Article

Soluble receptor for advanced glycation end products and risk of liver cancer

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Nation Institutes of Health, Bethesda, Maryland. .
Hepatology (Impact Factor: 11.06). 06/2013; 57(6). DOI: 10.1002/hep.26264
Source: PubMed
ABSTRACT
Unlabelled:
Binding of advanced glycation end products (AGEs) to their receptor (RAGE) increases oxidative stress and inflammation and may be involved in liver injury and subsequent carcinogenesis. Soluble RAGE (sRAGE) may neutralize the effects mediated by the AGE/RAGE complex. Epidemiologic studies examining sRAGE or AGEs in association with liver cancer are lacking. We examined the associations between prediagnostic serum concentrations of sRAGE or Nϵ-(carboxymethyl)-lysine (CML)-AGE and hepatocellular carcinoma in a case-cohort study within a cohort of 29,133 Finnish male smokers who completed questionnaires and provided a fasting blood sample between 1985 and 1988. During follow-up beginning 5 years after enrollment through April 2006, 145 liver cancers occurred. Serum concentrations of sRAGE, CML-AGE, glucose, and insulin were measured in case subjects and 485 randomly sampled cohort participants. Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) were available in most cases and in a subset of the study population. Weighted Cox proportional hazards regression was used to calculate relative risks (RR) and 95% confidence intervals (CI) adjusted for age, years of smoking, and body mass index. sRAGE and CML-AGE concentrations were inversely associated with liver cancer. Further adjustment for glucose and insulin or exclusion of case subjects with chronic HBV or HCV did not change the associations.

Conclusion:
Our results support the hypothesis that sRAGE is inversely associated with liver cancer. The findings need confirmation, particularly in populations that include women and nonsmokers. (HEPATOLOGY 2013 ).

Full-text

Available from: Neal Freedman, Mar 09, 2016
Soluble Receptor for Advanced Glycation End Products
and Risk of Liver Cancer
Kristin A. Moy,
1
Li Jiao,
2
Neal D. Freedman,
1
Stephanie J. Weinstein,
1
Rashmi Sinha,
1
Jarmo Virtamo,
3
Demetrius Albanes,
1
and Rachael Z. Stolzenberg-Solomon
1
Binding of advanced glycation end products (AGEs) to their receptor (RAGE) increases oxi-
dative stress and inflammation and may be involved in liver injury and subsequent carcino-
genesis. Soluble RAGE (sRAGE) may neutralize the effects mediated by the AGE/RAGE
complex. Epidemiologic studies examining sRAGE or AGEs in association with liver cancer
are lacking. We examined the associations between prediagnostic serum concentrations of
sRAGE or N-(carboxymethyl)-lysine (CML)-AGE and hepatocellular carcinoma in a case-
cohort study within a cohort of 29,133 Finnish male smokers who completed questionnaires
and provided a fasting blood sample between 1985 and 1988. During follow-up beginning
5 y ears after enrollment through April 2006, 145 liv er cancers occurred. Serum concentra-
tions of sRAGE, CML-AGE, glucose, and insulin were measured in case subjects and 485
randomly sampled cohort participants. Chronic hepatitis B virus (HBV) and hepatitis C vi-
rus (HCV) were available in most cases and in a subset of the study population. Weighted
Cox proportional hazards regression was used to calculate relative risks (RR) and 95% confi-
dence intervals (CI) adjusted for age, years of smoking, and body mass index. sRAGE and
CML-AGE concentrations were inversely associated with liver cancer. Further adjustment
for glucose and insulin or exclusion of case subjects with chronic HBV or HCV did not
change the associations. Conclusion: Our results support the hypothesis that sRAGE is inver-
sely associated with liver cancer. The findings need confirmation, particularly in populations
that include women and nonsmokers. (H
EPATOLOGY 2013;57:2338-2345)
W
orldwide, primary liver cancer is the sixth
most commonly occurring cancer and the
third most common cause of cancer-related
deaths.
1,2
Established risk factors for hepatocellular
carcinoma or HCC, the most common type, include
aflatoxin B exposure, chronic infection with hepatitis
B virus (HBV) or hepatitis C virus (HCV), excessive
alcohol consumption, and obesity and diabetes, which
increase the risk of nonalcoholic steatohepatitis
(NASH).
3,4
These risk factors are typified by their
ability to cause chronic inflammation in the liver,
which is associated with subsequent carcinogenesis.
5
Advanced glycation end products (AGE) and their re-
ceptor (RAGE) are implicated in both inflammation
and cancer (reviewed by Riehl et al.,
6
Singh et al.,
7
and Sparvero et al.
8
). However, the potential role of
the AGE-RAGE axis in the development of HCC is
unknown.
AGEs are heterogeneous irreversible adducts formed
by the nonenzymatic glycation of proteins, lipids, and
nucleic acids.
7
The two major sources of AGEs are en-
dogenous AGEs that form during normal metabolism
and exogenous AGEs derived from tobacco smoke or
food.
7,9
Dietary AGEs are formed when food is proc-
essed at high temperatures using methods such as deep
frying, broiling, and grilling.
10
AGEs accumulate in tis-
sues, and the rate of accumulation increases with aging
and under hyperglycemic conditions.
7
Of the approxi-
mately 20 different AGEs identified, N
-(carboxy-
methyl)-lysine (CML)-AGE is the best characterized.
11,12
Abbreviations: AGE, advanced glycation end product; anti-HBc, antibody to hepatitis B core antigen; anti-HBV, antibody to hepatitis B virus; ATBC, Alpha-
Tocopherol, Beta-Carotene Cancer Prevention Study; BMI, body mass index; CI, confidence interval; CML, N-(carboxymethyl)-lysine; HBsAg, hepatitis B surface
antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; RAGE, receptor for advanced glycation end product; RR, relative risks;
sRAGE, soluble receptor for advanced glycation end product; NASH, nonalcoholic steatohepatitis.
From the
1
Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda,
MD;
2
Section of Gastroenterology and Hepatology, Department of Medicine, Baylor College of Medicine, Houston, TX; and
3
Department of Chronic Disease
Prevention, National Institute for Health and Welfare, Helsinki, Finland.
Received August 27, 2012; accepted December 27, 2012.
Supported by the Intramural Research Program of the National Institutes of Health, Division of Cancer Epidemiology and Genetics, National Cancer Institute,
National Institutes of Health, Department of Health and Human Services, Bethesda, MD.
2338
Page 1
AGEs up-regulate inflammation by binding their full-
length membrane-bound receptor RAGE.
7,13
RAGE is
a multiligand receptor that belongs to the immuno-
globulin superfamily. Binding ligand triggers the acti-
vation of cell signaling pathways such as p38 and p44/
42 mitogen-activated protein kinase, as well as nuclear
factor kappa B, generating reactive oxygen species and
the production of proinflammatory cytokines (reviewed
by Riehl et al.,
6
and Bierhaus and Nawroth
14
). Because
the liver is important for the clearance and catabolism
of circulating AGEs (e.g., removing >90% of intrave-
nously injected AGEs via endocytosis has been shown
in rats
13,15
), the AGE-RAGE axis may be particularly
important for liver carcinogenesis and chronic liver
diseases, including NASH and liver cirrhosis.
16-18
In addition to the full-length receptor for AGEs,
RAGE has truncated soluble isoforms (sRAGE) contain-
ing only the RAGE extracellular domain,
13,15
including
a splice variant of the full-length receptor, endogenous
secretory RAGE, and an isoform formed by proteolytic
cleavage.
19,20
Both forms can be detected in human
serum, are capable of binding ligands, and are thought
to bind free AGEs, exerting a cytoprotective effect by
preventing ligands from binding to cell surface
RAGE.
8,15
In vitro and experimental studies suggest a
protective role of sRAGE in hepatocellular injury.
21-27
Although several hospital-based studies have found
sRAGE levels to be lower in lung, breast, and pancre-
atic cancer case subjects compared with healthy con-
trols,
28-30
only three prospective epidemiologic studies
have examined the potential association of AGEs with
cancer, finding no significant associations between
CML-AGE and pancreatic
31,32
or colorectal cancers.
33
In the current study, we examined the associations
of serum levels of sRAGE and CML-AGE with liver
cancer risk in the Alpha-Tocopherol, Beta-Carotene
Cancer Prevention (ATBC) Study.
34
We hypothesized
that lower levels of sRAGE or higher levels of CML-
AGE are associated with increased risk.
Subjects and Methods
Study Population. The design of the ATBC Study
has been described in detail elsewhere.
34
Briefly, the
ATBC Study was a randomized, double-blind, pla-
cebo-controlled, primary prevention trial conducted in
southwest Finland to determine the effects of supple-
mentation with a-tochopherol and b-carotene on can-
cer incidence among male smokers. Between 1985 and
1988, a total of 29,133 Caucasian men aged 50 to 69
years who smoked at least five cigarettes per day were
randomized to receive an active supplement or a pla-
cebo. Potential participants were excluded from the
trial if they reported malignancy other than nonmela-
noma skin cancer or carcinoma in situ, severe angina
on exertion, chronic renal insufficiency, liver cirrhosis,
chronic alcoholism, receiving anticoagulant therapy,
other medical problems that might limit participation
for 6 years, or current use of supplements containing
vitamin E, vitamin A, or b-carotene in excess of prede-
fined doses.
34
The trial ended in April 1993, but par-
ticipants continued to be followed for health outcomes
through national registries. The ATBC Study was
approved by the institutional review boards of both
the US National Cancer Institute and the National
Public Health Institute of Finland (now the National
Institute for Health and Welfare, Helsinki, Finland).
All participants provided written informed consent
before randomization.
At enrollment, participants completed a self-admin-
istered questionnaire that assessed demographics,
including medical, smoking, and occupational histor-
ies. Dietary intake during the previous year was also
assessed using a validated self-administered dietary his-
tory questionnaire. Height and weight were measured
by trained nurses. Body mass index (BMI) was calcu-
lated as weight in kilograms per height in meters
squared.
34
Study participants also provided venous
blood samples after an overnight fast at baseline, and
serum was aliquoted and stored at 70
C.
Cases and Subcohort. The present study is a case-
cohort study within the parent study. To reduce to the
potential effect of subclinical disease on serum levels of
CML-AGE or sRAGE in samples collected at baseline
prior to randomization, subjects in this study were
drawn from cohort members who were alive and can-
cer-free as of the sixth year of follow-up (N ¼
24,708).
34
Thus, follow-up began 5 years after blood
collection at randomization and ended at date of liver
cancer diagnosis, death, or on April 30, 2006,
Address reprint requests to: Kristin A. Moy, Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120
Executive Boulevard, Suite 320, Rockville, MD 20852, Fax: (301) 496-6829. E-mail: moyka@mail.nih.gov.
Copyright
V
C
2013 by the American Association for the Study of Liver Diseases.
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.26264
Potential conflict of interest: Nothing to report.
HEPATOLOGY, Vol. 57, No. 6, 2013 MOY ET AL. 2339
Page 2
whichever occurred first. A total of 146 incident liver
cancer cases (defined based on the International Classi-
fication of Diseases 9; codes 155.0, 155.1, and 155.2)
were identified from the Finnish Cancer Registry.
35
From the remaining eligible cohort members, 500 sub-
jects were randomly selected as the reference group.
After excluding 1 case and 15 subcohort participants
with missing data on one or more of the serological
biomarkers, the present analysis included 145 liver
cancer cases and 485 subcohort participants.
Laboratory Methods. Serum sRAGE and CML-
AGE were measured in duplicate by Microcoat
Biotechnologie Company using the human sRAGE
Quantikine ELISA kit (R&D Systems, Inc.) and the
AGE-CML-ELISA kit (Microcoat Biotechnologie
Company), respectively. The AGE-CML-ELISA kit
uses a CML-specific monocolonal antibody (mouse
monocolonal 4G9; Alteon Inc).
36
The sRAGE Quanti-
kine ELISA kit detects a heterogeneous group of total
sRAGE proteins, including cleavage forms of mem-
brane-bound full-length RAGE,
19
endogenous secre-
tory RAGE,
20
and other splice variant forms of
RAGE. Case and subcohort samples were randomly
ordered in each batch along with 10% blinded quality
control samples from a single pooled serum sample.
The intrabatch coefficients of variation for sRAGE and
CML-AGE were 3% and 7%, respectively, and the
corresponding interbatch coefficients of variation were
6% and 14%. Serum concentrations of glucose and in-
sulin were measured previously for 51 cases and 406
subcohort participants.
37
In the present study, serum
glucose and insulin were determined on an additional
95 cases that occurred after 2001 and on an additional
94 subcohort participants using the same method in
the same laboratory as the earlier study.
Statistical Analysis. The Wilcoxon rank sum test
for continuous variables and the v
2
test for categorical
variables were used to compare the distributions of
selected demographics, including dietary and other
characteristics between the cases and the subcohort.
Dietary variables were adjusted for total energy intake
using the residuals method.
38
Selected demographic
and dietary variables were examined across tertiles of
serum CML-AGE and sRAGE using the analysis of
covariance method.
Standard statistical methods for case-cohort studies
were used in the present study. Weighted Cox propor-
tional hazards regression was used to calculate relative
risks (RR) and their corresponding 95% confidence
intervals (CIs) and P values.
39
Study subjects
were grouped by tertiles of sRAGE and CML-AGE
defined according to the distribution in the subcohort.
Continuous RRs were calculated to the average size of
the central tertile to represent the change in risk of
liver cancer per tertile, and P values for linear trends
across tertiles were calculated using a score variable
based on the median value of each tertile. Follow-up
time was used as the underlying time metric. We used
forward and backward stepwise regression to evaluate
potential confounding. Factors evaluated as potential
confounders of the sRAGE or CML-AGE and liver
cancer associations include the variables listed in
Table 1. A variable was defined as a confounding fac-
tor if it was significantly associated with liver cancer
risk and sRAGE or CML-AGE, and inclusion of the
variable in the model changed the risk estimate for
sRAGE or CML-AGE by more than 10%. Although
Table 1. Baseline Characteristics of Liver Cancer Case
Subjects and Subcohort Participants, ATBC Study (1985-
2006)
Characteristic
Case
Subjects
(n ¼ 145)
Subcohort
Participants
(n ¼ 485) P*
Age, years, mean (SD) 58.1 (4.8) 56.4 (5.0) 0.0001
BMI, kg/m
2
, mean (SD) 27.6 (4.7) 26.7 (4.0) 0.03
Years of smoking, mean (SD) 37.2 (8.3) 35.2 (8.2) 0.0002
No. of cigarettes per
day, mean (SD)
21.5 (9.3) 20.8 (8.5) 0.50
Clinical diabetes, n (%)
Glucose <126 mg/dL 125 (86.2) 461 (95.0) 0.0002
Glucose 126 mg/dL 20 (13.8) 24 (5.0)
Dietary or nutrient
intake/day,† mean (SD)
Total meat, g 197 (73.3) 194 (68.8) 0.65
Red meat 73.1 (30.0) 68.2 (28.2) 0.06
Processed meat 69.4 (46.4) 74.3 (52.4) 0.59
Alcohol, g 27.7 (33.1) 18.9 (21.7) 0.01
Coffee, g 502 (337) 595 (354) 0.002
Total fat, g 119 (22.0) 122 (18.0) 0.28
Saturated fat, g 48.9 (15.0) 52.8 (13.9) 0.01
Protein, g 92.3 (13.2) 94.4 (12.7) 0.14
Carbohydrates, g 257 (38.5) 264 (39.1) 0.03
Glucose, g 8.69 (4.37) 9.30 (5.76) 0.69
Sucrose, g 48.9 (26.1) 51.5 (24.1) 0.15
Iron, mg 17.6 (3.87) 17.9 (3.43) 0.29
sRAGE
Mean (SD) 561 (262) 615 (307) 0.09
Median (IQR) 520 (377-706) 571 (413-738)
CML-AGE
Mean (SD) 484 (131) 565 (168) <0.0001
Median (IQR) 480 (404-680) 561 (471-668)
Treatment arm, n (%)
Placebo 35 (24.1) 110 (22.7) 0.48
BC only 40 (27.6) 116 (23.9)
AT only 33 (22.8) 131 (27.0)
AT þ BC 37 (25.5) 128 (26.4)
Abbreviations: AT, a-tocopherol; BC, b-carotene; IQR, interquartile range.
*Two-sided
P
values are based on Wilcoxon rank sum tests for continuous
variables and v
2
tests for categorical variables.
†Food and nutrient variables were adjusted for total energy intake. Informa-
tion was available for 133 liver cancer cases and 465 subcohort participants.
2340 MOY ET AL. HEPATOLOGY, June 2013
Page 3
none of the variables (Table 1) met the latter criterion,
final multivariate models included age at randomiza-
tion, years of smoking, and BMI. Further adjustment
for number of cigarettes per day, trial treatment arm,
or other variables associated with sRAGE or CML-
AGE (Table 2) did not change risk estimates more
than 10% and were not included in the final models.
To assess whether the associations between sRAGE or
CML-AGE and liver cancer were independent of other
serum analytes, insulin and glucose concentration were
added into the models individually, but because their
inclusion did not change the risk estimates more than
10%, they were not retained in the final models.
Effect modification of sRAGE and CML-AGE by
BMI (continuous and <25 kg/m
2
versus 25 kg/m
2
),
number of years of smoking (continuous), number of
cigarettes per day (continuous), serum glucose (contin-
uous), clinical diabetes (yes or no), energy-adjusted red
meat intake (continuous), coffee intake (continuous),
and trial intervention (placebo, b-carotene only, a-to-
copherol only, b-carotene plus a-tocopherol) was tested
by including cross-product terms in multivariate mod-
els and testing for statistical significance using the
Wald test. Additionally, the joint effects of sRAGE and
CML-AGE (both dichotomous) on the risk of liver
cancer were examined. To examine potential differen-
tial effects of sRAGE and CML-AGE at different
stages of liver carcinogenesis, we examined the associa-
tions between sRAGE or CML-AGE and liver cancer
stratified by follow-up time (subjects with 5-9 years of
follow-up and subjects with 10 years of follow-up).
Exposure to HBV and HCV infection serologic sta-
tus was available for 135 cases and 26 subcohort sub-
jects in the present study. Sensitivity analyses excluding
participants who were positive for antibody to hepatitis
B core antigen (anti-HBc), hepatitis B surface antigen
(HBsAg), or antibody to HCV (anti-HCV) were con-
ducted. Statistical analysis was performed using SAS
software version 9.1 (SAS Institute, Cary, NC) and
SUDAAN software (RTI, Research Triangle Park,
NC). All statistical tests were two-sided, and P < 0.05
was considered statistically significant.
Results
In the present study, the average time (6 SD)
between serum collection and liver cancer diagnosis
was 12.2 (6 4.2) years, ranging from 5 to 21 years.
The mean age (6 SD) at liver cancer diagnosis was
70.3 (6 5.4) years.
Compared with subcohort participants, liver cancer
case subjects were slightly older at time of randomiza-
tion, had a higher BMI, and reported smoking more
cigarettes per day for a longer period (Table 1). Case
subjects were also more likely to report having been
diagnosed with diabetes and consuming more red
meat and alcohol. Case subjects also consumed less
coffee, saturated fat, and carbohydrates than their sub-
cohort counterparts. Case subjects had significantly
lower serum levels of CML-AGE and borderline signif-
icantly lower serum levels of sRAGE compared with
the subcohort population (Table 1).
Table 2 shows the age-adjusted means of selected
baseline characteristics associated with liver cancer or
AGEs of the subcohort participants according to ter-
tiles of serum sRAGE and CML-AGE. There was
higher carbohydrate and sucrose intake across tertiles
Table 2. Selected Age-Adjusted Baseline Characteristics by
Tertiles of sRAGE and CML-AGE in Subcohort Participants,
ATBC Study (1985-2006)
Characteristic Tertile 1 Tertile 2 Tertile 3 P
trend
sRAGE
Age at randomization* 56.5 56.2 56.5 0.97
BMI, kg/m
2
27.1 26.7 26.4 0.12
Years of smoking† 35.2 35.6 34.7 0.57
No. of cigarettes/day 21.5 20.8 20.0 0.09
Total meat, g/day†,‡ 196.7 193.9 190.5 0.42
Red meat, g/day†,‡ 67.7 68.0 68.9 0.71
Processed meat, g/day†,‡ 77.4 75.1 70.1 0.22
Alcohol, g/day†,‡ 19.9 20.1 16.6 0.30
Coffee, g/day†,‡ 590.0 567.3 629.8 0.33
Total fat, g/day†,‡ 123.4 122.3 120.4 0.15
Saturated fat, g/day†,‡ 53.1 52.5 52.8 0.86
Carbohydrates, g/day†,‡ 258.7 262.1 271.0 0.006
Glucose intake, g/day†,‡ 8.9 9.4 9.6 0.27
Sucrose intake, g/day†,‡ 48.4 51.1 55.3 0.01
Iron, mg/day†,‡ 18.0 17.2 18.3 0.46
Serum insulin, lU/mL†,‡ 5.2 5.2 5.0 0.75
Serum glucose, mg/dL†,‡ 102.7 97.5 98.0 0.10
CML-AGE
Age at randomization* 56.8 55.8 56.6 0.74
BMI, kg/m
2
27.3 26.9 26.0 0.004
Years of smoking† 35.3 35.5 34.8 0.50
No. of cigarettes/day 21.1 20.4 10.7 0.70
Total meat, g/day†,‡ 193.5 189.8 197.9 0.57
Red meat, g/day†,‡ 69.6 66.6 68.3 0.70
Processed meat, g/day†,‡ 75.6 73.1 74.2 0.82
Alcohol, g/day†,‡ 18.5 18.7 19.5 0.67
Coffee, g/day†,‡ 566.7 636.3 582.7 0.69
Total fat, g/day†,‡ 126.1 122.0 118.0 <0.0001
Saturated fat, g/day†,‡ 55.5 53.4 49.5 0.0001
Carbohydrates, g/day†,‡ 255.2 263.2 273.1 <0.0001
Glucose intake, g/day†,‡ 8.3 8.6 11.0 <0.0001
Sucrose intake, g/day†,‡ 48.7 51.7 54.2 0.04
Iron, mg/day†,‡ 17.6 18.0 18.4 0.04
Serum insulin, lU/mL†,‡ 5.5 5.0 4.8 0.12
Serum glucose, mg/dL†,‡ 99.6 97.3 101.4 0.50
Data are expressed as means.
*Unadjusted.
†Analysis of covariance, adjusted for age at randomization.
‡Dietary variables were adjusted for total energy intake (n ¼ 465).
HEPATOLOGY, Vol. 57, No. 6, 2013 MOY ET AL. 2341
Page 4
of sRAGE (P 0.01). With increasing tertiles of
CML-AGE, mean BMI and total and saturated fat
intake were significantly lower (P 0.004), whereas
intake of carbohydrate, glucose, sucrose, and iron all
were significantly higher (P 0.04).
Serum sRAGE concentrations were associated with a
statistically significant reduction in risk of liver cancer
when examined as a continuous variable (RR, 0.86;
95% CI, 0.75-0.99; P ¼ 0.02) and nonsignificant
reduced risk comparing the highest and lowest tertile
(RR, 0.77; 95% CI, 0.48-1.24; P for trend ¼ 0.28)
(Table 3). Higher CML-AGE concentrations were sig-
nificantly associated with reduced risk of liver cancer
when examined as a continuous variable standardized
to the central tertile (RR, 0.74; 95% CI, 0.65-0.84; P
< 0.0001) and in a dose-dependent manner when
examined categorically (T3 versus T1, RR, 0.19; 95%
CI, 0.10-0.35; P for trend < 0.0001). Further adjust-
ment for serum glucose and insulin or trial interven-
tion did not materially change these associations (data
not shown).
There was no evidence of effect modification of the
sRAGE or CML-AGE and liver cancer associations by
BMI, smoking duration and intensity, serum glucose,
diabetes; intake of energy-adjusted red meat, alcohol,
or coffee; or trial intervention arm (P for interaction
> 0.10), nor the joint effect of serum sRAGE and
CML-AGE (P for interaction ¼ 0.34). We stratified
the associations by follow-up time (subjects with 5-9
years of follow-up and subjects with 10 years of fol-
low-up, based on 52 and 93 cases, respectively) and
observed no evidence that the associations differed by
time from blood collection to diagnosis (P for interac-
tion > 0.14, data not shown).
As part of another nested case-control study on liver
cancer within the ATBC cohort, information on hepa-
titis B and C titers were measured. Within that study,
the prevalence of HBsAg was 1.2% among liver cancer
cases (2/167) and 0.7% among controls (6/817),
whereas the prevalence of anti-HBc was 15% among
cases (25/167) and 7% among controls (57/817) and
the prevalence of hepatitis C exposure (anti-HCV) was
5% among cases (8/167) and 0.6% (5/817) among
controls (Neal Freedman, personal communication). In
the present study, 135 cases and 26 subcohort mem-
bers had hepatitis B and C information. Only 1 subco-
hort member and 18 case subjects tested positive for
anti-HBc, and one case subject and no subcohort
members were positive for HBsAg. Similarly, four case
subjects and two subcohort members were positive for
anti-HCV. Because hepatitis infection is an important
risk factor for liver cancer, though clearly not in our
study population, we repeated the analyses after
excluding the 19 case subjects and two subcohort
members who tested positive for HBsAg, anti-HBc, or
anti-HCV, and the associations did not change.
Discussion
In our prospective study of male Finnish smokers
with a low prevalence of chronic HBV and HCV
infections, serum levels of sRAGE were associated with
a modest reduction in risk of liver cancer. A borderline
statistically significant inverse association between con-
tinuous sRAGE and liver cancer risk suggests that the
soluble receptor of AGEs may protect against the
inflammatory effects caused by RAGE activation. We
also observed an unexpected, highly statistically
Table 3. Serum CML-AGE and sRAGE in Relation to Risk of Liver Cancer, ATBC Study (1985-2006)
Tertiles
P
trend
* Continuous† P12 3
sRAGE
Range, pg/mL <465.4 465.4 to <670.9 670.9
Case/subcohort, n 54/162 47/162 44/161
Model 1, HR (95% CI)‡ 1.00 0.89 (0.55-1.43) 0.74 (0.46-1.18) 0.21 0.84 (0.73-0.97) 0.02
Model 2, HR (95% CI)§ 1.00 0.91 (0.56-1.47) 0.77 (0.48-1.24) 0.28 0.86 (0.75-0.99) 0.04
CML-AGE
Range, ng/mL <505.4 505.4 to <616.9 616.9
Case/subcohort, n 85/162 45/162 15/162
Model 1, HR (95% CI)‡ 1.00 0.50 (0.32-0.79) 0.17 (0.09-0.32) <0.0001 0.73 (0.64-0.82) <0.0001
Model 2, HR (95% CI)§ 1.00 0.52 (0.33-0.81) 0.19 (0.10-0.35) <0.0001 0.74 (0.65-0.84) <0.0001
Abbreviation: HR, hazard ratio.
*Trend test based on median values of each tertile.
†HRs for continuous sRAGE and CML-AGE were standardized to the average size of the central tertile (sRAGE, 205.5 pg/mL; CML-AGE, 111.4 ng/mL).
‡Adjusted for age.
§Adjusted for age, BMI, and years of smoking.
2342 MOY ET AL. HEPATOLOGY, June 2013
Page 5
significant, inverse association between serum CML-
AGE and liver cancer.
Experimental studies suggest the critical role of
RAGE activation in liver injury and furthermore indi-
cate that blocking RAGE may mitigate liver injury.
For example, in a mouse model of total hepatic ische-
mia/reperfusion, blockade of RAGE with sRAGE
administered via intraperitoneal injections improved
survival, protected against hepatocellular necrosis, and
enhanced expression of proregenerative cytokine tumor
necrosis factor-a.
27
In another study, blockade of
RAGE with sRAGE attenuated liver injur y caused by
toxic doses of acetaminophen, and a similar increase in
the proregenerative cytokines tumor necrosis factor-a
and interleukin-6 was observed.
24
These and other
rodent models (reviewed by Basta et al.
15
) support the
role of RAGE in liver fibrosis and that blocking
RAGE activation may prevent the progression of liver
fibrosis, indirectly preventing liver carcinogenesis.
Several human studies have investigated the poten-
tial roles of RAGE and sRAGE in inflammation. In
the only study that includes HCC, expression of
RAGE messenger RNA is lower in normal liver than
in chronic hepatitis and is the highest in HCC, sug-
gesting that RAGE activation may be involved in
HCC etiology
16
; this study, however, was cross-sec-
tional, and temporality cannot be determined. In a
second study, patients with NASH had significantly
lower circulating levels of sRAGE compared with
healthy controls with normal liver function tests and
liver sonograms.
26
Obese Caucasian prepubertal chil-
dren with nonalcoholic fatty liver disease also had sig-
nificantly lower sRAGE levels compared with obese
but otherwise healthy children.
40
Because NASH may
precede liver cancer, our borderline statistically signifi-
cant, inverse association between serum sRAGE and
liver cancer is in line with these previous studies. To-
gether, these results suggest that sRAGE may act as a
decoy receptor, binding free AGEs and other RAGE
ligands, and perhaps mitigating the effects of RAGE
activation in the liver.
Because CML-AGE is one of the most abundant
AGEs and binds readily with full-length RAGE, we
hypothesized that higher serum levels of CML-AGE
would be associated with increased risk of liver cancer.
Instead, we observed an inverse association. Explana-
tions for these results are unclear. Serum CML-AGE
may not be the optimal marker of AGEs or RAGE
activation in the liver, because CML-AGE is only one
of many RAGE ligands. For example, high mobility
group box-1 is another ligand of RAGE that activates
the proinflammatory cell signaling cascade and plays a
critical role in the mechanisms leading to liver
injury.
6,8,15,41
One possible explanation for our inverse
association is confounding, if levels of CML-AGE
were associated with another liver cancer risk factor.
For example, we and others
42
have shown CML-AGE
to be inversely associated with body fat in older adults
and preferentially deposited in fat tissues. It is possible
that the inverse association between CML-AGE and
liver cancer might be explained by body fat. Adjust-
ments for BMI did not affect our results; however,
BMI is an imperfect proxy for body fat deposition,
particularly in the liver.
As with all observational studies, the present investi-
gation has some limitations. First, the study findings
in Finnish male smokers may not be generalizable to
other populations that include women and non-
smokers. Nevertheless, the modest inverse association
between serum sRAGE and liver cancer is both biolog-
ically plausible and in line with laboratory and limited
clinical data. Although the ATBC study population
included Finnish male smokers, previously observed
associations in this population have been replicated in
populations that include women and nonsmokers.
43,44
The present study also did not examine other AGEs
beside CML-AGE, which may limit our ability to
tease out the true role of total AGEs in RAGE activa-
tion and subsequent liver cancer etiology. Other
RAGE ligands that were not measured in this study,
such as high mobility group box-1, may be associated
with liver cancer risk. The use of a single measurement
of biomarkers at only one point in time may not char-
acterize long-term concentrations, whereas repeated
measurements within subjects over time may provide a
more accurate assessment of exposure. However, it is
rarely feasible to assess multiple time points due to the
high cost and logistical complexity of collecting bio-
specimens from a large number of participants in a
cohort study. Although the single, prediagnostic meas-
urements of sRAGE and CML-AGE limits our ability
to draw conclusions of the role of the AGE-RAGE
axis in the etiology of liver cancer, our study is the first
to examine sRAGE and CML-AGE in association with
incident liver cancer.
The strengths of our study include the prospective
nature and the availability of fasting blood samples
collected at least 5 years before the diagnosis of liver
cancer, as well as the long follow-up, which reduces
the possibility of reverse causality. However, we note
that chronic liver disease progression and carcinogene-
sis can occur over a very long period, and we lacked
information on preexisting liver disease at baseline.
The low prevalence of HBV and HCV in our
HEPATOLOGY, Vol. 57, No. 6, 2013 MOY ET AL. 2343
Page 6
population (14% among cases) diminishes the possibil-
ity that HBV/HCV status, the primary risk factor of
liver cancer in other populations, may confound our
results. Moreover, when we performed sensitivity anal-
yses excluding the few subjects who were positive for
chronic infection of HBV and HCV, the associations
between serum CML-AGE or sRAGE and liver cancer
did not change. Finland is known to have among the
lowest prevalence of HBV and HCV in the world. In
the general population of Finland, the prevalence of
HBV and HCV are 0.2% and <2%, respectively,
45
which is similar to that of the ATBC study.
In conclusion, the findings from our prospective
study among Finnish male smokers support the hy-
pothesis that sRAGE may be protective against liver
cancer. Our results, particularly the unexpected inverse
association observed with serum levels of CML-AGE,
warrant examination in other populations.
References
1. Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide
incidence and trends. Gastroenterology 2004;127:S5-S16.
2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer
J Clin 2012;62:10-29.
3. Alkofer B, Lepennec V, Chiche L. Hepatocellular cancer in the non-
cirrhotic liver. J Visc Surg 2011;148:3-11.
4. McGlynn KA, London WT. The global epidemiology of hepatocellular
carcinoma: present and future. Clin Liver Dis 2011;15:223-243.
5. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology
and molecular carcinogenesis. Gastroenterology 2007;132:2557-2576.
6. Riehl A, Nemeth J, Angel P, Hess J. The receptor RAGE: bridging
inflammation and cancer. Cell Commun Signal 2009;7:12.
7. Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-prod-
ucts: a review. Diabetologia 2001;44:129-146.
8. Sparvero LJ, Asafu-Adjei D, Kang R, Tang D, Amin N, Im J, et al.
RAGE (receptor for advanced glycation endproducts), RAGE ligands,
and their role in cancer and inflammation. J Transl Med 2009;7:17.
9. Abe R, Yamagishi S. AGE-RAGE system and carcinogenesis. Curr
Pharm Des 2008;14:940-945.
10. Uribarri J, Woodruff S, Goodman S, Cai W, Chen X, Pyzik R, et al.
Advanced glycation end products in foods and a practical guide to their
reduction in the diet. J Am Diet Assoc 2010;110:911-916.
11. Semba RD, Nicklett EJ, Ferrucci L. Does accumulation of advanced
glycation end products contribute to the aging phenotype? J Gerontol
A Biol Sci Med Sci 2010;65:963-975.
12. Semba RD, Ang A, Talegawkar S, Crasto C, Dalal M, Jardack P, et al.
Dietary intake associated with serum versus urinary carboxymethyl-ly-
sine, a major advanced glycation end product, in adults: the Energetics
Study. Eur J Clin Nutr 2012;66:3-9.
13. Hyogo H, Yamagishi S. Advanced glycation end products (AGEs) and
their involvement in liver disease. Curr Pharm Des 2008;14:969-972.
14. Bierhaus A, Nawroth PP. Multiple levels of regulation determine the
role of the receptor for AGE (RAGE) as common soil in inflammation,
immune responses and diabetes mellitus and its complications. Diabe-
tologia 2009;52:2251-2263.
15. Basta G, Navarra T, De Simone P, Del Turco S, Gastaldelli A, Fili-
pponi F. What is the role of the receptor for advanced glycation end
products-ligand axis in liver injury? Liver Transpl 2011;17:633-640.
16. Hiwatashi K, Ueno S, Abeyama K, Kubo F, Sakoda M, Maruyama I,
et al. A novel function of the receptor for advanced glycation end-prod-
ucts (RAGE) in association with tumorigenesis and tumor differentia-
tion of HCC. Ann Surg Oncol 2008;15:923-933.
17. Yagmur E, Tacke F, Weiss C, Lahme B, Manns MP, Kiefer P, et al.
Elevation of Nepsilon-(carboxymethyl)lysine-modified advanced
glycation end products in chronic liver disease is an indicator of liver
cirrhosis. Clin Biochem 2006;39:39-45.
18. Hyogo H, Yamagishi S, Iwamoto K, Arihiro K, Takeuchi M, Sato T,
et al. Elevated levels of serum advanced glycation end products in
patients with non-alcoholic steatohepatitis. J Gastroenterol Hepatol
2007;22:1112-1119.
19. Raucci A, Cugusi S, Antonelli A, Barabino SM, Monti L, Bierhaus A,
et al. A soluble form of the receptor for advanced glycation endprod-
ucts (RAGE) is produced by proteolytic cleavage of the membrane-
bound form by the sheddase a disintegrin and metalloprotease 10
(ADAM10). FASEB J 2008;22:3716-3727.
20. Yonekura H, Yamamoto Y, Sakurai S, Petrova RG, Abedin MJ, Li H,
et al. Novel splice variants of the receptor for advanced glycation end-
products expressed in human vascular endothelial cells and pericytes,
and their putative roles in diabetes-induced vascular injury. Biochem J
2003;370:1097-1109.
21. Butscheid M, Schafer C, Brenner S, Alscher D, Murdter T, Niwa T,
et al. Unchanged serum levels of advanced glycation endproducts in
patients with liver disease. Naunyn Schmiedebergs Arch Pharmacol
2007;375:401-406.
22. Butscheid M, Hauptvogel P, Fritz P, Klotz U, Alscher DM. Hepatic
expression of galectin-3 and receptor for advanced glycation end prod-
ucts in patients with liver disease. J Clin Pathol 2007;60:415-418.
23. Cataldegirmen G, Zeng S, Feirt N, Ippagunta N, Dun H, Qu W,
et al. RAGE limits regeneration after massive liver injury by coordi-
nated suppression of TNF-alpha and NF-kappaB. J Exp Med 2005;
201:473-484.
24. Ekong U, Zeng S, Dun H, Feirt N, Guo J, Ippagunta N, et al. Block-
ade of the receptor for advanced glycation end products attenuates acet-
aminophen-induced hepatotoxicity in mice. J Gastroenterol Hepatol
2006;21:682-688.
25. Sebekova K, Kupcova V, Schinzel R, Heidland A. Markedly elevated
levels of plasma advanced glycation end products in patients with liver
cirrhosis—amelioration by liver transplantation. J Hepatol 2002;36:
66-71.
26. Yilmaz Y, Ulukaya E, Gul OO, Arabul M, Gul CB, Atug O, et al.
Decreased plasma levels of soluble receptor for advanced glycation end-
products (sRAGE) in patients with nonalcoholic fatty liver disease.
Clin Biochem 2009;42:802-807.
27. Zeng S, Feirt N, Goldstein M, Guarrera J, Ippagunta N, Ekong U,
et al. Blockade of receptor for advanced glycation end product (RAGE)
attenuates ischemia and reperfusion injury to the liver in mice. H
EPATO-
LOGY
2004;39:422-432.
28. Tesarova P, Kalousova M, Jachymova M, Mestek O, Petruzelka L, Zima
T. Receptor for advanced glycation end products (RAGE)—soluble
form (sRAGE) and gene polymorphisms in patients with breast cancer.
Cancer Invest 2007;25:720-725.
29. Krechler T, Jachymova M, Mestek O, Zak A, Zima T, Kalousova M.
Soluble receptor for advanced glycation end-products (sRAGE) and
polymorphisms of RAGE and glyoxalase I genes in patients with pan-
creas cancer. Clin Biochem 2010;43:882-886.
30. Jing R, Cui M, Wang J, Wang H. Receptor for advanced glycation end
products (RAGE) soluble form (sRAGE): a new biomarker for lung
cancer. Neoplasma 2010;57:55-61.
31. Grote VA, Nieters A, Kaaks R, Tjonneland A, Roswall N, Overvad K,
et al. The associations of advanced glycation end products and its solu-
ble receptor with pancreatic cancer risk: a case-control study within the
prospective EPIC cohort. Cancer Epidemiol Biomarkers Prev 2012;21:
619-628.
32. Jiao L, Weinstein SJ, Albanes D, Taylor PR, Graubard BI, Virtamo J,
et al. Evidence that serum levels of the soluble receptor for advanced
glycation end products are inversely associated with pancreatic cancer
risk: a prospective study. Cancer Res 2011;71:3582-3589.
2344 MOY ET AL. HEPATOLOGY, June 2013
Page 7
33. Jiao L, Taylor PR, Weinstein SJ, Graubard BI, Virtamo J, Albanes D,
et al. Advanced glycation end products, soluble receptor for advanced
glycation end products, and risk of colorectal cancer. Cancer Epidemiol
Biomarkers Prev 2011;20:1430-1438.
34. The alpha-tocopherol, beta-carotene lung cancer prevention study,:
designmethods participant characteristics, and compliance. The ATBC
Cancer Prevention Study Group. Ann Epidemiol 1994;4:1-10.
35. Korhonen P, Malila N, Pukkala E, Teppo L, Albanes D, Virtamo J.
The Finnish Cancer Registry as follow-up source of a large trial
cohort—accuracy and delay. Acta Oncol 2002;41:381-388.
36. Boehm BO, Schilling S, Rosinger S, Lang GE, Lang GK, Kientsch-
Engel R, et al. Elevated serum levels of N(epsilon)-carboxymethyl-
lysine, an advanced glycation end product, are associated with prolifera-
tive diabetic retinopathy and macular oedema. Diabetologia 2004;47:
1376-1379.
37. Limburg PJ, Stolzenberg-Solomon RZ, Vierkant RA, Roberts K, Sellers
TA, Taylor PR, et al. Insulin, glucose, insulin resistance, and incident
colorectal cancer in male smokers. Clin Gastroenterol Hepatol 2006;4:
1514-1521.
38. Hu FB, Stampfer MJ, Rimm E, Ascherio A, Rosner BA, Spiegelman
D, et al. Dietary fat and coronary heart disease: a comparison of
approaches for adjusting for total energy intake and modeling repeated
dietary measurements. Am J Epidemiol 1999;149:531-540.
39. Kulathinal S, Karvanen J, Saarela O, Kuulasmaa K. Case-cohort design
in practice—experiences from the MORGAM Project. Epidemiol Per-
spect Innov 2007;4:15.
40. D’Adamo E, Giannini C, Chiavaroli V, de Giorgis T, Verrotti A,
Chiarelli F, et al. What is the significance of soluble and endogenous
secretory receptor for advanced glycation end products in liver steatosis
in obese prepubertal children? Antioxid Redox Signal 2011;14:
1167-1172.
41. Albayrak A, Uyanik MH, Cerrah S, Altas S, Dursun H, Demir M,
et al. Is HMGB1 a new indirect marker for revealing fibrosis in chronic
hepatitis and a new therapeutic target in treatment? Viral Immunol
2010;23:633-638.
42. Semba RD, Arab L, Sun K, Nicklett EJ, Ferrucci L. Fat mass is inver-
sely associated with serum carboxymethyl-lysine, an advanced glycation
end product, in adults. J Nutr 2011;141:1726-1730.
43. Stolzenberg-Solomon RZ, Pietinen P, Taylor PR, Virtamo J, Albanes
D. Prospective study of diet and pancreatic cancer in male smokers.
Am J Epidemiol 2002;155:783-792.
44. Thiebaut AC, Jiao L, Silverman DT, Cross AJ, Thompson FE, Subar
AF, et al. Dietary fatty acids and pancreatic cancer in the NIH-AARP
diet and health study. J Natl Cancer Inst 2009;101:1001-1011.
45. Global surveillance and control of hepatitis C. Report of a WHO Con-
sultation organized in collaboration with the Viral Hepatitis Prevention
Board, Antwerp, Belgium. J Viral Hepat 1999;6:35-47.
HEPATOLOGY, Vol. 57, No. 6, 2013 MOY ET AL. 2345
Page 8
  • Source
    • "Only a few studies have described the relationship between the OPG/RANKL/RANK system and pregnancy-induced hypertension, preeclampsia, and intrauterine growth re- striction505152535455. Negative RAGE isoforms can inhibit endogenous inflammation and their protective function has been confirmed in diabetes mellitus, some cardiovascular diseases, atherosclerosis, and in some types of neo- plasms56575859606162. Only a few studies have assessed the importance of RAGE for preterm labor39404142 63]. "
    [Show abstract] [Hide abstract] ABSTRACT: This study aimed to determine the relationships between secretory and endogenous secretory receptors for advanced glycation end products (sRAGE, esRAGE), sRANKL, osteoprotegerin and the interval from diagnosis of threatened premature labor or premature rupture of the fetal membranes to delivery, and to evaluate the prognostic values of the assessed parameters for preterm birth. Ninety women between 22 and 36 weeks' gestation were included and divided into two groups: group A comprised 41 women at 22 to 36 weeks' gestation who were suffering from threatened premature labor; and group B comprised 49 women at 22 to 36 weeks' gestation with preterm premature rupture of the membranes. Levels of sRAGE, esRAGE, sRANKL, and osteoprotegerin were measured. The Mann-Whitney test was used to assess differences in parameters between the groups. For statistical analysis of relationships, correlation coefficients were estimated using Spearman's test. Receiver operating characteristics were used to determine the cut-off point and predictive values. In group A, sRAGE and sRANKL levels were correlated with the latent time from symptoms until delivery (r = 0.422; r = -0.341, respectively). The sensitivities of sRANKL and sRAGE levels for predicting preterm delivery were 0.895 and 0.929 with a negative predictive value (NPV) of 0.857 and 0.929, respectively. In group B, sRAGE and sRANKL levels were correlated with the latent time from pPROM until delivery (r = 0.381; r = -0.439). The sensitivity of sRANKL and sRAGE for predicting delivery within 24 h after pPROM was 0.682 and 0.318, with NPVs of 0.741 and 0.625, respectively. Levels of esRAGE and sRANKL were lower in group A than in group B (median = 490.2 vs 541.1 pg/mL; median = 6425.0 vs 11362.5 pg/mL, respectively). Correlations between sRAGE, sRANKL, and pregnancy duration after the onset of symptoms suggest their role in preterm delivery. The high prognostic values of these biomarkers indicate their usefulness in diagnosis of pregnancies with threatened premature labor.
    Full-text · Article · Jun 2015 · BMC Pregnancy and Childbirth
  • Source
    • "Secreted HMGB1 can be neutralized by administering soluble RAGE as discussed for controlling atherosclerosis [8] and inflammation [96]. Interestingly, the endogenous levels of soluble RAGE are downregulated in patients with liver cancer [97], colorectal adenoma [98], pancreatic cancer [99, 100], lung cancer [101], and breast carcinoma [102]. However, the status of soluble RAGE in PCa patients is not yet known and warrants further investigation for PCa treatment. "
    [Show abstract] [Hide abstract] ABSTRACT: High mobility group box 1 (HMGB1) was originally discovered as a chromatin-binding protein several decades ago. It is now increasingly evident that HMGB1 plays a major role in several disease conditions such as atherosclerosis, diabetes, arthritis, sepsis, and cancer. It is intriguing how deregulation of HMGB1 can result in a myriad of disease conditions. Interestingly, HMGB1 is involved in cell proliferation, angiogenesis, and metastasis during cancer progression. Furthermore, HMGB1 has been demonstrated to exert intracellular and extracellular functions, activating key oncogenic signaling pathways. This paper focuses on the role of HMGB1 in prostate cancer development and highlights the potential of HMGB1 to serve as a key target for prostate cancer treatment.
    Full-text · Article · May 2013
  • [Show abstract] [Hide abstract] ABSTRACT: RAGE regulates cellular proliferation in hepatocellular carcinoma (HCC) and the aim of this study was to test the in vitro effect of YHK, a nutraceutical with prior data suggesting its hepatocyte protecting role, in regulating RAGE in the proliferation of HCC cell line HuH7 as well checking also its potential modulation in the expression of the transcriptional factor NF-κB p65. Our study showed that YHK significantly reduced cellular growth in the HuH7 cell line (p<0.05). Moreover, this phytocompound partly reduced gene expression of NF-κB p65 (by 35%, p<0.05). These data suggest that YHK has a potential role as a modulator of RAGE and RAGE-ligands for potential healthy-liver intervention in HCC prevention strategies.
    No preview · Article · Sep 2013 · Rejuvenation Research
Show more