Tumor Biol.

(2015) 36:35733582
DOI 10.1007/s13277-014-2994-6

RESEARCH ARTICLE

Oxidized low-density lipoprotein is associated

with advanced-stage prostate cancer
Fangning Wan & Xiaojian Qin & Guiming Zhang &
Xiaolin Lu & Yao Zhu & Hailiang Zhang & Bo Dai &
Guohai Shi & Dingwei Ye

Received: 17 October 2014 / Accepted: 17 December 2014 / Published online: 8 January 2015
# International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract Clinical and epidemiological data suggest coronary
artery disease shares etiology with prostate cancer (PCa). The
aim of this work was to assess the effects of several serum
markers reported in cardiovascular disease on PCa. Serum
markers (oxidized low-density lipoprotein [ox-LDL], apoli-
poprotein [apo] B100, and apoB48) in peripheral blood sam-
ples from 50 patients from Fudan University Shanghai Cancer
Center (FUSCC) with localized or lymph node metastatic PCa
were investigated in this study. Twenty-five samples from
normal individuals were set as controls. We first conducted
enzyme-linked immunosorbent assay analysis to select candi-
date markers that were significantly different between these
patients and controls. Then, the clinical relevance between
OLR1 (the ox-LDL receptor) expression and PCa was ana-
lyzed in The Cancer Genome Atlas (TCGA) cohort. We also
investigated the function of ox-LDL in PCa cell lines in vitro.
Phosphorylation protein chips were used to analyze cell sig-
naling pathways in ox-LDL-treated PC-3 cells. The ox-LDL
level was found to be significantly correlated with N stage of
prostate cancer. OLR1 expression was correlated with lymph
node metastasis in the TCGA cohort. In vitro, ox-LDL stim-
ulated the proliferation, migration, and invasion of LNCaP
Fangning Wan and Xiaojian Qin contributed equally to this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s13277-014-2994-6) contains supplementary material,
which is available to authorized users.
F. Wan : X. Qin : G. Zhang : X. Lu : Y. Zhu : H. Zhang : B. Dai :
G. Shi : D. Ye (*)
Department of Urology, Fudan University Shanghai Cancer Center,
Shanghai, Peoples Republic of China
e-mail: dwyeli@163.com
F. Wan : X. Qin : G. Zhang : X. Lu : Y. Zhu : H. Zhang : B. Dai :
G. Shi : D. Ye
Department of Oncology, Shanghai Medical College, Fudan
University, No. 270, Dongan Road, Shanghai 200032, Peoples
Republic of China
and PC-3 in a dose-dependent manner. The results of phos-
phoprotein microarray illustrated that ox-LDL could influence
multiple signaling pathways of PC-3. Activation of prolifera-
tion promoting signaling pathways (including -catenin,
cMyc, NF-B, STAT1, STAT3) as well as apoptosis-
associating signaling pathways (including p27, caspase-3)
demonstrated that ox-LDL had complicated effects on pros-
tate cancer. Increased serum ox-LDL level and OLR1 expres-
sion may indicate advanced-stage PCa and lymph node me-
tastasis. Moreover, ox-LDL could stimulate PCa proliferation,
migration, and invasion in vitro.
Keywords Ox-LDL . OLR1 . Cell migration . Prostate
cancer . Proliferation
Introduction
In developed countries, prostate cancer (PCa) is the most
common malignancy in men and the second leading cause
of cancer-related mortality [1]. In recent years, the incidence
of PCa has gradually increased in the Peoples Republic of
China. According to the latest Chinese Cancer Registry An-
nual Report (2012), PCa has become the sixth most prevalent
cancer and the ninth leading cause of cancer-related mortality
in men, especially in urban areas [2].
Large cohort studies and meta-analyses suggest a poten-
tially beneficial effect of statins on PCa patients treated with
radiotherapy. Statin usage was associated with improved free-
dom from biochemical failure, freedom from salvage andro-
gen deprivation therapy, and relapse-free survival in these
patients [3, 4]. Data show that men with coronary artery
disease were more likely to have diabetes, hypertension, and
hypercholesterolemia, and this was associated with a 35 %
3574
increased risk of PCa diagnosis, so it is reasonable to speculate
that coronary artery disease shares etiology with PCa [5].
Oxidative stress is often cited as playing an important role
in cardiovascular outcomes [68]. In PCa, oxidative stress, an
innate and key event characterized by supraphysiological
concentrations of reactive oxygen species, has been identified
as one of the hallmarks of the aggressive disease phenotype.
Specifically, oxidative stress is associated with PCa develop-
ment, progression, and response to therapy [911].
Oxidized low-density lipoprotein (ox-LDL) is an important
prognostic marker in the serum of atherosclerosis patients
[12]. It is suggested that ox-LDL plays a more important role
in the genesis and progression of atherosclerosis than native
LDL [4, 13], and ox-LDL is a biomarker associated with
oxidative stress in pathophysiological processes and is linked
to atherogenesis and tumorigenesis [14, 15]. However, there
are insufficient studies on ox-LDL and its relevance with
respect to PCa behavior.
Materials and methods
Patients and samples
This study received Institutional Review Board approval from
Fudan University Shanghai Cancer Center (FUSCC), and
written informed consent was obtained from all subjects. A
total of 75 patients including 50 PCa patients and 25 benign
prostatic hyperplasia (BPH) patients were retrospectively col-
lected from 2008 to 2011. Pathology diagnoses were con-
firmed by experienced pathologists. Baseline characteristics,
such as age, history of hypertension or diabetes mellitus, body
mass index (BMI), smoking status, lipid profiles, statin usage,
prostate-specific antigen (PSA) levels, Gleason score (GS),
and stage at diagnosis (tumor, node, metastasis [TNM] clas-
sification), were obtained from electronic records and medical
charts. Blood samples of these patients were obtained from
routine tests, and sera were separated by centrifugation and
immediately stored at 70 C in the tissue bank of FUSCC.
Sandwich enzyme-linked immunosorbent assay (ELISA) kits
(ABIN417290, ABIN1371105, ABIN456069, ABIN414494,
Antibodies-Online Inc., Atlanta, GA, USA) were used to
detect apoprotein B48 (APOB48), apoprotein B100
(APOB100), anti-oxidized LDL antibody (OLAb), and ox-
LDL levels in blood serum with protocols and reagents sup-
plied by the manufacturer.
OLR1 expression and clinical data were acquired from The
Cancer Genome Atlas (TCGA) database. The data are avail-
able from the Cancer Genomics Browser of the University of
California, Santa Cruz (UCSC) (https://genome-cancer.ucsc.
edu/). In total, 256 primary prostate tumors from male patients
with detailed OLR1 expression data were chosen from the
updated TCGA database according to parameters mentioned
Tumor Biol. (2015) 36:35733582
in a previous study [16]. Only patients with intact tumor
expression data were included in this study. Detailed
demographics of these patients, characterized by the TCGA
consortium, are shown in Table 1.
In vitro studies
Cell lines
Prostate cancer cell lines, LNCaP (CRL-1740) and PC-3
(CLR-1435), were purchased from American Type Culture
Collection (Manassas, VA, USA). LNCaP and PC-3 were
respectively cultured in RPMI 1640 (L0495, Biowest LCC,
Kansas City, MI, USA) and F-12 K (21127-022, Life
Technologies, Carlsbad, CA, USA) with 10 % fetal bovine
serum (S181P, Biowest LCC) at 37 C in an atmosphere of
5 % CO2.
Western blot
Cell lysates of LNCaP and PC-3 were harvested following 0-,
6-, 12-, 24-, 48-, and 96-h treatments with ox-LDL. Western
blot analysis of OLR1 expression was performed as per de-
scribed methods [17]. Primary antibodies including anti-
OLR1 (ab85839, Abcam, Cambridge, UK), anti-GAPDH
(AP7873a, Abgent Inc., San Diego, CA, USA), and anti-
rabbit IgG peroxidase-conjugated antibody (LP1001b,
Abgent Inc.) were used for western blot analysis.
Cell viability assays
The effects of ox-LDL on cell viability were assessed by Cell
Counting Kit-8 (CCK-8) assays [17]. Cells were plated on 96-
well plates at a concentration of 5000 cells per well in tripli-
cate. After seeding for 12 h, cells were exposed to a series of
concentrations of ox-LDL diluted in complete medium (0, 4,
20, and 100 g/ml). Next, 10 l of CCK-8 reagent was added
to each well 24, 48, and 96 h after adding ox-LDL. Cells were
incubated at 37 C for 2 h and measured at 450 nm on a plate
reader. Results were calculated and plotted with GraphPad
Prism software (GraphPad Software Inc., version 5.01, La
Jolla, CA, USA). Experiments were repeated a minimum of
three times and representative results are shown.
Cell migration and invasion
Transwell assays were performed to evaluate cell migration
and invasion [9]. For invasion analysis, 60 l of Matrigel (BD
Biosciences, Mississauga, ON, Canada) diluted in a serum-
free medium (1:5) was added to the upper compartment of the
chamber. Next, 5104 LNCaP and PC-3 cells were suspended
in 200 l of serum-free medium with a series of ox-LDL
concentrations (0, 4, 20, and 100 g/ml) and seeded into the
3574
increased risk of PCa diagnosis, so it is reasonable to speculate
that coronary artery disease shares etiology with PCa [5].
Oxidative stress is often cited as playing an important role
in cardiovascular outcomes [68]. In PCa, oxidative stress, an
innate and key event characterized by supraphysiological
concentrations of reactive oxygen species, has been identified
as one of the hallmarks of the aggressive disease phenotype.
Specifically, oxidative stress is associated with PCa develop-
ment, progression, and response to therapy [911].
Oxidized low-density lipoprotein (ox-LDL) is an important
prognostic marker in the serum of atherosclerosis patients
[12]. It is suggested that ox-LDL plays a more important role
in the genesis and progression of atherosclerosis than native
LDL [4, 13], and ox-LDL is a biomarker associated with
oxidative stress in pathophysiological processes and is linked
to atherogenesis and tumorigenesis [14, 15]. However, there
are insufficient studies on ox-LDL and its relevance with
respect to PCa behavior.
Materials and methods
Patients and samples
This study received Institutional Review Board approval from
Fudan University Shanghai Cancer Center (FUSCC), and
written informed consent was obtained from all subjects. A
total of 75 patients including 50 PCa patients and 25 benign
prostatic hyperplasia (BPH) patients were retrospectively col-
lected from 2008 to 2011. Pathology diagnoses were con-
firmed by experienced pathologists. Baseline characteristics,
such as age, history of hypertension or diabetes mellitus, body
mass index (BMI), smoking status, lipid profiles, statin usage,
prostate-specific antigen (PSA) levels, Gleason score (GS),
and stage at diagnosis (tumor, node, metastasis [TNM] clas-
sification), were obtained from electronic records and medical
charts. Blood samples of these patients were obtained from
routine tests, and sera were separated by centrifugation and
immediately stored at 70 C in the tissue bank of FUSCC.
Sandwich enzyme-linked immunosorbent assay (ELISA) kits
(ABIN417290, ABIN1371105, ABIN456069, ABIN414494,
Antibodies-Online Inc., Atlanta, GA, USA) were used to
detect apoprotein B48 (APOB48), apoprotein B100
(APOB100), anti-oxidized LDL antibody (OLAb), and ox-
LDL levels in blood serum with protocols and reagents sup-
plied by the manufacturer.
OLR1 expression and clinical data were acquired from The
Cancer Genome Atlas (TCGA) database. The data are avail-
able from the Cancer Genomics Browser of the University of
California, Santa Cruz (UCSC) (https://genome-cancer.ucsc.
edu/). In total, 256 primary prostate tumors from male patients
with detailed OLR1 expression data were chosen from the
updated TCGA database according to parameters mentioned
Tumor Biol. (2015) 36:35733582
in a previous study [16]. Only patients with intact tumor
expression data were included in this study. Detailed
demographics of these patients, characterized by the TCGA
consortium, are shown in Table 1.
In vitro studies
Cell lines
Prostate cancer cell lines, LNCaP (CRL-1740) and PC-3
(CLR-1435), were purchased from American Type Culture
Collection (Manassas, VA, USA). LNCaP and PC-3 were
respectively cultured in RPMI 1640 (L0495, Biowest LCC,
Kansas City, MI, USA) and F-12 K (21127-022, Life
Technologies, Carlsbad, CA, USA) with 10 % fetal bovine
serum (S181P, Biowest LCC) at 37 C in an atmosphere of
5 % CO2.
Western blot
Cell lysates of LNCaP and PC-3 were harvested following 0-,
6-, 12-, 24-, 48-, and 96-h treatments with ox-LDL. Western
blot analysis of OLR1 expression was performed as per de-
scribed methods [17]. Primary antibodies including anti-
OLR1 (ab85839, Abcam, Cambridge, UK), anti-GAPDH
(AP7873a, Abgent Inc., San Diego, CA, USA), and anti-
rabbit IgG peroxidase-conjugated antibody (LP1001b,
Abgent Inc.) were used for western blot analysis.
Cell viability assays
The effects of ox-LDL on cell viability were assessed by Cell
Counting Kit-8 (CCK-8) assays [17]. Cells were plated on 96-
well plates at a concentration of 5000 cells per well in tripli-
cate. After seeding for 12 h, cells were exposed to a series of
concentrations of ox-LDL diluted in complete medium (0, 4,
20, and 100 g/ml). Next, 10 l of CCK-8 reagent was added
to each well 24, 48, and 96 h after adding ox-LDL. Cells were
incubated at 37 C for 2 h and measured at 450 nm on a plate
reader. Results were calculated and plotted with GraphPad
Prism software (GraphPad Software Inc., version 5.01, La
Jolla, CA, USA). Experiments were repeated a minimum of
three times and representative results are shown.
Cell migration and invasion
Transwell assays were performed to evaluate cell migration
and invasion [9]. For invasion analysis, 60 l of Matrigel (BD
Biosciences, Mississauga, ON, Canada) diluted in a serum-
free medium (1:5) was added to the upper compartment of the
chamber. Next, 5104 LNCaP and PC-3 cells were suspended
in 200 l of serum-free medium with a series of ox-LDL
concentrations (0, 4, 20, and 100 g/ml) and seeded into the
Tumor Biol. (2015) 36:35733582 3575

Table 1 Demographic and clinical characteristics of PCa patients Table 1 (continued)

investigated in the TCGA and FUSCC cohorts
Variables TCGA cohort FUSCC cohort FUSCC cohort
Variables TCGA cohort FUSCC cohort FUSCC cohort (PCa) (N=256) (PCa) N=50 (BPH) (N=25)
(PCa) (N=256) (PCa) N=50 (BPH) (N=25)
pN stage
Age, years 0 207 (80.9) 21 (42.0) NA
Median (range) 61 (4477) 66.5 (5186) 67.0 (4880) 1 16 (6.3) 29 (58.0)
Weight (kg) mean NA 69.5 70.1 NA 33 (12.9) 0 (0.0)
BMI (kg/m2) NA M stage
Normal (<24) 18 (36.0) 9 (36.0) 0 156 (60.9) 21 (42.0) NA
Overweight (24) 32 (64.0) 16 (64.0) 1 0 (0.0) 29 (58.0)
Smoking status NA NA 100 (39.1) 0 (0.0)
Ever 13 (26.0) 4 (16.0)
Percentages in parentheses unless indicated otherwise
Never 37 (74.0) 21 (84.0)
TCGA The Cancer Genome Atlas, FUSCC Fudan University Shanghai
Diabetes NA Cancer Center, NA not applicable, PCa prostate cancer, PSA prostate-
Yes 15 (30.0) 6 (24.0) specific antigen, BMI body mass index, TC total cholesterol, TG triglyc-
No 35 (70.0) 19 (76.0) eride, LDL low-density lipoprotein, HDL high-density lipoprotein, GS
Gleason score
Atherosclerosis NA
Yes 8 (16.0) 3 (12.0)
No 42 (78.0) 22 (88.0) upper chamber. The chamber was then placed in a 24-well
Hypertension NA plate that contained 750 l of complete medium per well.
Yes 8 (16.0) 4 (16.0) After 48 h of incubation at 37 C, the cells were fixed with
No 42 (84.0) 21 (84.0) 10 % trichloroacetic acid at room temperature for 30 min,
TC (mg/dl) NA followed by staining using crystal violet (0.25 %) for 20 min
Normal(<200) 28 (56.0) 21 (84.0) at room temperature. Chambers were then washed three times
Abnormal(200) 22 (44.0) 4 (16.0) with phosphate-buffered saline (PBS) in new 24 plates, and
TG (mg/dl) NA the upper surfaces were wiped with cotton swabs. The number
Normal (<150) 31 (68.0) 19 (76.0) of invading cells was counted by a microscope. Random
Abnormal (150) 16 (32.0) 6 (24.0) pictures were taken and representative images are shown. Cell
LDL (mg/dl) NA migration analysis was similar to invasion analysis without the
Normal (<130) 27 (54.0) 17 (68.0) addition of Matrigel.
Abnormal (130) 23 (46.0) 8 (32.0)
HDL (mg/dl) NA Wound healing assay
Normal (40) 45 (90.0) 22 (88.0)
Abnormal (<40) 5 (10.0) 3 (12.0) A monolayer of PC-3 cells cultured in a 6-well plate was
Stain usage NA treated with a series concentration of ox-LDL for 48 h. Next,
Yes 8 (16.0) 3 (12.0) the cells were manually scratched with a blue pipette tip to
No 42 (78.0) 22 (88.0) form a wound. Images were taken at 0 and 12 h after
PSA scratching. Each well was imaged three times at different
010 148 (57.8) 17 (34.0) 18 (72.0) places and representative images are shown.
10.120 8 (3.1) 7 (14.0) 7 (28.0)
20.1100 1 (0.4) 1 (2.0) 0 (0.0) Protein microarray analysis
>100 0 (0.0) 25 (50.0) 0 (0.0)
NA 99 (38.7) 0 (0.0) 0 (0.0) The Phospho Explorer Antibody Microarray was conducted
GS by Full Moon BioSystems Inc. (Sunnyvale, CA, USA).
7 136 (53.1) 21 (42.0) NA
Whole-cell lysates from PC-3 treated with ox-LDL (20 g/ml)
8 38 (14.8) 29 (58.0) or untreated controls were harvested using Protein Extraction
NA 82 (32.0) 0 (0.0) Buffer (Full Moon BioSystems Inc.) and were immediately
pT stage frozen in liquid nitrogen. The samples were transported to Full
T2c 75 (29.3) 23 (46.0) NA Moon BioSystems Inc. on dry ice. The array included 1318
T3a 98 (38.3) 27 (54.0) phospho-protein antibodies, and the microarray analysis was
NA 83 (32.4) 0 (0.0) carried out as previously described [18]. Fold change was
calculated as phosphorylation of ox-LDL-treated cells/
3576 Tumor Biol. (2015) 36:35733582

phosphorylation of untreated cells. This experiment was car-
ried out once.
Statistical analyses
Statistical analyses were performed with SPSS software (ver-
sion 17.0, IBM Corp., Armonk, NY, USA) using independent
t tests (for continuous variables) and Pearsons 2 tests (for
categorical variables). Logistic regression was used to deter-
mine the correlation between OLR1 expression level and
clinicopathological characteristics in the TCGA cohorts. Sta-
tistical significance was based on two-sided p values of <0.05.
two groups (all p > 0.05). However, abnormal TC was
more commonly seen in PCa patients (p<0.05). Besides,
we screened four more relative biomarkers in patient
serum. ApoB48 and ApoB100 were found to be strongly
co-expressed, but did not show a significant increase in
primary or metastatic PCa. Similarly, OLAB was not
associated with PCa. However, ox-LDL was significantly
increased in PCa patients and even higher in metastatic
PCa patients (Fig. 1).
OLR1 was associated with high Gleason score and lymph
node metastasis

Oxidized low-density lipoprotein (lectin-like) receptor

Results
Ox-LDL was elevated in primary and metastatic PCa
A total of 50 PCa patients and 25 BPH controls were
enrolled in this study; the mean age was 66.5 and
67.0 years, respectively. Detailed demographic and clini-
copathological characteristics of the 75 patients are shown
in Table 1. Age, weight, BMI, smoking habits, diabetes,
atherosclerosis, hypertension, statin usage, TG, LDL, and
HDL did not show a significant difference between the
(OLR1) expression data were available for a total of 256
PCa patients in the TCGA cohort; clinical data are listed in
Table 1. According to univariate analysis, the likelihood of
positive lymph nodes increased dramatically when OLR1
expression increased, as well as with high Gleason scores
(>7) and a high serum PSA level. In multivariate analysis,
high Gleason score and high PSA level were independent
predictors of morbidity (Table 2). In univariate analysis, the
likelihood of high Gleason score (>7) increased as OLR1
increased (pT >2 and pN1). After adjusting for other risk
factors, positive lymph nodes, high T stage, and OLR1

Fig. 1 Ox-LDL level was

elevated in metastatic prostate
cancer. a There was no significant
difference in serum APOB48
distribution within FUSCC
cohorts. b The median serum
APOB100 level was higher in
metastatic prostate cancer but
failed to achieve statistical
significance. c Serum OLAb level
increased in primary metastatic
prostate cancer patients; however,
no statistical significance was
observed. d Serum ox-LDL levels
increased in primary metastatic
prostate cancer patients;
metastatic patients had
significantly higher ox-LDL
levels than benign prostate
hypertrophy control (p<0.05)
Tumor Biol. (2015) 36:35733582 3577

Table 2 Univariate and

multivariate analyses for Variable Lymph node positive
evaluation of the associations
between tested parameters and N Univariate Multivariate
stage in the TCGA cohort
OR 95 % CI p OR 95 % CI p

Age 0.998 0.928 to 1.073 0.955 1.018 0.921 to 1.126 0.720

pT(3)a 6.083 1.338 to 27.660 0.190 7.974 0.823 to 77.237 0.073
a
Categorical variables: pT >2 or Gleason scorea 14.412 4.052 to 51.253 0.000 5.891 1.462 to 23.738 0.013
otherwise; Gleason score >7 or PSAa 1.097 1.005 to 1.199 0.039 2.405 0.459 to 12.587 0.039
otherwise; prostate-specific OLR1 1.860 1.178 to 2.937 0.008 1.649 0.796 to 3.415 0.178
antigen 10, 20, 100, or >100

were found to be independent predictors of high Gleason Ox-LDL increased the migration and invasion capabilities
score (Table 3). of LNCaP and PC-3

Cell migration and invasion capabilities are closely related

Ox-LDL increased the proliferation of LNCaP and PC-3
through OLR1
Western blot analysis showed that PC-3 expressed more
OLR1 protein than LNCaP (Fig. 1a). In addition, ox-LDL
stimulated PC-3 proliferation more remarkably than LNCaP at
48 h. We also analyzed the effects of ox-LDL on LNCaP and
PC-3 cell cycle and apoptosis. There were significant differ-
ences in high-dose (100 g/ml) ox-LDL for LNCaP with
respect to G1 and S phase distributions, but this was not
significant with medium (20 g/ml) or low (4 g/ml) doses.
However, PC-3 cells were more sensitive than LNCaP cells,
and high doses (100 g/ml) of ox-LDL caused G1 arrest. Ox-
LDL decreased apoptosis of LNCaP cells but increased the
apoptosis rate of PC-3 in a concentration-dependent manner.
Dose-response experiments showed that ox-LDL was also
able to increase OLR1 protein expression in a dose-dependent
manner. OLR1 started to increase with an ox-LDL dose of
4 g/ml and reached maximum levels at 100 g/ml ox-LDL
(Fig. 2c). Time course experiments with 20 g/ml ox-LDL
treatment showed OLR1 protein expression increased after
12 h and peaked after 48 h (Fig. 2c). A 48-h incubation time
was therefore used in subsequent studies.
with cancer metastasis. Therefore, we compared the effects of
ox-LDL on the cell migration capabilities of PC-3 cells by
wound healing assay in a dose-dependent manner. Twelve
hours after scratching, the higher doses of ox-LDL promoted
cell migration (Fig. 3a, b). In the transwell assay, ox-LDL
stimulated LNCaP and PC-3 invasion in a dose-dependent
manner (Fig. 4a, b).
Ox-LDL can affect signaling pathways of PC-3
in a complicated manner
The results of our protein microarray indicate that multiple
signal pathways were changed in ox-LDL-treated PC-3 cells
(Supplementary Table 1). Caveolin-1 (phospho-Tyr14, fold
change 3.0) was activated after ox-LDL treatment. Activated
-catenin (phospho-Thr41/phospho-Ser45, fold change 3.68)
and downstream c-Myc (phospho-Ser62, fold change 2.26) as
well as NF-B-p65 (phospho-Ser529, fold change 1.69) could
promote proliferation and migration. Decreased Akt
(phospho-Thr308, fold change 0.80) activity led to activation
of p27Kip1 (phospho-Ser10, fold change 1.89) which could
inhibit proliferation. STAT1 (phospho-Tyr701, fold change

Table 3 Univariate and

multivariate analyses for the Variable High Gleason scoreb
evaluation of the association
between tested parameters and high Univariate Multivariate
Gleason score in the TCGA cohort
OR 95 % CI p OR 95 % CI p

Age 1.011 0.951 to 1.073 0.733 0.961 0.887 to 1.042 0.333

a
Categorical variables: pT >2 pT (3)a 7.843 2.220 to 27.706 0.001 9.303 2.186 to 39.538 0.003
or otherwise; pN >0; prostate- pNa 14.412 4.052 to 51.253 0.000 5.468 1.378 to 21.693 0.016
specific antigen 10, 20, 100, PSAa 2.939 0.772 to 11.193 0.114 1.641 0.325 to 8.278 0.548
or >100
b
OLR1 2.076 1.330 to 3.239 0.001 2.468 1.368 to 4.453 0.003
Gleason score >7
3578 Tumor Biol. (2015) 36:35733582

Fig. 2 Ox-LDL increased the

proliferation and apoptosis of
LNCaP and PC-3. a OLR1, the
ox-LDL receptor, was highly
expressed in PC-3 compared with
LNCaP cells. b Ox-LDL can
promote proliferation of LNCaP
and PC-3 in a dose-dependent
manner in CCK-8 assays at 48 h.
c Ox-LDL decreased G1 phase
and promoted S phase fractions in
LNCaP and PC-3 cells in a dose-
dependent manner; however,
higher concentrations of ox-LDL
(100 l LDL) induced G1 arrest
in PC-3 cells. d Ox-LDL inhibited
apoptosis of LNCaP but increased
apoptosis of PC-3 cells. *p<0.05

2.45) and STAT3 (phospho-Tyr705, fold change 1.63) activa-
tion could stimulate expression of downstream genes respon-
sible for progressive PCa. Apoptosis-associated protein
caspase-3 (phospho-Ser150, fold change 1.99) is activated in
ox-LDL-treated cells (Fig. 5). Activation of these signaling
pathways might be responsible for ox-LDL-induced tumor
proliferation, migration, and apoptosis.
Discussion
Our study revealed that the ox-LDL level in serum was
associated with PCa aggressiveness. In addition, overexpres-
sion of OLR1 was associated with higher Gleason score and
lymph node metastasis. Moreover, we found that ox-LDL
might promote PC-3 and LNCaP proliferation, migration,
and invasion and stimulate OLR1 expression in a dose- and
time-dependent manner in PC-3 cells. Taken together, our
study demonstrated that ox-LDL could be a pivotal factor
for advanced PCa and also a hallmark of PCa progression
and prognosis. Furthermore, patients with high expression of
OLR1 are more likely to have lymph node metastasis; thus,
extended lymph node dissection and closer examination of
surgical margins should be performed for patients with high
serum ox-LDL levels.
A recent study linked metabolic disorders to the sophisti-
cated nature of cancer phenotypes. Thanks to previous ath-
erosclerosis and hyperlipidemia studies, our study on ox-LDL
and PCa was inspired. Ox-LDL promoted proliferation,
Tumor Biol. (2015) 36:35733582
Fig. 3 Ox-LDL promoted PC-3 migration and stimulated the expression
of OLR1. a Representative images of the wound healing assay showed
that ox-LDL could promote the migration capabilities of PC-3 in a dose-
accelerated cell cycle, stimulated cell migration, and promoted
PCa in our study. To our knowledge, few studies have ex-
plored the role of ox-LDL and its mechanisms on PCa. OLR1,
the ox-LDL receptor, was highly expressed in PC-3 compared
with LNCaP cells. Interestingly, PC-3 cells are more aggres-
sive than LNCaP with respect to many biological behaviors,
such as proliferation [19, 20] and drug resistance to docetaxel
[21]. This phenomenon inspired us to investigate OLR1 ex-
pression with respect to PCa invasion and migration.
OLR1, a lectin-like scavenger receptor, is capable of rec-
ognizing several ligands, including the ox-LDL protein moi-
ety [22]. Overexpression of OLR1 has already been found in
the cellular components of atherosclerotic lesions [23]. Recent
studies also showed that OLR1 might act as an oncogene by
activating of NF-kB target genes responsible for proliferation,
migration, and inhibition of apoptosis [22] and analogous to
our microarray findings. In addition to NF-kB activation, in
our study, exogenous ox-LDL increased cellular P27, c-Myc,
-catenin, and caspase-3 levels. These findings illustrate that
3579
dependent manner. b Wound healing percentages were normalized to
controls and plotted. c Ox-LDL can increase OLR1 levels in a dose-
and time-dependent manner
ox-LDL can stimulate OLR1 expression and is responsible for
proliferation, migration, and apoptosis.
High Gleason score, lymph node metastasis, and bone
metastasis are typically seen in aggressive PCa, implying
decreased survival and poor quality of life. As a complex
process, PCa metastasis begins with a cell tropism to invasion
and is driven by extracellular signals [24]. Ox-LDL level is
always high in obese and atherosclerotic patients [25], who
also suffer from metabolic syndromes, such as hypertension,
diabetes mellitus, and dyslipidemia. The association between
metabolic syndrome and the risk of biochemical recurrence
after radical prostatectomy in patients with PCa is well
established [26]. These results may explain why statin usage
was reported to be beneficial for PCa patients [3, 13].
In this study, we show, for the first time, that ox-LDL is a
new marker for metastatic PCa. Khaidakov reported that ox-
LDL triggers pro-oncogenic signaling in human breast mam-
mary epithelial cells via stimulation of MiR-21 [27]. Ox-LDL
has also been reported as an independent factor differentiating
3580 Tumor Biol. (2015) 36:35733582

Fig. 4 Ox-LDL promoted PC-3

invasion in vitro. a Representative
images of the transwell chamber
assays indicated that ox-LDL
could promote the invasion
capabilities of LNCaP and PC-3
cells in a dose-dependent manner.
b Invading cells were counted in
each well and plotted

Fig. 5 Treated with 20 g/ml of

ox-LDL, PC-3 cells were
harvested and analyzed by protein
microarray. The positive findings
are plotted and shown. Detail data
are shown in Supplementary
Table 1
Tumor Biol. (2015) 36:35733582
patients with pancreatic cancer from patients with chronic
pancreatitis [28]. In this study, we discovered that ox-LDL
might promote proliferation through stimulating cell cycle and
did not interfere with apoptosis at low concentrations. Al-
though p-Akt decreased in ox-LDL-treated PC-3 cells, p-Akt
inhibited P27 increases, and the MAPK/c-Myc pathway and
NF-B were activated.
There are several limitations to this study. For instance, this
was a single-center research study with limited cases, and
thus, possible selection bias was unavoidable. Furthermore,
the TCGA cohort of PCa patients is incomplete because many
data are currently unavailable. Therefore, further validation of
our work at other centers, including patients of other races and
re-analysis with updated TCGA data, is needed. Moreover,
further validation of our protein microarray results in other
PCa cell lines and studies evaluating crosstalk after ox-LDL
treatment are required. The role of ox-LDL in apoptosis of
PCa cells was not clearly defined yet. However, this study
revealed a novel serum protein marker related to obesity and
oxidization stress that contributes to PCa metastasis. Our
research may facilitate further research on how ox-LDL can
trigger PCa invasion or metastasis and elucidate the underly-
ing mechanisms of this relationship.
Conclusion
Increased serum ox-LDL levels and OLR1 expression may
indicate advanced-stage PCa and lymph node metastasis. Ox-
LDL can stimulate prostate cancer proliferation, migration,
and invasion in vitro, and exogenous ox-LDL can activate
multiple signaling pathways in PC-3 cells in vitro.
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