Abstract: BPH could be considered the most common benign tumor for men between 60 and 75 years of age. PSA is an unsatisfactory biomarker to define BPH progression to cancer. Recently, sigma-2 receptors have been recognized in several prostate cancer cell lines such as PC-3, DU-145 and LNCaP. It is reported that, in some cases, BPH could progress to malignancy and this progression can not easily be monitored by biomarkers such as PSA. In this paper we investigated 10 specimens from TURP, finding overexpression of sigma-2 receptors in two of these specimens (specimen 1 and 2) accompanied by 4 ng/mL PSA values. We hypothesize that the presence of sigma-2 receptors is related to a BPH progressing in prostate cancer. A possible correlation between sigma-2 receptors and PSA values could be useful to identify this pathological progression

Arcangelo Pagliarulo

Carmela Inglese

Francesco Berardi

Marialessandra Contino

Michele Bottalico

Nicola Antonio Colabufo

Pasquale Saponaro

Roberto Perrone

Vincenzo Pagliarulo

CiteSeerX

 The Open Biomarkers Journal, 2009, 2, 11-13 11 
 
 1875-3183/09 2009 Bentham Open 
Open Access 
Sigma-2 Receptors as Potential Novel Biomarkers During the Progression 
of Benign Prostatic Hypertrophy (BPH) into Prostate Cancer 
Nicola Antonio Colabufo*
,a
, Pasquale Saponaro
b,c
, Michele Bottalico
b,c
, Marialessandra Contino
a
, 
Carmela Inglese
a
, Vincenzo Pagliarulo
c
, Arcangelo Pagliarulo
c
, Francesco Berardi
a
 and Roberto Perrone
a
 
a
Dipartimento Farmacochimico, Facoltá di Farmacia, Universitá degli Studi di Bari, via Orabona, 4, 70125, Bari, Italy 
b
Unità Operativa di Urologia, Casa di Cura Santa Maria, SPA, Via De Ferrariis 18/D, 70124 Bari, Italy 
c
Dipartimento Emergenza e Trapianti d’Organo, Sezione di Urologia, Universitá degli Studi di Bari, Piazza G. Cesare, 
11, 70124, Bari, Itay 
Abstract: BPH could be considered the most common benign tumor for men between 60 and 75 years of age. PSA is an 
unsatisfactory biomarker to define BPH progression to cancer. Recently, sigma-2 receptors have been recognized in sev-
eral prostate cancer cell lines such as PC-3, DU-145 and LNCaP. It is reported that, in some cases, BPH could progress to 
malignancy and this progression can not easily be monitored by biomarkers such as PSA. In this paper we investigated 10 
specimens from TURP, finding overexpression of sigma-2 receptors in two of these specimens (specimen 1 and 2) ac-
companied by 4 ng/mL PSA values. We hypothesize that the presence of sigma-2 receptors is related to a BPH progress-
ing in prostate cancer. A possible correlation between sigma-2 receptors and PSA values could be useful to identify this 
pathological progression. 
Keywords: Sigma-2 receptors, benign prostate hypertrophy, PSA. 
INTRODUCTION 
 Benign Prostatic Hypertrophy (BPH) is characterized by 
hyperplasia of prostatic stromal and epithelial cells, resulting 
in the formation of large, fairly discrete nodules in the peri-
urethral region of the prostate [1, 2]. These nodules, if suffi-
ciently large, compress the urethral canal causing partial or 
complete obstruction of the urethra, modulating the normal 
urine flow. These epithelial cells actively proliferate but do 
not frequently progress to malignancy, so that BPH could be 
considered the most common benign tumor in men between 
60 and 75 years of age.  
 Prostate Specific Antigen (PSA), synthesized only by 
prostatic gland cells, is a sensitive and specific marker for 
prostatic diseases, including Benign Prostatic Hypertrophy 
(BPH), prostatitis and cancer. Clinically, total PSA values 
> 4 ng/mL are considered pathological, whilst values > 10 
ng/mL, are strongly suggestive of malignancy. PSA levels 
may be elevated in these patients because of increased organ 
volume and inflammation due to urinary tract infections [3-
6], thus BPH can not be always considered a premalignant 
lesion [7]. FDA approved PSA test in conjunction with a 
digital rectal examination to detect prostate cancer. However, 
much remains unknown about the interpretation of PSA lev-
els and hence they can not be considered a definitive test. 
For this reason, after transurethral resection of the prostate 
(TURP), (the most common surgery for BPH, [8]), the re-
moved tissue is subjected to histological and immunohisto-
chemical analysis. However, these investigations discrimi- 
 
 
*Address correspondence to this author at the Dipartimento Farmacochimi-
co, Facoltá di Farmacia, Universitá degli Studi di Bari, via Orabona, 4, 
70125, Bari, Italy; E-mail: colabufo@farmchim.uniba.it  
nate only between BPH and prostate cancer, whilst the pro-
gression of BPH to malignancy can not be detected [9]. 
 Sigma receptors are endocellular sites involved in tumor 
progression; in fact it has been demonstrated that these re-
ceptors are overexpressed in several tumor cell lines and 
tissues [10]. Recently, our research group demonstrated that 
sigma-2 and sigma-1 receptors are overexpressed in tumor 
cell lines such as rat C6 glioma, MCF-7 human breast ade-
nocarcinoma, SK-N-SH human neuroblastoma, murine 
mammarian sarcoma EMT-6 and in TRAnsgenic Mouse 
Prostate (TRAMP) cells [11]. 
 In particular, we have recognized these receptors in hu-
man bladder carcinoma, thereby establishing a correlation 
between sigma-2 receptor expression level and tumor staging 
and grading [12]. By contrast, a correlation between sigma-1 
subtype and tumor staging and grading has not been demon-
strated. Moreover, it has been reported that human PC-3 an-
drogen-insensitive [13] and LnCap androgen-sensitive pros-
tate tumour cell lines overexpress sigma-2 receptors [14].  
 The aim of the present work is to verify if sigma-2 recep-
tors, could be additional biomarkers to characterize BPH, in 
particular for specimens displaying ambiguous PSA level. 
Our objective is to identify the progression of BPH to malig-
nancy where other diagnostic tools have failed. 
 Following our protocol for the characterization of sigma 
receptor recognition in human bladder tumor, the stage and 
grade of the tumor was correlated with the expression of 
these receptors [12]. Ten specimens from TURP were exam-
ined in order to establish a possible correlation between BPH 
and sigma receptor density. 
376
12    The Open Biomarkers Journal, 2009, Volume 2 Colabufo et al. 
MATERIALS AND METHODS 
Radioligand and Drugs 
 The radioligand [
3
H]-DTG (56 Ci/mmol), (+)-[
3
H]-
pentazocine (32 Ci/mmol) were purchased from PerkinElmer 
Life Sciences (Zavantem, Belgium). (+)-Pentazocine was 
obtained from Sigma-Aldrich s.r.l. (Milan, Italy). DTG was 
purchased from Tocris Cookson Ltd., UK. 
Membrane Preparation 
 The membrane preparations were carried out as described 
by Colabufo et al. with minor modifications [12]. Briefly, 
500 mg human prostate tissue was homogenized with a 
Brinkman polytron (setting 5 for 3  15 s) in ice-cold-buffer 
(20 mL) containing 0.32 M sucrose and 10 mM Tris pH 7.4. 
The homogenate was centrifuged at 4 °C at 1,000  g for 10 
minutes and the supernatant saved. The pellet was resus-
pended in 20 mL Tris-sucrose buffer and centrifuged again 
at 4 °C at 1,000  g for 10 minutes. The supernatants from 
both 1,000  g spins were combined and ultra-centrifuged at 
4 °C at 31,000  g for 15 minutes. The pellet was resus-
pended in 20 mL of 10 mM Tris (pH 7.4). The homogenate 
was incubated at 25 °C for 30 minutes then centrifuged at 
31,000  g for 15 minutes. The final pellet was resuspended 
in 10 mM Tris-HCl (pH 7.4) and stored at –80 °C until use. 
For each membrane preparation the protein content was de-
termined by the Lowry method.  
Saturation Binding Assay 
 The saturation experiments were carried out as described 
by Colabufo et al. with minor modifications [12]. Sigma-2 
receptors in prostate specimens membranes were radiola-
beled using [
3
H]-DTG ligand, with concentrations ranging 
1.0-150 nM. Samples containing 250 μg of membrane pro-
tein, radioligand, 10 μM DTG (to determine non-specific 
binding) and 1 μM (+)-pentazocine (to mask sigma-1 recep-
tors), were equilibrated in a final volume of 250 μL of 50 
mM Tris pH 8.0 for 120 minutes at 25 °C. Incubations were 
stopped by addition of 5 mL of ice-cold buffer, and then the 
suspension was filtered through GF/B pre-soaked in 0.3 % 
poly(ethylenimine) for at least 60 minutes prior to use. The 
filters were washed twice with 5 mL of ice-cold buffer. 
 The saturation experiments for labeling sigma-1 recep-
tors were carried out employing the same procedure except-
ing for (+)-[
3
H]-pentazocine to radiolabel sigma-1 receptors 
(concentrations ranging 1.0-15 nM). The non-specific bind-
ing has been determined with 10 μM (+)-pentazocine. 
RESULTS 
 The experimental data was derived from ten men (age 
60-75 years) receiving pharmacological treatment (alpha 
adrenergic antagonist such as alphuzosin and tamsulosin). 
 The presence of sigma-1 and sigma-2 receptors in each 
specimen was recognized by saturation analysis with (+)-
[
3
H]-pentazocine and [
3
H]-DTG, specific radioligands target-
ing these receptors. In this assay, several concentrations of 
radioligand were added to constitute the receptor-radioligand 
complex. Two parameters were calculated by saturation 
analysis: Kd and Bmax. The Kd value indicates the radioligand 
concentration, at equilibrium, that binds 50% of total sigma-
2 receptors present in the biological sample. This value could 
vary from 10 nM to 30 nM for [
3
H]-DTG and from 1 nM to 
5 nM for (+)-[
3
H]-pentazocine, depending on the tissues and 
the cells employed. Moreover, through this analysis, sigma-2 
receptors population is quantified by the Bmax value. This 
value indicates the receptor density and it is expressed as 
fmol/mg of protein. 
 All specimens have been studied by saturation analysis 
with radioligand, but only two of these specimens, namely 1 
and 2, overexpressed sigma-2 receptors. Unfortunately, in 
the same specimens sigma-1 receptors have not been de-
tected because of high non specific binding. In the other 
samples, Kd and Bmax values were not determined because of 
the low expression of sigma-2 receptors. 
 In Fig. (1A) the saturation binding assay of specimen 1 is 
reported. In this sample sigma-2 receptors are overexpressed 
(Bmax = 6600 ± 320 fmol/mg) and Kd value was 12.3 ± 0.60 
nM. Analogously, in specimen 2 Kd = 22.59 ± 1.10 nM and 
Bmax = 6500 ± 325 fmol/mg of protein have been found (Fig. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. (1). Representative radioligand saturation analysis curve for recognition of sigma-2 receptors in Specimen 1 (A) and in Specimen 2 (B). 
A 
Specimen 1 (PSA = 4 ng/mL)
0 20 40 60
0
2500
5000
7500
Bmax = 6600  320 fmol/mg of protein
Kd = 12.3  0.60 nM
[
3
H]DTG (nM)
B
o
u
n
d
(f
m
o
l/
m
g
 o
f 
p
ro
te
in
)
 
B 
Specimen 2 (PSA = 4 ng/mL)
0 10 20 30 40 50 60
0
2500
5000
7500
Bmax = 6500 325 fmol/mg of protein
Kd = 22.59  1.10  nM
[
3
H]DTG (nM)
B
o
u
n
d
 (
fm
o
l/
m
g
 o
f 
p
ro
te
in
)
 
377
Sigma-2 Receptors as Potential Novel Biomarkers The Open Biomarkers Journal, 2009, Volume 2    13 
1B). These results demonstrated that Kd values of each 
specimen were consistent with the Kd range reported above. 
Moreover, in both biological samples, similar sigma-2 recep-
tor overexpression was found. Before TURP, 8 patients dis-
played total PSA levels  2 ng/ml and specimens 1 and 2 
displayed a PSA level of 4 ng/ml. 
DISCUSSION 
 The prostate pathology can be divided in three progres-
sive steps: (1) BPH in a non progressive state; (2) BPH pro-
gressing into prostate cancer; (3) prostate cancer. PSA is a 
robust diagnostic tool for prostatic cancer, nevertheless it can 
not be considered a definitive test.  
 It has been demonstrated that sigma-2 receptors are over-
expressed in several prostate tumor cell lines such as PC-3 
and DU-145 (androgen-independent cells) and in LNCaP 
(androgen-dependent cells) [14]. Therefore, sigma-2 recep-
tors as well as PSA displayed high sensitivity in prostate 
cancer (step 3) but only sigma-2 receptors are sensitive to 
step 2 as found in the present paper. The combination of 
these two biomarkers can identify steps 2 and 3 however 
sigma-2 receptors are unable to characterize (or diagnose) 
BPH in non progressive state. 
CONCLUSION 
 The present study is a contribution to better characterize 
BPH progression into prostate cancer where PSA alone is 
considered an unsatisfactory biomarker. Although these 
studies are focused on few samples it is possible to hypothe-
sise that the detection of PSA > 4 ng/mL accomplished with 
high sigma-2 receptor density could led to a BPH progres-
sion into prostate cancer. By contrast the role of sigma 1 
receptors should be elucidated yet. 
ABBREVIATIONS 
BPH = Benign Prostatic Hypertrophy 
PSA = Prostate Specific Antigen 
PC-3 = Human androgen-insensitive prostate tumor 
cell line 
DU-145 = Human androgen-insensitive prostate tumor 
cell line 
LnCap = Human androgen-sensitive prostate tumor 
cell line 
TURP = Transurethral resection of the prostate 
SK-N-SH = Human neuroblastoma cell line 
EMT-6 = Murine mammarian Sarcoma 
MCF-7 = Human breast adenocarcinoma 
Kd = Dissociation constant of radioligand-receptor 
complex at the equilibrium. 
Bmax = Receptor density expressed as fmol/mg of the 
protein 
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Received: February 05, 2009 Revised: March 20, 2009 Accepted: March 23, 2009 
 
© Colabufo et al.; Licensee Bentham Open. 
 
This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License 
(http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the 
work is properly cited. 
 
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Sigma-2 Receptors as Potential Novel Biomarkers During the Progression of Benign Prostatic Hypertrophy (BPH) into Prostate Cancer

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Sigma-2 Receptors as Potential Novel Biomarkers During the Progression of Benign Prostatic Hypertrophy (BPH) into Prostate Cancer

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