Unconjugated bilirubin is a novel prognostic biomarker for...

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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,


5. Department of Radiation Oncology, Sun Yat-sen University Cancer Center,


6. Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer

Center, Guangzhou 510060, China.

Cancer Research. on July 5, 2018. © 2015 American Association forcancerpreventionresearch.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 25, 2015; DOI: 10.1158/1940-6207.CAPR-15-0257

http://cancerpreventionresearch.aacrjournals.org/

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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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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




25

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




26

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.




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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