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- Original Article -
Clinical significance of the leptin and leptin receptor expressions in prostate tissues
Jung Hoon Kim1, Shin Young
Lee1, Soon Chul Myung1, Young Sun
Kim1, Tae-Hyoung Kim1, Mi Kyung
Kim2
Departments of 1Urology and
2Pathology, College of Medicine, Chung-Ang University, Seoul 140-757, Korea
Abstract
Aim: To evaluate the expression of leptin and leptin receptor in benign prostatic hyperplasia (BPH) and prostate
cancer (PCa), and to investigate whether they are associated with the development and progression of
PCa. Methods: Immunohistochemical staining was performed to examine the expression of leptin and leptin receptor in BPH and PCa.
PCa was divided into three groups: localized PCa, locally advanced PCa and metastatic PCa. The positive staining was
identified and the percentage of the positive staining was graded. We also assessed the relationship between both the
Gleason score and body mass index (BMI) and PCa.
Results: The percentage of the leptin expression in PCa was
significantly higher than that in BPH (P < 0.01). For the PCa group, the expressed levels of leptin showed a
considerable correlation with localized PCa and metastatic PCa
(P < 0.05). Leptin receptor, however, did not reveal a definite
difference between BPH and PCa. The expression of leptin indicated a significant difference between
well-differentiated PCa (Gleason score ¡Ü 6) and poorly differentiated PCa (Gleason score 8_10)
(P < 0.05). The relation between the leptin expression level in PCa and the BMI was not remarkable
(P = 0.447). Conclusion: Our results suggest that
leptin might have a promoting effect on the carcinogenesis and progression of PCa.
(Asian J Androl 2008 Nov; 10: 923_928)
Keywords: leptin; leptin receptor; prostatic neoplasms
Correspondence to: Dr Tae-Hyoung Kim, Department of Urology, College of Medicine, Chung-Ang University, 65-207, Hankang-Ro 3
Ka, Yong-san Ku, Seoul 140-757, Korea.
Tel: +82-2-748-9715 ext.9579 Fax: +82-2-798-8577
E-mail: kthlmk@hanafos.com
Received 2008-04-22 Accepted 2008-06-30
DOI: 10.1111/j.1745-7262.2008.00438.x
1 Introduction
As people's diets and lifestyles have changed in the
process of development, obesity has become a
significant issue, in Asia as well as in the rest of the world.
Obesity is related to health care as well as to social
problems. It causes many pathophysiological problems
in the human body, and it is closely linked to coronary
arterial diseases, diabetes, hypertension, malignancy, as
well as various other diseases [1, 2]. Adipose tissue
produces diverse cytokines and hormones; therefore, it
influences the metabolism of lipids and carbohydrates.
Cytokines secreted from adipose tissue, such as leptin,
adiponection or resistin, are called adipokines [3]. It is
well known that obesity and insulin resistance cause
metabolic syndrome. Moreover, several studies concerned with the relation between other diseases and the
secretion of cytokines and hormones are now underway.
Leptin is a hormone secreted from adipose tissue
that affects the ingestion of food and body weight [4].
Furthermore, it plays a role in keeping the energy
balance in the body by stimulating the generation of heat through
activation of the sympathetic nerve system [5]. It also
has a role in reproduction, hematogenesis, neovas-cularization, wound healing and secretion of insulin, and
it increases the blood concentration of male hormones,
such as testosterone [6, 7]. According to a recent study,
the higher the blood leptin concentration, the greater the
negative effect on cellular differentiation and cancer
progression in prostate cancer (PCa) [8]. It is also known
that leptin stimulates the cellular proliferation of benign
prostatic hyperplasia (BPH).
It has been reported that the blood concentration of
leptin, as well as the amount of leptin in the tissue, is
related to the progression of several cancers [9, 10]. It
was revealed in a study regarding leptin and leptin
receptor expression in breast cancer and stomach cancer
tissues that leptin is connected with the progression and
metastasis of cancer. However, there is no study
considering how leptin and leptin receptor in BPH tissue and PCa
tissue are expressed, and what functions leptin and leptin
receptor perform on the progression of cancer.
There-fore, in the present study, we investigate the expression
of leptin and leptin receptor using BPH tissue and PCa
tissue that were acquired from operations or biopsies.
2 Materials and methods
2.1 Patients
The present study was conducted on patients who
visited the Chung-Ang University Hospital in Korea from
November 2005 to October 2006. A total of 26 patients
were diagnosed with BPH and 30 patients were diagnosed with PCa. The prostate-specific antigen (PSA)
blood concentration of the patients with BPH was 4
ng/mL and below. Tissues of BPH and PCa were obtained by
performing transurethral prostatectomy or suprapubic
prostatectomy. The PCa patients were divided into three
groups. Those patients with PCa without capsular
invasion and no lymphatic or bone metastasis were
categorized as the localized PCa group. The second group was
defined as those patients with locally advanced PCa: this
group did not show lymphatic or other organ metastasis;
however, their cancer extended beyond the prostate
capsule and it had invaded the tissues around it. The last
group included those patients for whom their PCa was
found to metastasize to other organs, such as lymphatic
glands, bones and lungs, regardless of the capsule
invasion of cancer: this was the metastatic PCa group. The
number of patients with local PCa was 12. Of these, six
had locally advanced PCa. The tissue acquired from
radical prostatectomy or prostate checkup was used for
the experiments. Twelve patients with metastatic PCa
were not operable for operation, so their tissue samples
were acquired from prostate biopsies, and they were
diagnosed at that time.
2.2 Immunohistochemistry
Imunohistochemistry for leptin and leptin receptor
was conducted on the tissue samples of the study patients.
Sections that were 4_5 µm thick were cut from each
paraffin-embedded tissue block and then the sections
were deparaffined. In turn, they were dipped in a series
of graded anhydrous alcohol solutions (90%, 75% and
50%) and then washed with distilled water for 5 min.
To suppress the endogenous peroxidase activity, the
sections were dipped in hydrogen peroxide and washed in
distilled water for 10 min. For serotype retrieval, the
sections were dipped in a buffer solution of 10 mmol/L
citrate (pH 6.0) and eradiated in microwaves 2 times
for 5 min, cooled at room temperature and washed in
50 mmol/L of Tris buffer solution (TBS, pH 7.5). To
stop nonspecific immunohistochemistry reactions, the
sections were treated with chlorine serum for 30 min
and then washed to remove the rest of the solution. The
sections were treated with antibody to leptin (A-20; Santa
Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of
1:150 and leptin receptor Ob-R (M18; Santa Cruz Biotechnology) at a dilution of 1:30 at room temperature
for 1 night and the sections were then washed out with
TBS three times for 5 min each time. After that, the
sections were reacted with the second antibody (Zymed,
San Francisco, CA USA), attached to biotin for 20 min,
and peroxide enzyme complex combined with streptoavidin was added for 20 min. Finally, coloring
was done with AEC chromogen (3-amino-9-ethylcarbazole) solution and the results were observed
with an optical microscope after contrast dyeing with
Mayer hematoxylin stain.
2.3 Reaction and analysis of results
The leptin staining was deemed negative when there
were no cells dyed a red-brown color among all the cells.
The other staining was defined as + if < 25% of the cells
were stained positive, as ++ if 25%_50% of the cells were
stained positive, as +++ if 50%_75% of the cells were
stained positive and ++++ if > 75% of the cells were
stained positive (Figure 1). The same method was
applied to the results of the leptin receptor staining (Figure 2).
We conducted the immunohistochemistry using the
above method and we determined the degree of the leptin
and leptin receptor expression in the BPH and PCa tissues.
After that, we analyzed the differences of expression in
each patient group, and we tried to verify the relation
between the degree of leptin expression and the body
mass index (BMI), which is strongly associated with the
amount of adipose tissue that secretes leptin.
Following the Korean BMI standards, the patients were
divided into groups: those who had a normal weight that
was less 25 kg/m2, those who were overweight at
25_30 kg/m2 and those who were obese at over 30
kg/m2. We then compared the difference of the expressions of
leptin and leptin receptor in each group.
We also analyzed and compared the degree of
malignancy of cancer cells with the expression of leptin by
classifying the PCa patients according to their Gleason
score. We categorized the patients into three groups: the
first had a Gleason score of 6 or below, the second had
a Gleason score of 7, which indicates moderate
differen-tiation, and the third group had a Gleason score of 8,
which indicates a high a degree of differentiation. In
addition, we compared the degree of expression of leptin
and leptin receptor.
2.4 Statistical analysis
The SPSS program version 12.0 (SPSS Inc., Chicago,
IL, USA) was used for the statistical analysis. The
difference between the leptin and leptin receptor
expressions in BPH and PCa tissues was statistically analyzed
using unpaired t-test and analysis of variance.
P < 0.05 was regarded as statistically significant.
3 Results
3.1 Characteristics of the patients
The average age of the BPH patients was 67.92 ±
8.27 years and that for the PCa patients was 68.23 ± 7.24
years, which showed no statistical difference. The
average volume of the prostate of the BPH patients, as
measured by transrectal ultrasonography, was 40.28
± 20.09 mL and that of the PCa patients was 42.30 ± 19.37
mL, which showed no significant difference. In terms of
the BMI of the two patients groups, the BPH patients'
BMI was 24.05 ± 2.62 kg/m2 and PCa patients' BMI was
23.92 ± 2.48 kg/m2: there was no distinctive difference
(Table 1).
3.2 BPH and PCa
When comparing the expressions of leptin and leptin
receptor in the BPH and PCa patients, in terms of leptin,
there were five cases who reacted as negative, 10 as +,
eight as ++ and three as +++ among the 26 BPH patients.
In contrast, for the PCa patients, one reacted as negative,
four as +, 10 as ++ and +++ and five as ++++, which
means the expression of leptin was much stronger in the
PCa patients (P < 0.01). In regard to the expression of
leptin receptor in the BPH patients, 13 patients were
negative, 10 were +, three were ++ and none were +++
or ++++. In the case of the PCa patients, 11 cases were
negative, 13 were +, five were ++ and one was +++;
therefore, there were no statistically significant
differences between the groups (P = 0.226) (Table 2).
3.3 PCa
The tissue staining conducted by dividing the PCa
patients into three groups showed that the patients with
metastatic PCa had a higher degree of leptin expression
in their tissue compared with that of the localized PCa
patients (P < 0.05). The expression of leptin receptor
did indicate the differentiation according to the degree of
the progression of cancer (P = 0.816) (Table 3).
3.4 Classification of PCa by the Gleason score
When the tissue was classified according to the
Gleason score regardless of the progress of cancer, the
group with a high Gleason score (a Gleason score 8_10)
had higher leptin expression than the low Gleason score
group (Gleason score ¡Ü 6) (P < 0.05) (Table 4). In this
case, there was no difference between the two groups,
as was the case with expression of leptin receptor.
3.5 Classification of PCa using the BMI
In regard to the BMI, there were 19 cases who had
normal weight (< 25 kg/m2), 11 were overweight
(25_30 kg/m2), and nobody was obese
(¡Ý 30 kg/m2) among the PCa patients. There was also no
statistically significant difference in leptin expression between the normal
weight and overweight patients (P = 0.447) (Table 5).
4 Discussion
Many recent studies have focused on the role of leptin
in the body, and, in particular, have examined the
relation between leptin and cancer. In one previous study
regarding breast cancer, leptin and leptin receptor were
more expressed in breast cancer than in normal breast
tissue [9]. That study indicated that the degree of the
expressions of leptin and leptin receptor was significantly
related to distant metastasis of breast cancer. In the
case of stomach cancer, it has been reported that as leptin
and leptin receptor are highly expressed, the occurrence
of cancer and the malignant degree of it are also greatly
affected [10]. In a study of renal cell carcinoma, it is
revealed that the increase of the leptin concentration and
the expressed degree of leptin receptor in blood is
extraordinarily increased with tissue invasion of renal cell
carcinoma [11].
There have been studies focusing on the relation
between prostate disease and leptin. Lagiou et
al. [12] reported that there is no special relation between the leptin
concentration in blood and the occurrence of BPH and
the degree of PCa. In contrast, there is another study
that finds that leptin concentration in blood is associated
with the testosterone and PSA levels of PCa patients.
Yet another report determines that the increase in leptin
is related to the occurrence and progress of PCa
[13_15]. Stattin et al. [16] measured the concentration of
leptin and leptin receptor in blood and the expression of
leptin and leptin receptor in PCa tissue, and proved that
they are all related to the progression of PCa. The present
study shows that the expressed degree of leptin is higher
in PCa tissue than in BPH tissue. This leads us to believe
that leptin possibly has an influence on the occurrence
of PCa. However, in the case of leptin receptor, there
was no difference of the expressed degree of leptin
receptor between the BPH tissue and the PCa tissue, which
is a different result from the previous study [16]. In the
present study, the number of patients was small and
immunohistochemical staining of normal prostate tissue was
not performed. Hence, future studies are needed that
include a greater number of patients. In addition, normal
prostate tissue should be included in the research.
Although subject to debate, according to a previous
epidemiologic study, an increase in BMI affects the
increased occurrence of PCa [17]. It is already known
that leptin is associated with adipocytes, which are the
cells in which leptin is directly formed, and the blood
concentration of leptin is different according to the range
of obesity. In the present study, although the result was
restricted because there were no obese patients included,
and there was not much statistical difference of the leptin
expression between the normal weight patients and the
overweight patients, as measured by BMI. This indicates
that the expression of leptin in PCa tissue is not
influenced by the range of obesity, which is unlike the leptin
concentration in blood.
The Gleason score divides PCa into five grades
according to the range of tissue differentiation, and this is
shown in the present study as the sum of the two grades
that were most prevalent. The Gleason score is a
valuable prognosis factor that can predict capsular invasion
of PCa, metastasis to lymph nodes and so on. The
tissue with a higher Gleason score is more likely to have a
higher expressed level of leptin among the samples of
PCa tissue. In contrast, when measuring the degree of
leptin expression as categorized into three groups
according to the progress of PCa, the metastatic PCa
tissue expressed leptin more than that in the localized PCa
tissue. These two results suggest that the degree of leptin
expression is closely linked with the progression and the
degree of malignancy of PCa.
Therefore, the expression of leptin is higher in PCa
tissue than in BPH tissue, and for PCa tissue, the lower
the degree of differentiation and progression of PCa, the
higher the expression of leptin.
The results of this study suggest that an increase in
lepin expression in prostate tissue is related to the
progression and the degree of malignancy of PCa. These
findings support the idea that further studies are needed
that focus not only on leptin, but also on other adipokines
in regard to the occurrence and progression of BPH and
PCa.
Acknowledgment
This Research was supported by Chung-Ang University Research Grants (Seoul, Korea) in 2008.
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