Chen), Research Award Program for Outstanding Young Teachers in Higher
Education Institutions (15ykpy38; to M. Xu) and Guangdong Provincial Natural
Science Foundation of China (2015133; to M. Xu).
Corresponding author:
Yi-Xin Zeng, Department of Experimental Research, Sun Yat-sen University Cancer
Center, 651 Dongfeng Road East, Guangzhou 510060, China. Tel: +86-20-8734-3333;
Fax: +86-20-8734-3171. E-mail: [email protected]
Ming-Yuan Chen, Department of Nasopharyngeal Carcinoma, Sun Yat-sen
University Cancer Center, 651 Dongfeng Road East, Guangzhou 510060, China. Tel:
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+86-20-87342422; Fax: +86-20-8734-3171.E-mail: [email protected]
Conflict of interest:The authors disclose no potential conflicts of interest.
Word count: 4751 words
Figures: 4 Tables: 2
Supplementary figures: 6 Supplementary tables: 3
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Abstract
Distant metastasis is the most common cause of treatment failure and mortality
in nasopharyngeal carcinoma (NPC) patients. Thus, it is important to understand the
mechanism of NPC metastasis and identify reliable prognostic factors. In this study,
we investigated the prognostic value of unconjugated bilirubin (UCB), which was
previously considered a byproduct of heme catabolism, in NPC patients and examined
the effects of UCB on NPC metastasis. The Receiver operating characteristic (ROC)
analysis–generated UCB cutoff point for DMFS was 9.7 μM. We found that higher
UCB levels were significantly associated with favorable distant metastasis-free
survival (DMFS, 93.3% vs. 84.2%, P<0.001) in NPC patients and was an independent
predictor for DMFS [Hazard Ratio (HR) = 0.416; 95% Confidence Interval (CI) =
0.280-0.618; P < 0.001]. We next found that UCB treatment impaired the invasion
capability of NPC cells and potently inhibited lung metastasis of NPC cells in nude
mice. Further investigation showed that UCB inhibited reactive oxygen species (ROS)
production, which is involved in the repression of ERK1/2 activation and matrix
metalloproteinase-2 (MMP-2) expression. Moreover, lower levels of ERK1/2
phosphorylation and MMP-2 expression were observed in the NPC lung metastases of
nude mice administered UCB. Taken together, our results indicate that UCB is a
significantly favorable factor for DMFS in NPC patients and may play important role
in NPC chemoprevention.
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1. Introduction
Nasopharyngeal carcinoma (NPC) is a common malignancy in South China, North
Africa, and Alaska (1-3). In South China, the annual incidence of NPC was reported
25–30 per 100,000 individuals (1). NPC is highly malignant and often invades
adjacent regions and metastasizes to regional lymph nodes or distant organs. Although
early-stage NPC patients are sensitive to radiotherapy and chemotherapy, treatment
failure is common in late-stage patients due to distant metastases, which are the key
contributors to NPC mortality and highlight the need for both further understanding of
NPC metastasis and novel drugs to treat it (4, 5).
Bilirubin, a major product of heme catabolism, is generated by the
hemeoxygenase–mediated oxidation of heme during the production of biliverdin.
Unconjugated bilirubin (UCB) is produced by biliverdin reductase-catalyzed
reduction. UCB is cleared from the circulation by the liver, where it is conjugated
with glucuronic acid by the enzyme bilirubin UDP-glucuronosyl transferase
(UGT1A1) to form conjugated bilirubin, which enters the small intestine as a
component of bile. Until the discovery of its antioxidant effects, bilirubin was
considered a waste product of the body (6). Currently, an increasing number of studies
have suggested the potential protective effects of bilirubin against oxidative
stress-related diseases, including stroke, carotid artery atherosclerosis, coronary heart
disease, respiratory disease and cancer (7-17). However, few studies have investigated
the effects of bilirubin on tumor metastasis.
Free radicals and reactive molecules containing oxygen are known as reactive
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oxygen species (ROS) and induce oxidative stress in cells (18). Extensive research
has revealed that ROS and oxidative stress may mediate many behaviors of cancers,
including transformation, survival, proliferation, chemoresistance, radioresistance,
angiogenesis and metastasis (19, 20). Thus, ROS inhibition is important in cancer
chemoprevention. Oxidative stress can activate many molecules, such as Ras,
PI3K/Akt, ERK1/2, p38 MAPK, and JNK1/2, and inactivate the phosphatases that
regulate these proteins (21). Activation of these molecules often leads to up-regulation
of the activity and expression of matrix metalloproteinase (MMP) proteins, which are
associated with the invasion and metastasis of malignant tumors of various
histogenetic origins (22, 23).
In this study, we used a large-scale, retrospective cohort study method and in
vitro and in vivo NPC metastasis models to examine the association between bilirubin
levels and NPC metastasis. We found that UCB is a favorable prognosis biomarker for
DMFS in NPC patients and that it can inhibit the metastasis of NPC cells both in vitro
and in vivo by repressing ERK1/2 activation and MMP-2 expression via its
anti-oxidation activity. Our results indicate for the first time that UCB can inhibit the
metastasis of nasopharyngeal carcinoma and may play important role in
chemoprevention.
2. Materials and Methods
2.1 Patients’ recruitment and data collection
In total, 1327 histologically diagnosed non-metastatic NPC patients at Sun Yat-sen
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University Cancer Center (SYSUCC) between January 2005 and December 2007
were included in the current study. Fifteen patients with unknown personal
information, such as gender, age, and TNM classification, and 50 patients with liver
dysfunction were excluded. The clinical characteristics are summarized in
Supplementary Table S1. All patients were staged according to the 6th edition of the
UICC/AJCC TNM classification system. Informed consent was obtained, and the
study was approved by the institutional ethical board at SYSUCC. Baseline bilirubin
levels were obtained before treatment and determined by an automated
immunoturbidmetric analyzer 7600-020 (Hitachi, High-Technologies, Tokyo, Japan).
2.2 Cell lines
The NPC cell line 5-8F and two single-cell clones derived from the NPC cell line
CNE-2, CNE-2-S18 (S18) and CNE-2-S26 (S26), were kindly provided by Dr.
Chao-Nan Qian, Sun Yat-sen University Cancer Center, China (24). The other NPC
cell line, HONE-1, was also obtained from SYSUCC. All cell lines were thawed from
early passage stocks and passaged for less than 6 months. Each cell line was
authenticated in March 2013 and was periodically monitored for mycoplasma using
Hoechst staining. Cells were maintained in Dulbecco’s Modified Eagle’s Medium
(Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at
37 °C and 5% CO2.
2.3 Reagents
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Unconjugated bilirubin, cisplatin (DDP) and dimethyl sulfoxide (DMSO) were
purchased from Sigma (St. Louis, MO). UCB was freshly prepared in DMSO prior to
each experiment and all experiments were carried out avoiding direct light. The final
concentration of DMSO was no more than 0.5% v/v. When bilirubin was used, the
corresponding amount of DMSO was added to the cultures for appropriate control.
Bovine serum albumin (BSA) was from Merck Millipore (Bedford, MA, USA). When
UCB was used, BSA was added to the culture medium at a UCB/BSA ratio of 1. The
MEK1/2 inhibitor U0126 and reactive oxygen species assay kits were purchased from
Beyotime (China).
2.4 Migration and invasion assays
Migration and invasion assays were performed as previously described (25). S18 cells
(2×104 cells/well for migration assays and 5×104 cells/well for invasion assays) and
HONE-1 cells (5×104 cells/well for migration assays and 8×104 cells/well for invasion
assays) were plated on the inserts and cultured in the upper chambers at 37 °C for 24
h. The number of migrated and invaded cells in five random optical fields (×100
magnification) from triplicate filters was counted and averaged.
2.5 In Vivo metastasis and antitumor assay
All in vivo experiments were approved by the Institutional Animal Care and Use
Committee of Sun Yat-sen University. For the metastasis assay, S18 cells were
injected into the tail veins of BALB/c nude mice (1×106 cells/0.1 ml DMEM). Daily
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treatment by intraperitoneal injection of 20 mg/kg or 30 mg/kg body weight UCB or
DMSO began three days before cells were injected and lasted 7 weeks. Then, the
animals were euthanized and the lungs were collected, weighed and separated into
two parts. Total RNA was extracted from one part and used to detect human HPRT
expression as a measurement for human cell metastasis. The remaining lung sections
were fixed and embedded in paraffin for hematoxylin and eosin staining.
For the antitumor assay, S18 cells (1 × 106 in 100 μl DMEM) were injected near
the scapula of 4- to 6-week-old nude mice. The treatment regimen was the same as in
the in vivo metastasis assay and lasted approximately 1 month. Tumor length and
width were measured with a vernier caliper every other day. Tumor volume was
calculated using the formula V = 0.5 × (length×width2).
2.6 Colony formation assay
Colony formation assays were performed as previously described (26). S18 and
HONE-1 cells were counted and plated in triplicate at 200 cells per well in 6-well
plates and cultured for approximately 10 days. The colony formation efficiency was
the ratio of the number of colonies formed to the number of cells plated.
2.7 Cell apoptosis assay
Cell apoptosis was determined using an Annexin V-FITC apoptosis detection kit
(KGA107, Keygen, China) following the manufacturer’s instructions. Cells were
incubated with 20 μM UCB or DMSO for 24 h. Then, the cells were harvested,
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washed twice with cold PBS, and resuspended with 500 μl of binding buffer. Cells
were stained for 15 min at room temperature in the dark with Annexin V-FITC and
propidium iodide (PI) and then analyzed by flow cytometry (Beckman Coulter).
Cisplatin (DDP), a common clinical drug for NPC, served as a positive control.
2.8 Real-time quantitative PCR
Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden,
Germany), and first-strand cDNA was synthesized using Maxima First Strand cDNA
Synthesis Kits (Thermo Scientific) following the manufacturer’s protocol.
Quantitative determination of RNA levels was performed in triplicate in three
independent experiments. Real-time PCR and data collection were performed with a
Bio-Rad CFX96 real-time PCR detection system, and analyses were performed with
the Biorad CFX manager 2.1 software. The housekeeping gene β-actin was used as an
internal control to normalize expression levels. The primers used for the amplification
of the indicated genes are listed in Supplementary Table S2.
2.9 Western blot
Western blotting was performed as previously described (27). Briefly, cells were lysed
in RIPA lysis buffer on ice, and the clarified lysates were resolved by SDS-PAGE and
transferred to polyvinylidenedifluoride (PVDF) membranes for western blotting using
ECL detection reagents (Beyotime). Antibodies against MMP-9, p-p38, p-JNK1/2,
p-ERK1/2, and ERK1/2 were obtained from Cell Signaling Technology (Danvers,
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MA). The antibody against MMP-2 was from Merck Millipore (Bedford, MA, USA),
and the antibody against α-tubulin was from Santa Cruz Biotechnology (Santa Cruz,
CA).
2.10 Gelatin zymography assay
MMP-2 and MMP-9 protease activities in the concentrated supernatant medium of
S18 or HONE-1 cells were detected by zymography assay. Briefly, 10% SDS-PAGE
gels containing 0.1% gelatin were used to separate the proteins by electrophoresis
under non-reducing conditions. Gelatin zymography was performed using an MMP
zymography assay kit (Applygen Technologies Inc, Beijing, China) according to the
manufacturer’s instructions. To activate proteinases, the gels were incubated at 37 °C
for 40 h in an incubation buffer containing 50 mM Tris–HCl (pH 7.5), 10 mM CaCl2,
and 0.02 mM NaN3. The gels were subsequently fixed and stained with 0.25%
Coomassie brilliant blue R-250 for 1 h and washed in 30% methanol and 10% acetic
acid to visualize the bands of proteolytic activity.
2.11 Measurement of intracellular ROS levels
Changes in the intracellular ROS levels were determined by measuring the oxidative
conversion of cell permeable DCFH-DA to fluorescent dichlorofluorescein (DCF)
using flow cytometry. Following pretreatment with different concentrations of UCB
or DMSO for 24 h, the cells were collected and adjusted to 1×106 cells/mL and
incubated with DCFH-DA at 37 °C for 20 min. Following the incubation, cells were
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washed 3 times with PBS. The signal intensity of DCF was detected with flow
cytometry at an excitation wavelength of 488 nm and an emission wavelength of 535
nm.
2.12 Statistical analysis
Receiver operating characteristic (ROC) curve analysis was performed to select the
most appropriate cut-off point of UCB levels to stratify patients at a high risk of
metastasis. The score closest to the point with both maximum sensitivity and
specificity was selected as the cut-off value. The following endpoints were assessed:
distant metastasis-free survival (DMFS) and overall survival (OS). The events for
DMFS and OS were distant metastasis and death by any cause, respectively. The
duration for each endpoint was defined as the time period from the date of diagnosis
to the occurrence of the event, or the censored date of follow-up or the date of the
final follow-up. These endpoints were analyzed and compared using the
Kaplan-Meier method and log-rank tests. Multivariate analyses using the Cox
proportional hazards model were performed to test the independence, significance,
hazard consistency and the hazard discrimination. A 2-tailed P value < 0.05 was
considered statistically significant. The analyses were performed using SPSS
statistical software (version 16.0, SPSS Inc, Chicago, IL).
3. Results
3.1 Univariate analysis of UCB as a prognostic factor for OS and DMFS
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The patients’ characteristics were summarized in Supplementary Table S1. The 5-year
OS and DMFS of all patients were 88.2% and 87.7%, respectively. The median of
UCB level was 8.7 μM (inter-quartile range: 6.8-11.3 μM). The cut-off point of UCB
level for survival outcomes was determined by ROC curve analyses (Supplementary
Fig. S1), and a UCB value of 9.7 μM resulted in the most appropriate sensitivity and
specificity for DMFS. To identify valuable prognostic factors for NPC, 1327
histologically diagnosed non-metastatic NPC patients were recruited for this study.
Univariate analysis was used to determine prognostic factors for OS and DMFS in
NPC. The analysis revealed that a higher UCB level (≥ 9.7 μM) was associated with
superior OS and DMFS in nasopharyngeal carcinoma (P = 0.023 and P < 0.001,
respectively) (Table 1 and Fig. 1A).
To find whether there’s difference between the UCB levels of males and females,
we took Student’s t-test to compare the UCB levels of males and females. The mean
of UCB for males was 9.5 μM, while the mean of UCB for females was 8.8 μM.
Significant between-group difference was observed (P = 0.004). Serum UCB levels
are significantly higher in men than in women. This was consistent with prior studies
(15). Then we took separate survival analysis in the two groups. After stratification by
genders, UCB levels remained a clinically and statistically significant predictor of
DMFS in both groups (Supplementary Table S3). It indicated that biological effects of
UCB were similar between both genders.
3.2 Multivariate analysis indicates that UCB is an independent prognostic factor for
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DMFS
We next performed multivariate analysis to investigate whether UCB was an
independent predictor for DMFS in NPC. The characteristics, including gender, age, T
stage, N stage, overall stage, treatment modality and serum UCB level were recruited
in the multivariate analysis (Table 2). The results showed that UCB ≥ 9.7 μM was a
significantly independent predictor for the favorable DMFS [hazard ratio (or HR) =
0.416, 95% confidence interval (or 95% CI) = 0.280-0.618; P < 0.001)], while UCB ≥
9.7 μM was not a significantly independent predictor for the favorable OS (HR =
0.733, 95% CI= 0.520-1.033; P=0.076). Taken together, our results indicate that UCB
is a favorable prognosis marker leading to better DMFS for NPC patients.
3.3 UCB impairs the invasion abilities of NPC cells
These results obtained from the clinical analysis encouraged us to investigate whether
UCB can inhibit the metastasis of nasopharyngeal carcinoma. To elucidate the effect
of UCB on NPC metastasis in vitro, we tested whether UCB could inhibit the
metastasis of the NPC cell lines S18 and HONE-1. Boyden chamber assays showed
that 20 μM UCB treatment led to a significant reduction in the number of cells that
invaded through the membrane compared with the vehicle control in both S18 and
HONE-1 cells (Fig. 1B). However, the migration of NPC cells was not significantly
impaired by UCB (Fig. 1B), suggesting that UCB inhibits NPC metastasis by
inhibiting NPC cell invasion rather than migration. The inhibition of invasion was
also observed in two other NPC cell lines, 5-8F and S26 (Supplementary Fig. S2).
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We next evaluated the in vivo effects of UCB on NPC cell metastasis.
Intraperitoneal injections of 20 mg/kg or 30 mg/kg bilirubin into BALB/c mice each
day resulted in an increase of serum UCB levels to 7-10 μM, measured at 30 minutes
and 6 hours after injection (Supplementary Fig. S3). After 3 days of administration of
bilirubin, we injected S18 cells into the lateral tail vein of 8-week-old nude mice and
evaluated cancer metastasis to the lungs. Hematoxylin and eosin staining showed that
intraperitoneal injection of UCB at a dosage of 20 mg/kg or 30 mg/kg per day for 7
consecutive weeks significantly reduced lung metastasis and average lung weight
compared with the massive amount of metastasis observed in the DMSO injection
control mice (Fig. 1C). Correspondingly, the average lung weight of the UCB group
was decreased compared with the control group (Fig. 1C). Quantification of human
HPRT mRNA further showed that UCB reduced the number of metastatic NPC cells
in the lungs of the mice (Fig. 1D). Collectively, these data suggest that UCB inhibits
the metastasis of NPC cells both in vitro and in vivo.
Previous studies suggested that UCB may inhibit cell growth and induce
apoptosis in cancer cells (28, 29). However, our results suggested that 20μM UCB
does not significantly affect the growth and apoptosis of NPC cells both in vitro and
in vivo (Supplementary Fig. S4). These results indicated that the anti-metastasis effect
of UCB didn’t result from change of cell growth rate.
3.4 UCB down-regulates the expression of MMP-2
To explore the mechanism by which UCB inhibits the metastasis of NPC cells, we
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next tested the effect of UCB on the expression of two matrix metalloproteinases
(MMPs) that are crucial to cellular invasion, MMP-2 and MMP-9 (23,30). Real-time
quantitative PCR and western blot analysis showed that UCB significantly decreased
the expression of MMP-2 in S18 and HONE-1 cells but it did not significantly change
the expression of MMP-9 (Fig. 2A and B). Zymography assays of the cultured media
of S18 and HONE-1 cells treated with vehicle or UCB revealed that UCB treatment
caused a significant reduction in the activity of MMP-2, but not MMP-9, in the
medium (Fig. 2C). These results suggest that UCB inhibits the expression of MMP-2
in NPC cells.
3.5 UCB down-regulates MMP-2 expression by inhibiting the phosphorylation of
ERK
Because the mitogen-activated protein kinase (MAPK) pathway is critical for the
stimulation of cell invasion and MMP-2 expression (22, 31), we next evaluated the
effect of UCB on the signal transducers of the MAPK pathway. As shown in Fig. 3A,
treatment of UCB suppressed the phosphorylation of ERK1/2, although it did not
affect p38 MAPK or JNK1/2 phosphorylation.
Inhibition of ERK1/2 phosphorylation down-regulates the expression of MMP-2
in many cancers (22), thus we next explored whether the down-regulation of MMP-2
by UCB in NPC was mediated through the inhibition of ERK1/2. As shown in Fig. 3B,
both 0.25 μM U0126, a specific MEK inhibitor (Supplementary Fig. S5A), and 20 μM
UCB significantly inhibited the expression of MMP-2, and this effect increased when
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U0126 and UCB treatments were combined. Zymography assays of the cultured
media of S18 and HONE-1 cells revealed that both U0126 and UCB treatment caused
a significant reduction in the activity of MMP-2, and this effect became more distinct
when U0126 and UCB treatments were combined (Supplementary Fig. S5B).
Moreover, lower levels of ERK1/2 phosphorylation and MMP-2 expression were
observed in the NPC lung metastases of nude mice administered UCB (Fig. 3C).
Boyden chamber assays showed that the inhibition of ERK1/2 phosphorylation by
U0126 decreased the invasion of NPC cells and that co-administration of 20 μM UCB
enhanced this effect (Fig. 3D). The decrease in NPC invasion was not due to an
inhibition of proliferation because 0.25 μM U0126 did not change the growth rate of
S18 or HONE-1 cells (Supplementary Fig. S5C).
3.6 UCB inhibits the invasion of NPC by decreasing the levels of intracellular reactive
oxygen species
Previous studies have suggested that UCB is an antioxidant. Thus, we examined the
production of reactive oxygen species (ROS) in NPC cells in the absence and
presence of UCB treatment. We found that the ROS levels in CNE2-S18 and HONE-1
cells were markedly decreased following 20 μM UCB treatment (Fig. 4A). ROS can
promote the phosphorylation of ERK1/2 and the invasion of cancers (32). To
determine if the inhibition of the invasiveness of NPC cells by UCB was due to a
reduction in ROS, we used hydrogen peroxide (H2O2) to increase ROS levels in NPC
cells treated with UCB and examined whether the invasive potential of the NPC cells
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recovered. Cell counting assays indicated that low concentrations of H2O2 (<50 μM)
did not significantly change the growth rate of S18 and HONE-1 cells (Supplementary
Fig. S6A). ROS analysis showed that 10 μM H2O2 treatment could recover the ROS
levels of NPC cells treated with 20 μM UCB (Fig. 4B and Supplemental Fig. S6B).
Exposure of NPC cells to 20 μM UCB plus 10 μM H2O2 led to a remarkable increase
in cell invasion compared with 20 μM UCB alone (Fig. 4C). Western blot and
zymography analysis also showed that treating the NPC cells with 10 μM H2O2
restored the expression and activity of MMP-2 and ERK1/2 phosphorylation (Fig. 4D
and Supplemental Fig. S6C). Taken together, these results indicate that UCB inhibits
the invasion of NPC cells by decreasing intracellular ROS levels and repressing
ERK1/2 activation and MMP-2 expression (Supplemental Fig. S6D).
4. Discussion
This study demonstrates for the first time that bilirubin is a favorable and
independent prognostic factor for distant metastasis-free survival in NPC patients.
Further study revealed that bilirubin can inhibit the metastasis of NPC cells by
repressing the phosphorylation of ERK1/2 and subsequent MMP-2 expression via its
anti-oxidation activity.
Until the discovery of its anti-oxidation capability, bilirubin had long been
regarded as just a byproduct of heme metabolism. Extremely high levels of serum
bilirubin lead to its accumulation in the brain, causing kernicterus in newborns (33-
35). However, moderately high levels of bilirubin (17.1 µM to 34.2 µM) are probably
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beneficial for many diseases, including cancer (13, 16). A case control study that
evaluated the relationship between antioxidant levels and breast cancer risk identified
that elevated serum bilirubin levels were correlated with reductions in breast cancer
risk (36). In addition, a large cohort study based on a Belgian population noted that
higher serum bilirubin levels were associated with lower cancer mortality and
concluded that serum bilirubin levels might estimate cancer risk (37). Furthermore,
data from the Third National Health and Nutrition Examination Survey of the United
States population revealed that serum bilirubin levels had a negative correlation with a
history of colorectal cancer (15). All of these studies indicate that higher levels of
bilirubin are associated with lower cancer risk and cancer-related mortality. However,
few studies evaluated serum bilirubin as a prognostic marker in patients. To our
knowledge, this is the first study that evaluated the association between bilirubin and
metastasis.
Because bilirubin is an antioxidant and ROS play an important role in tumor
metastasis, we hypothesized that bilirubin inhibited the metastasis of NPC cells
through its anti-oxidation activity. As early as the 1950s, bilirubin was reported to
protect against the oxidation of lipids such as vitamin A (38, 39). Then, Stocker et al
confirmed that bilirubin was an important physiological antioxidant under physical
concentrations (6, 40). As an antioxidant, bilirubin is itself oxidized to biliverdin and
then recycled back to bilirubin by biliverdin reductase, reflecting an amplification
cycle to control the primary physiological function of bilirubin (41). Bilirubin can
protect against 10,000-fold higher concentrations of H2O2, which may be attributed to
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the role of biliverdin reductase (41). Consistent with previous reports, our results
indicate that bilirubin can decrease the ROS levels in NPC cells. Because ROS have
been reported to promote metastasis by activating the mitogen-activated protein
kinase (MAPK) pathway (22, 31), we next explored the effect of UCB on MAPK. We
found that UCB decreased MMP-2 expression mainly by suppressing the
phosphorylation of ERK1/2, other than phosphorylation of p38 and JNK1/2.
Importantly, increasing the ROS levels in cells by adding H2O2 following UCB
treatment restored the expression of MMP-2 and phosphorylation of ERK1/2. Taken
together, our results indicate that UCB decreases MMP-2 expression and ERK1/2
phosphorylation by reducing ROS levels.
Metastases represent the end products of a multistep cell-biological process that
includes invasion, intravasation, survival in the circulation, arrest at a distant organ
site, extravasation, micrometastasis formation and metastatic colonization (42, 43).
Any of these steps can become the therapeutic target in treating tumor metastasis. As
an important metabolite in blood circulation, UCB may affect the metastasis of NPC
cells at multiple steps. In fact, ROS and MMPs have been reported to be associated
with many stages of tumor metastasis, including invasion, intravasation and
extravasation (20, 23). Which steps are affected when UCB impairs the metastasis of
NPC cells remains to be explored.
A small number of studies have suggested that UCB may inhibit proliferation
and induce apoptosis in cancer cells (28, 29). However, we did not observe an effect
on NPC cells until the concentration of UCB reached 100 μM (Supplementary Fig.
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21
S4). We speculate the reason for this result is that the sensitivity to the toxicity of
UCB is different among tumors. Even within one tumor type, different cell lines are
reported to have disparate sensitivities (29). Further studies need to be performed to
determine why different tumors have disparate sensitivities to UCB.
In addition to NPC, we also found that elevated UCB at diagnosis was a
significant favorable prognostic biomarker correlated with better DMFS in lung
cancer (data not shown). Given that ROS and MMPs are important factors in the
metastasis of many cancers and that UCB is a vital antioxidant in blood circulation,
these results suggest that UCB may play an anti-metastasis role in many types of
tumors.
In conclusion, our results suggest that UCB is a favorable prognostic biomarker
correlated with better DMFS in NPC patients and that UCB can inhibit the metastasis
of NPC cells both in vitro and in vivo via its anti-oxidation activity. Elucidating the
mechanisms by which UCB inhibits tumor metastasis and researching the potential
roles of other antioxidants in blood circulation will provide valuable insight towards
understanding tumor metastasis and discovering novel strategy for the
chemoprevention of NPC.
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Figure legends
Figure 1. Unconjugated bilirubin (UCB) is a favorable prognostic marker and
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can inhibit the invasion of NPC cells. (A) The overall survival (OS) rate and the
distant metastasis-free survival (DMFS) rate were significantly higher in the
high-UCB group. (B) UCB dramatically reduced the invasive abilities of S18 and
HONE-1 cells as determined using Boyden chamber assays. Scale bars: 50 μm. Bars
denote the standard deviation (n = 3). *P<0.05 and ***P<0.001. (C) Representative
H&E staining and wet weight of lungs from mice 7 weeks after tail vein injection of
S18 cells. Scale bars: 500 μm (upper panel) and 100 μm (lower panel). *P<0.05. (D)
Expression of human HPRT mRNA relative to 18S rRNA in the lungs of
tumor-bearing mice. Data are normalized to DMSO-treated mice. Bars denote the
standard deviation (n = 6). *P<0.05.
Figure 2. UCB inhibits the expression of MMP-2. (A-B) Real-time quantitative
PCR (A) and western blot (B) analyses showed that 20 μM UCB markedly decreased
the expression of MMP-2 in S18 and HONE-1 cells but did not significantly change
the expression of MMP-9. Bars denote the standard deviation (n = 3). *P<0.05 and
**P < 0.01. (C) Zymography indicated that 20 μM UCB inhibited the activity of
MMP-2 in the culture medium of S18 and HONE-1 cells.
Figure 3. UCB down-regulates the expression of MMP-2 by inhibiting the
phosphorylation of ERK1/2. (A) S18 (left panel) and HONE-1 (right panel) cells
were treated with various concentrations (0, 5, 10 and 20 μM) of UCB for 48 h, and
the phosphorylated levels of ERK1/2, p38 and JNK1/2 were determined by western
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blot. (B) S18 (left panel) and HONE-1 (right panel) cells were treated with UCB,
ERK1/2 inhibitor U0126 or a combination of UCB and U0126 as indicated. The
phosphorylated levels of ERK1/2 and MMP-2 expression were determined by western
blot. (C) Immunohistochemical analysis of MMP-2 and p-ERK expression in the
lungs of mice 7 weeks after tail vein injection of S18 cells. Scale bars: 100 μm. (D)
S18 and HONE-1 cells were treated as indicated, and the invasive abilities of the cells
were determined using Boyden chamber assays. Scale bars: 50 μm. Bars denote the
standard deviation (n = 3). *P<0.05, **P < 0.01 ***P < 0.001.
Figure 4. UCB decreases intracellular reactive oxygen species levels of NPC cells.
(A) S18 and HONE-1 cells were treated with DMSO or 20 μM UCB. Intracellular
ROS were determined by measuring the oxidative conversion of cell permeable
DCFH-DA to fluorescent dichlorofluorescein (DCF) using either flow cytometry
(upper panel) or fluorescence microscopy (lower panel). Scale bars: 50μm. (B) S18
and HONE-1 cells were treated with DMSO, 20 μM UCB or a combination of 20 μM
UCB and 10 μM H2O2. Levels of intracellular ROS were determined by flow
cytometry. (C) S18 and HONE-1 cells were treated as indicated, and the invasive
abilities of the cells were determined using Boyden chamber assays. Scale bars: 50
μm. Bars denote the standard deviation (n = 3). *P<0.05 and ***P < 0.001. (D) S18
and HONE-1 cells were treated as indicated, and the phosphorylated levels of ERK1/2
and MMP-2 expression were determined via western blot.
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Table 1.Univariate analysis of prognostic factors for patients with nasopharyngeal carcinoma.
Factors Overall Survival Distant Metastasis-Free Survival
5-year (%) HR (95%CI) P-value 5-year (%) HR (95%CI) P-value
Gender
Female 92.7 reference 86.2 reference
Male 85.6 2.005 (1.316-3.054) 0.001 91.6 1.714 (1.144-2.568) 0.009
Age(years)
≤ 52 90.0 reference 89.5 reference
> 52 85.0 1.604(1.166-2.205) 0.004 85.7 1.379 (1.003-1.897) 0.048
T status
T1/T2 94.3 reference 93.0 reference
T3/T4 85.7 2.425(1.620-3.630) <0.001 86.2 1.999 (1.360-2.939) <0.001
N status
N0/N1 90.7 reference 91.5 reference
N2/N3 86.3 1.614(1.177-2.213) 0.003 85.0 1.827(1.324-2.521) <0.001
Overall stage
I/II 94.2 reference 93.8 reference
III/IV 85.7 2.561(1.711-3.834) <0.001 85.7 2.475(1.652-3.709) <0.001
Treatment
RT 90.6 reference 91.7 reference
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CRT 86.2 1.459(1.019-2.090) 0.039 85.8 1.834(1.247-2.695) 0.002
UCB (μmol/L)
< 9.7 86.0 reference 84.2 reference
≥ 9.7 90.1 0.676(0.481-0.950) 0.023 93.3 0.387(0.261-0.574) <0.001
Abbreviations: RT, radiotherapy; CRT, chemoradiotherapy; UCB, unconjugated bilirubin.
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Table 2.Multivariate analysis of prognostic factors for patients with nasopharyngeal carcinoma.
Abbreviations: CRT, chemoradiotherapy; RT, radiotherapy; UCB, unconjugated bilirubin.
Factors Overall Survival Distant Metastasis-Free Survival
HR (95% CI) P-value HR (95% CI) P-value
Gender (male vs. female) 2.087(1.356-3.190) 0.001 1.819(1.211-2.733) 0.004
Age (≥ vs. <52 years) 1.569(1.137-2.164) 0.006 1.385(1.004-1.911) 0.047
T status (T3/T4 vs. T1/T2) 1.869(1.194-2.926) 0.006 1.482(0.969-2.265) 0.069
N status (N2/N3 vs. N0/N1) 1.119(0.760-1.648) 0.570 1.247(0.829-1.874) 0.289
Overall stage (III/IV vs. I/II) 1.977(1.170-3.340) 0.011 1.626(0.944-2.802) 0.080
Treatment (CRT vs. RT) 0.927(0.629-1.366) 0.702 1.241(0.820-1.879) 0.307
UCB (≥ vs.<9.7μmol/L) 0.733(0.520-1.033) 0.076 0.416(0.280-0.618) <0.001
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Published OnlineFirst September 25, 2015.Cancer Prev Res Cheng-Cheng Deng, Miao Xu, Jing Li, et al. anti-oxidation activitynasopharyngeal carcinoma and inhibits its metastasis via Unconjugated bilirubin is a novel prognostic biomarker for
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