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- Original Article -
Reduced expression of α-tocopherol-associated protein is associated with tumor cell proliferation and the increased
risk of prostate cancer recurrence
Xing-Qiao Wen1, Xiao-Juan
Li2, Zu-Lan Su3, Yong
Liu3, Xiang-Fu Zhou1, Yu-Bin
Cai1, Wen-Tao Huang1, Xin
Gao1
1Department of Urology, 2Department of Health Care, 3Department of Pathology, Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, China
Abstract
Aim: To examine the impact and prognostic significance of
α-tocopherol associated protein (TAP) expression in a series
of prostate cancer patients. Methods: Tissues from 87 patients underwent radical prostatectomy were examined for
TAP expression by immunohistochemistry. The relationships of the staining results, the clinic pathological
characteristics and the recurrence times were analyzed.
Results: Compared with the adjacent areas of normal and benign glands,
immunoreactivity of TAP was reduced in areas of prostate cancer. A lower
TAP-positive cell number per mm2
of the largest cancer area (defined as TAP-PN) was associated with higher clinical stage
(r = -0.248, P = 0.0322). Inverse
associations were found among the TAP-PN and positive lymph nodes
(r = -0.231, P = 0.0325), preoperative
prostate-specific antigen (PSA) levels
(r = -0.423, P = 0.0043),
tumor size
(r = -0.315, P = 0.0210) and elevated tumor cell
proliferation, which was indicated by the staining of
Ki-67 (r = -0.308, P = 0.0026). TAP-PN was a significant
predictor of recurrence univariately
(P = 0.0006), as well as multivariately, adjusted for known markers including preoperative
PSA, clinical stage, Gleason score, surgical margin, extra-prostatic extension, seminal vesicle invasion and lymph node
metastasis (P = 0.0012).
Conclusion: Reduced expression of TAP was associated with the cell proliferation status of
prostate cancer, adverse pathological parameters and the increased risk of
recurrence. (Asian J Androl 2007 Mar; 9:
206_212)
Keywords: α-tocopherol associated protein; prostate neoplasms; recurrence; vitamin E
Correspondence to: Dr Xing-Qiao Wen, Department of Urology, Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630,
China.
Tel: +86-20-8551-6867 ext. 2052 Fax: +86-20-8758-3378
E-mail: xingqiaowen@yahoo.com
Received 2006-07-08 Accepted 2006-08-18
DOI: 10.1111/j.1745-7262.2007.00236.x
1 Introduction
The α-tocopherol associated protein (TAP) was first
identified as α tocopherol-binding protein from bovine
liver cytosol using a-tocopherol as the bait [1].
Northern blotting assays indicated that higher levels of TAP
mRNA were found mainly in the liver, brain and prostate.
TAP might play an important physiological role in the
prostate [1]. Our previous studies showed that unlike
other vitamin E-associated proteins, TAP facilitated the
antiproliferation effect of vitamin E and functioned like a
tumor suppressor gene to control cell viability in
prostate cancer [2]. Over-expression of the
TAP gene in prostate cancer cells significantly suppressed the
proliferation of the cells. Knockdown of endogenous TAP by
small interfering RNA (siRNA) in non-malignant
prostate HPr-1 cells promoted cell growth. However, little is
known about the clinical significance of the expression
of this novel vitamin E-binding protein in prostate cancer
tissues. The aim of the present study was to examine
the impact and prognostic significance of TAP
expression in a series of prostate cancer patients.
Oxidative stress (OS) has been implicated in the
development of many kinds of cancer, including prostate
cancer [3]. Induction of high levels of reactive oxygen
species (ROS) such as superoxide, hydrogen peroxide
(H2O2) and hydroxyl radicals can subject the cell to a
state of OS, which might damage cellular DNA, proteins
and lipids, and result in cell-cycle arrest, cell death and
the development of malignancy [4]. However, what causes the increase in the susceptibility of tissues to OS
is not well understood. Diet and environmental factors
play important roles in the pathogenesis of prostate
cancer [5]. Vitamin E is an antioxidant and had been
suggested by epidemiological studies to have a protective
effect against prostate cancer [6]. α-Tocopherol (also
known as α-vitamin E) is one of the compounds in
vitamin E group and plays an important role in the
suppression of lipid oxidation in the membranes of intracellular
organelles [7]. It has been shown that vitamin E could
inhibit the proliferation [8], suppress the function of
androgen receptor and the expression of prostate-specific
antigen [9], regulate cell cycle distribution and induce
apoptosis in prostate cancer cells [10]. As
a vitamin E binding protein, TAP might be involved in the procession
of anti-oxidative damage. It is interesting to investigate
the clinical significance of the expression of this vitamin
E binding protein in prostate cancer tissues.
In the present study, we identified a novel link
between a vitamin E binding protein gene
TAP, whose expression is associated with cell proliferation,
pathological parameters and the risk of recurrence in prostate
cancer.
2 Materials and methods
2.1 Patients and tissue processing
For the present study, 87 prostate cancer specimens
obtained at radical prostatectomy carried out between
2000 and 2005 were used. The patients were aged from
52 to 76 years (mean, 63.4 years). According to the
International Union Against Cancer (UICC) clinical staging
system (1992), the patients had cancer staged as T1a-c
(n = 27), T2a-c (n = 49) and T3a
(n = 11). The prostate
specimens were sliced into 5-mm thick tissue blocks.
The largest (dominant) focus of cancer was identified
by visual inspection of the images and was chosen for
subsequent immunological studies. The tissue blocks
were fixed in 10% formalin and embedded in paraffin.
Hematoxylin_eosin stained sections from each
tissue block were evaluated for tumor histological differential
status as shown by Gleason score and pathological stage
(Tumor-Node-Metastasis system). Clinical recurrence
was defined as a postoperative
prostate-specific antigen (PSA) level > 0.4 ng/mL (Hybritech, San
Diego, CA, USA) on two successive measurements with evidence of
locoregional recurrences or metastases.
2.2 Immunohistochemistry analysis
Immunohistochemical analysis was carried out in the
largest (dominant) focus of cancer tissue in the prostate.
Prostate cancer slides were used for antibodies against
TAP and Ki-67 by immunohistochemistry as described
previously [2]. The tissue sections were subjected to
heat-induced epitope retrieval in citrate buffer (pH 6.0)
using a microwave oven at 350 W for 5 min for TAP.
Microwave treatment for Ki-67 was carried out for 4 min
in an ethylenediaminetetraacetic acid (EDTA) buffer (pH
8.0). Endogenous peroxidase activity was blocked by
incubation with 3% H2O2 in methanol for 15 min,
followed by a protein block (Dako Cytomation, Carpinteria,
CA, USA). The primary antibodies to TAP (0.5 mg/mL,
produced as described previously [2]) and Ki-67 (1 mg/mL;
BioGenex, San Ramon, CA, USA) were added and incubated at room temperature for 1 h. Diaminobenzidine
(DAB) and 3-amino-9-ethylcarbazole (AEC) were used
as the chromogen for Ki-67 and TAP, respectively. Slides
in which the primary antibody was omitted served as
negative controls.
2.3 Histological analysis
Each slide was evaluated independently by two
pathologists in a blind manner. The mean of the results was
calculated and recorded. Immunohistochemical analysis
was carried out in the largest focus of cancer. For each
specimen, 20_30 ocular measuring fields each
composed of 100 grids and having a real
area of 0.0625 mm2 were randomly chosen under a microscope
at a power of × 400 within a cancer. The number of TAP-positive cells per
mm2 of cancer tissue area was defined as TAP-PN.
Ki-67 expression was estimated essentially in a similar way as
for TAP. The number of Ki-67-positive cells per
mm2 of total cancer tissue area was defined as Ki-67-PN.
2.4 Statistics
Analyses were carried out using the statistical
package SPSS (version 14.0, SPSS, Chicago, IL, USA). The
correlation of TAP-positive cells with pathological
and clinical variables was evaluated using
Spearman's correlation coefficient testing. Comparisons in
TAP-PN among normal and prostate
cancer specimens were made using Mann_Whitney
U-test. Univariate survival analysis of time to clinical recurrence was carried out using the
product-limit method (Kaplan_Meier), with the log-rank test
for differences between categories of each variable.
Multivariate analyses were carried out using the Cox
proportional hazard regression model. The hazard ratio and
its 95% confidence interval (CI) were recorded for each
marker.
3 Results
3.1 Staining pattern of TAP in prostate cancer tissues
Examples of immunostaining for TAP in the malignant prostate tissues are shown in Figure 1. If the
immunoreactivity was positive, TAP was stained red by the
chromogen of AEC in the tissues. TAP protein was mainly
expressed in the prostate epithelium, and positive TAP
staining was rarely seen in the stroma. In the epithelial
cells, TAP staining was mostly confined to the cytoplasma
compartment, not to the nuclear part. Compared with
the adjacent areas of normal and benign glands,
immunoreactivity of TAP was reduced in areas of prostate
cancer. In areas of normal glands and benign
hyperplastic glands, TAP showed strong positive
cytoplasm-staining (Figure 1A, 1B). As shown in Figure 1A, 1C,
prostate cancer tissues with a low Gleason grade were
weak-positive in the adenocarcinoma area. In Figure 1D,
normal prostate glands showed strong positive cytoplasm
staining (in red). Figure 1E shows a high-grade
adenocarcinoma area showing absence of TAP staining.
3.2 Correlation of TAP-positive cells with clinical
parameters and tumor pathology
All the 87 cases had complete pathological
and clinical follow-up information and were
used for analyses. This cohort consisted of 9 patients
(10.3%) having positive margins, 12 (13.8%) having positive extracapsular
extension, 13 (14.9%) having positive seminal vesicle
invasion and 10 (11.5%) having positive
lymph nodes. The clinical stages were distributed as 27 (31.0%) T1,
49 (56.3%) T2 and 11 (12.7%) T3a. Sixteen patients
(18.4%) had a Gleason score of less than 6, 25
(28.7%) had a Gleason score of 6, 31 (35.6%) had a Gleason
score of 7, and 15 (17.2%) had a Gleason score of more than 7.
Preoperative PSA levels ranged from 6_180 ng/mL with a
mean level of 13.5 ng/mL.
The number of TAP-positive cells per
mm2 of the cancer area was defined as TAP-PN. In general,
patients with cancers of a relatively advanced clinical stage, a higher
Gleason score, seminal vesicle invasion or lymph node
involvement tended to have relatively lower TAP-PN, and
some of these associations were found to be statistically
significant (Table 1). The Spearman correlation analyses
showed that a lower TAP-PN was associated with more
advanced clinical stage
(r = _0.248,
P = 0.0322). Inverse associations were also found between the TAP-PN and
positive lymph nodes (r = _0.231,
P = 0.0325), preoperative PSA levels
(r = _0.423, P = 0.0043),
tumor size (r = _0.315,
P = 0.0210; the tumor size refers to the
volume of the entire cancer area within each specimen).
3.3 Correlation of TAP-positive cells with Ki-67
antigen expression
Quantification of the proportion of cells with nuclear
Ki-67 antigen expression is a measure of proliferation
fraction and, hence, biological aggressiveness in
malignancy [11]. Expression of Ki-67 was detected in normal
prostate and prostate cancer specimens. As shown in
Figure 1F, normal prostate tissue stained with Ki-67
antibody shows a few cells that were positively stained in
the nucleus. In Figure 1G, much Ki-67 staining was seen
in the high-grade carcinoma area.
A simple regression analysis was used to determine
the relationship between the TAP and Ki-67 positive cells
per mm2 of cancer tissue. A scatterplot suggested that
the relationship between the TAP and Ki-67 positive cell
numbers could be modeled as linear. A significant
reverse correlation was observed between TAP-PN and
Ki-67-PN (r = _ 0.308, P = 0.0026).
3.4 TAP-PN is a predictor of clinical recurrence after
surgery
The recurrence-free follow-up time for the 87 cases
was 11.2_72.6 months (averaged 38.8 months). Twelve
of the 87 patients (13.8%) had clinical recurrences
(PSA > 0.4 ng/mL, on two successive measurements
with evidence of local recurrences or metastases)
during the follow-up. The value of TAP-PN as a
continuous predictive marker for recurrence was analyzed
separately in this set of patients using the Cox proportional
hazard regression model.
Univariately, TAP-PN was inversely associated with
clinical failure (hazard ratio [HR] = 0.823,
HR-1 = 1.215, P = 0.0006). The inversed HR of 1.215 means that for
every 10 units' reduction in TAP-PN, risk of
experiencing a recurrence (estimated by HR) during the follow-up
time increases by 21.5%.
The TAP-PN data of this cohort were additionally
stratified with the median value of
64.6/mm2 as a cut-off point, with
44 cases (50.6%) falling into the low category
(TAP-PN £ 64.6/mm2),
and 43 cases (49.4%) falling into the high
(TAP-PN > 64.6/mm2)
category. A highly significant association of the
low TAP-PN category with poor clinical recurrence was established
by the univariate analysis
(HR = 0.236, HR-1 = 4.23
, P = 0.0004). Thus, a patient with TAP-PN
£ 64.6/mm2 had 4.2 times
of more chances of experiencing a recurrence during
the follow-up than the patient with a high TAP-PN level.
This difference in risk can be also observed on the
Kaplan-Meier plot (Figure 2). These
results strongly support the hypothesis that a high number of
TAP-positive cells within the cancer area was
associated with a lower rate of recurrence after surgery. As expected, in this the present
series of patients, other known predictors of recurrence
including extracapsular extension, seminal vesicle
invasion, lymph node metastasis, surgical margin status,
preoperative PSA level, tumor volume and Gleason score
were all significant predictors in univariate analyses.
Multivariate analysis showed that
TAP-PN is a very strong independent
predictor of disease progression when used either as a continuous or
a grouped variable. For any two patients with identical
clinical/pathological characteristics, one with low levels
(64.6/mm2) of expression index had a 3.8 times greater chance of
experiencing a recurrence during the follow-up than the other
with high levels (HR = 0.265,
HR-1 = 3.77, P = 0.0012)
of TAP.
Other parameters such as extra-capsular extension
(HR-1 = 2.252, P = 0.0201), seminal vesicle invasion
(HR-1 = 1.424,
P = 0.0036), lymph node metastasis
(HR-1 = 1.932, P = 0.0431), surgical margin status
(HR-1 = 0.958, P = 0.0472),
preoperative PSA level (HR-1 = 1.716,
P = 0.0316), tumor volume
(HR-1 = 2.723, P = 0.0148),
and Gleason score (HR-1 = 3.051,
P = 0.0035), were all
significant predictors in multivariate analyses.
These results indicate that TAP-PN was an
independent predictor of recurrence rate after
surgery. We did not analyze the survival rate during the follow-up
because most of the patients were still alive when the data
was collected.
4 Discussion
In the present study, we found that expression of
TAP at protein levels was reduced in prostate cancer
tissues compared with the adjacent normal and benign
tissues, which was consistent with our previous finding
that both mRNA and protein levels of TAP were decreased
in prostate cancer tissues [2]. According to previous
studies, downregulation of the TAP gene might be
specific in prostate cancer, because the mRNA levels of
another two genes in the TAP family, CRALBP and
TTP, showed no significant difference between the cancers
and benign tissues in seven pairs of samples [2].
We also found a significant relationship between TAP
expression and the pathological parameters of prostate
cancer. Reduced expression of TAP in the cancer tissues
was associated with higher clinical stage, larger tumor size
and elevated pre-operative PSA levels in
prostate cancer patients. The number of TAP-positive cells per
mm2 of the cancer area was associated with elevated tumor cell
proliferation as indicated by the staining of Ki-67.
The patients with relatively low levels
of TAP-positive cells tended to develop lymph node metastasis and were
associated with the increased risk of cancer recurrence. Taken
together, these results agree with the suppressive role of
TAP in prostate cancer as discovered by our previous
in vitro and in vivo studies [2].
We presented the possible biological reasons as to
why TAP is downregulated in more advanced disease stages. Our previous study suggested that TAP
suppresses PI3K/Akt signaling in prostate cancer cells, and
it can control the homeostasis of phospholipid, interfere
with p110a-p85 complex formation and reduce Akt activity [2]. PI3K/Akt signaling is the major survival
pathway in prostate cancer cells and plays a variety of
physiologic roles, such as cell growth, cell cycle regulation
and survival. According to current findings, we
hypothesized that no expression of TAP or insufficient function
of TAP could lead to the loss of the control of PI3K/Akt
signaling in the normal prostate cells, allowing the cells
to transform into malignant cells, grow out of control
and progress rapidly towards the end. Whether TAP is
involved in other signaling pathways such as JNK or
MAPK [12] remains to be investigated.
Currently, the exact physiological role of TAP protein
in prostate cells is still not well-known. Yamauchi
et al. [13] concluded that TAP has the following properties:
(i) α-tocopherol specific binding; (ii)
α-tocopherol-dependent nuclear translocation; and (iii)
α-tocopherol-dependent transcriptional activation in mammalian cells.
It was reported that the α-tocopherol metabolite,
α-tocopherolquinone, has a higher affinity to TAP
compared with α-tocopherol [14]. We previously found that
TAP enhanced vitamin E function in the prostate by
improving vitamin E uptake [2]. As indicated in the present
study, in normal prostate tissue, TAP was highly expressed
in the epithelial cells, suggesting that it might facilitate
the transportation of vitamin E in the prostate tissue from
the plasma and retain the high concentration of vitamin E
within the cells. Indeed, TAP can facilitate the retention
of α, γ, δ-vitamin E, and α-vitamin E succinate in the
prostate cancer cells. TAP can also enhance the
anti-proliferation effects of α-vitamin E, γ-vitamin E,
and α-vitamin E succinate [2]. Wilson
et al. [15] reported that in the rat prostate, a vitamin E deficiency disrupts some
differentiation functions, such as delaying the secretion
of 26 kDa protease in the ventral prostate.
Plasma and tissue vitamin E concentrations are
remarkably stable in healthy humans. Three proteins,
namely tocopherol regulatory proteins (TRP), have been
identified and they specifically bind tocopherols. They
are tocopherol transfer protein (TTP), tocopherol
associated protein (TAP) and tocopherol-binding protein
(TBP). They have been shown to play important roles in
the tissue distribution and intracellular trafficking of
vitamin E [7]. TTP mediates the selective transfer of
α-tocopherol into plasma lipoproteins and plays an
important role in the body [7]. It has been shown that a
certain disease of familial isolated vitamin E deficiency
(FIVE), also called ataxia with vitamin E deficiency, is
caused by mutations in the gene for TTP [16]. If left
untreated, these patients have extraordinarily low plasma
concentrations of α-tocopherol (α-T), less than 1% of
normal. When they take α-T supplements, plasma α-T
concentrations reach normal levels within hours, but
when they stop taking supplements, plasma α-T concentrations fall to extraordinarily low levels within days.
Terasawa et al. [17] reported that in TTP knockout mice,
the brain was particularly susceptible to vitamin E
depletion; less than 2% of the total α-T in control mouse
brains was detected in knockout mouse brains.
Similarly as TTP, a good way to identify the role of TAP in
the prostate is to establish the TAP knockout mice and
carry out further studies in the animal model.
Increasing bodies of evidence have shown a
prominent role for ROS in the pathogenesis of carcinoma of
the prostate, which is initiated by OS [3].
However, what promotes the susceptibility of tissues to oxidative
stress is not well understood. Vitamin E is an antioxidant
and has been suggested in epidemiological studies to have
a protective effect against prostate cancer. The
incidence and mortality of prostate cancer were reduced
among men receiving 50 IU of vitamin E for up to
8 years in the α-tocopherol and β-carotene cancer prevention
study (ATBC Study) [6]. These findings were unexpected and prompted the National Cancer Institute
to sponsor a 12-year long-term study named Selenium and
Vitamin E Cancer Prevention Trial (SELECT), in which
32 000 people were involved [18] to further identify the
role of antioxidants in preventing prostate cancer.
As a vitamin E binding protein, TAP might be involved in the anti-oxidative reaction of vitamin E in the
prostate. Loss of TAP expression might increase
susceptibility of tissues to OS, insufficient function of TAP,
which lead to deficiency in facilitating vitamin E in the
body, contributing to the development and progression
of prostate cancer. At present, there is little effective
treatment for metastasis prostate cancer, especially for
the stage of androgen-independent prostate cancer [19,
20], therefore, whether or not TAP could be developed
as a valuable therapy target for prostate cancer needs to
be investigated. It is interesting to elucidate how TAP
exerts its effect in the reaction of anti-OS in prostate
cells.
In conclusion, we have identified that reduced
expression of α-TAP in prostate cancer tissues is
associated with adverse pathological parameters and the
increased risk of cancer recurrence. TAP might be
developed as a valuable marker for the prognosis of prostate
cancer.
Acknowledgment
This study was supported by the National Natural
Science Funding of China (No. 30600620) and
Guang-dong Natural Science Funding (No. 05001762), China.
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