APMIS 114: 893–8, 2006 C 2006 The Authors

Printed in Denmark . All rights reserved

Journal Compilation C 2006 APMIS
ISSN 0903-4641

Immunohistochemical analysis of Omi/HtrA2 expression

in prostate cancer and benign prostatic hyperplasia
XIAO-YONG HU,1 YUE-MIN XU,1 XIAO CHUN CHEN,2 HAO PING,2 ZHAO-HUI CHEN2
and FU-QING ZENG2
1
Department of Urology, Shanghai No. 6 People’s Hospital, Shanghai Jiaotong University, Shanghai, and
2
Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China

Hu XY, Xu YM, Chen XC, Ping H, Chen ZH, Zeng FQ. Immunohistochemical analysis of Omi/
HtrA2 expression in prostate cancer and benign prostatic hyperplasia. APMIS 2006;114:893–8.
The serine protease Omi/HtrA2 is released from mitochondria into the cytosol after apoptotic stimuli,
inducing apoptosis in a caspase-independent manner through its protease activity and in a caspase-
dependent manner by neutralizing the inhibition of inhibitor of apoptosis proteins (IAPs) on caspases.
Alteration of apoptosis is essential for cancer development, and cancer cell death by radiation and
chemotherapy is largely dependent upon apoptosis. Thus, analysis of the expression status of Omi/
HtrA2, a regulator of apoptosis, in cancer tissues is needed for an understanding of cancer develop-
ment. In the current study we analyzed the expression of Omi/HtrA2 in 65 prostate cancer, 40 benign
prostatic hyperplasia and 10 normal prostate specimens by immunohistochemistry. Omi/HtrA2
mRNA levels of in vivo prostate cancer and benign prostatic hyperplasia samples were also assayed
by semiquantitative reverse transcription-polymerase chain reaction. Immunopositivity (defined as
Ø30%) was observed for Omi/HtrA2 in most of the prostate cancers, and the positive rate of Omi/
HtrA2 was lower in the well-differentiated group than in the poorly and moderately differentiated
groups (p⬍0.005). By contrast, the cells in the normal prostate and benign prostatic hyperplasia
groups showed no or only weak expression of Omi/HtrA2. Meanwhile, the Omi/HtrA2 mRNA level
of prostate cancer is much higher than that of benign prostatic hyperplasia (p⬍0.001). Taken together,
these results suggest that prostate cancer cells in vivo may need Omi/HtrA2 expression for apoptosis,
and that Omi/HtrA2 expression might be involved in prostate cancer development.
Key words: Omi/HtrA2; serine protease; prostate cancer; benign prostatic hyperplasia; apoptosis.
Xiao-Yong Hu, Department of Urology, Shanghai No. 6 People’s Hospital, Shanghai Jiaotong Univer-
sity, Shanghai 200233, China. e-mail: urologist2010/yahoo.com.cn

It is now believed that clonal expansion and tu-
mor growth is the result of the deregulation of
intrinsic proliferation (cell division) and cell
death (apoptosis). Advanced prostate cancer is
resistant to many proapoptosis factors and
shows distinct apoptosis resistance, so the key is
to reverse its apoptosis resistance in the treat-
ment of advanced prostate cancer (1, 2). Omi/
HtrA2 is a serine protease (3–5) that is released
Received 20 May 2006.
Accepted 19 September 2006.
during apoptosis from mitochondria to cytosol
along with cytochrome C and Smac/DIABLO
(6, 7). Omi/HtrA2 can displace IAPs from casp-
ases and release a suppressive effect on caspase
activity (8, 9). Furthermore, Omi/HtrA2 can
also trigger apoptosis in a caspase-independent
pathway, which entirely depends on its serine
protease function (10, 11). Therefore, Omi/
HtrA2 might have promising applications in the
gene treatment of cancer. We assayed the expres-
sion of Omi/HtrA2 in prostate cancer (Cap), be-
nign prostatic hyperplasia (BPH) and normal
893
 HU et al.

prostate (NP) specimens by means of immuno-
histochemistry and reverse transcription poly-
merase chain reaction to ascertain the effect of
Omi/HtrA2 on prostate cancer pathogenesis
and development.
MATERIALS AND METHODS
Tissue specimens
All paraffin-embedded specimens of prostate can-
cer, benign prostatic hyperplasia and normal prostate
tissues were obtained from the pathology archives of
the Tongji and Union Hospitals of Huazhong Univer-
sity of Science and Technology. The specimens had
previously been fixed in 10% formaldehyde, following
established methods. According to the Jewett-Whit-
more-Prout staging system, the 65 prostate cancer
samples included A (5), B (22), C (27), and D (11)
stages. The prostate cancer samples were also distrib-
uted as well- (Gleason 3–4), moderately (Gleason 5–6),
moderately-poorly (Gleason 7–8), and poorly (Glea-
son 9–10) differentiated groups based on the Gleason
grading system. The age of prostate cancer patients
was between 57 and 78 years, with an average of 67.7
years. The oldest benign prostatic hyperplasia patient
was 65, while the youngest was 60, and the average age
was 62.3 years. All specimens were reviewed indepen-
dently by two pathologists. In vivo prostate cancer and
benign prostatic hyperplasia tissues came separately
from radical prostatectomy and suprapubic prostatec-
tomy specimens from Shanghai Jiaotong University
Affiliated No. 6 People’s Hospital.
Immunohistochemical procedure
The goat multiclonal antibody (Santa Cruz Bio-
technology, USA) contains amino acids 335–350 of
human Omi/HtrA2. In the manufacturer’s data sheet,
this antibody is able to detect two protein bands of
approximate molecular size 50 and 38 kDa, which
may represent the precursor and mature form of
Omi/HtrA2, respectively. To maximize the signal on
immunohistochemistry, two strategies were used in
the present study: antigen retrieval in citrate buffer
and signal amplification with biotinylated tyramide.
For the former, heat-induced epitope retrieval was
conducted by immersing the slides in Coplin jars
filled with 10 mmol/L citrate buffer (pH 6.0) and
boiling the buffer for 30 min in a pressure cooker
(Nordic Ware, Minneapolis, MN) inside a microwave
oven at 700 W; the jars were then cooled for 20 min.
For the latter, the Renaissance TSA indirect kit
(NEN Life Science, Boston, MA), which included
streptavidin-peroxidase and biotinylated tyramide,
was used. After rinsing with PBS, the slides were
treated with 1% H2O2 in PBS for 15 min at room
temperature to abolish endogenous peroxidase activ-
ity. After washing with TNT buffer (0.1 mol/L Tris-
HCl, pH 7.4, 0.15 mol/L NaCl and 0.05% Tween 20)
for 20 min, the slides were treated with TNB buffer
(0.1 mol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl and
0.5% blocking reagent). Sections were incubated
overnight at 4 æC with the antibody for Omi/HtrA2
(1/25 dilution). They were then incubated with bi-
otinylated secondary antibodies (Sigma, St. Louis,
MO) for 50 min at 37 æC; streptavidin-peroxidase was
applied for 30 min at room temperature, followed by
biotinylated tyramide treatment for 7 min, and
streptavidin-peroxidase treatment for 30 min at room
temperature. Each incubation step was followed by
three washes for 5 min in TNT buffer. The reaction
products were developed with diaminobenzidine
(Sigma, St. Louis, MO) and counterstained with
hematoxylin. Tumors were interpreted as positive by
immunohistochemistry when at least weak-to-moder-
ate cytoplasmic staining was seen in over 30% of the
neoplastic cells. The results were reviewed indepen-

TABLE 1. Omi/HtrA2 expression in CaP, BPH and N, and relationship between Omi/HtrA2 protein expression
and clinicopathological features of CaP
Item n Omi/HtrA2 staining intensity p(X2)
ª π ππ πππ
CaP 65 4 19 18 24
BPH 40 25 7 7 1 0.000*
NP 10 9 1 0 0 0.000*
Clinical stage (J-W-P)
AπB 27 7 16 3 1 0.005**
CπD 38 4 5 10 19
Prostate cancer (Gleason)
3–4 13 2 0 8 3
5–6 24 2 17 2 3 0.010***
7–8 20 0 2 8 10 0.006***
9–0 8 0 0 0 8 0.009***
Intensity of expression described as ª (negative), π (weakly positive), ππ (moderately positive), and πππ
(strongly positive). * compared with CaP group; ** compared between AπB and CπD groups; *** compared
with well-differentiated group.

894
 OMI/HTRA2 EXPRESSION IN PROSTATE CANCER

Fig. 1A. Visualization of Omi/HtrA2 in prostate can-

cers by immunohistochemistry. Antibodies were de-
tected using a diaminobenzidine method that pro-
duces a brown color. Counterstaining of nuclei was
done with hematoxylin (blue). Prostate cancer shows
strong immunoreactivity for Omi/HtrA2 in cyto-
plasm (A, ¿100).
Fig. 1B. Visualization of Omi/HtrA2 in benign pros-
tatic hyperplasia by immunohistochemistry. Anti-
bodies were detected using a diaminobenzidine
method that produces a brown color. Counterstain-
ing of nuclei was done with hematoxylin (blue). Be-
nign prostatic hyperplasia shows week immunoreac-
tivity for Omi/HtrA2 in cytoplasm (B, ¿100).
Fig. 1C. Visualization of Omi/HtrA2 in normal pros-
tate by immunohistochemistry. Antibodies were de-
tected using a diaminobenzidine method that pro-
duces a brown color. Counterstaining of nuclei was
done with hematoxylin (blue). Normal prostate
shows no immunoreactivity for Omi/HtrA2 in cyto-
plasm (C, ¿100).

dently by two pathologists. As negative controls, a

slide was treated by replacement of primary antibody
with non-immune serum.

Analysis of Omi/HtrA2 expression

The Leica QWin Image Processing and Analysis
System (Leica Imaging System Ltd., UK) was used
to analyze the immunohistochemical scope and inten-
sity of Omi/HtrA2. The immunoreactivity was
graded as πππ when strongly positive (scope¿in-
tensity ⬎20), ππ when moderately positive (10⬍
scope¿intensity Æ20), π when weakly positive (1⬍
scope¿intensity Æ10), and – when negative (scope¿
intensity Æ1). The results were reviewed indepen-
dently by two pathologists.

Semiquantitative RT-PCR procedure

Prostate cancer and benign prostatic hyperplasia
tissues were homogenized and total mRNA was iso-
lated using TRIZOLA reagent following the manu-
facturer’s instructions (Gibco BRL, Grand Island,
NY). RNA was treated with RNase-free DNase I
(Boehringer Mannheim, Indianapolis, IN) and run
on 0.8% agarose gel (Ultrapure Agarose Electro-
phoresis Grade, GIBCO BRL, Gaithersburg, MD)
with 1¿TBE buffer (10¿TBE buffer diluted with
dH2O, Life Technologies/GIBCO BRL, Gaithers-
burg, MD) to check for DNA contamination. RNA
concentration was determined by spectrophotometry
and stored at ª80 æC until use. Reagents for reverse
transcription-polymerase chain reaction (RT-PCR)
were obtained from Fermentas (MBI, USA). We gen-
erated the first-strand cDNA using total RNA (1 mg)
in a reaction volume (20 ml) following the manufac-
turer’s recommended protocol. The RT conditions
were as follows: reverse transcript (30 æC for 10 min),
annealing (42 æC for 30 min), and deactivation of re-
verse transcriptase (99 æC for 5 min and 5 æC for 5
min). The PCR (total volume 20 ml) was carried out
using 1 ml of RT product with Taq DNA polymerase
(Fermentas). The synthesized cDNAs were amplified
using specific sets of primers for Omi/HtrA2 (sense-
5ø-GACCGGCACCCTTTCTTG-3ø, antisense-5ø-G-
CCCCCACTGGTTCATTT-3ø) and b-actin (sense-
5ø-CTGTTCCAGCCTTCCTTC-3ø, antisense-5ø-T-
CCTGCTTGCTAATCCAC-3ø). The PCR procedure
consisted of 30 cycles of denaturation at 94 æC for 30
s, annealing at 60 æC for 30 s, extension at 72 æC for 60
s, with an initial denaturation of the sample cDNA
at 94 æC for 2 min before PCR. PCR products were
quantitated to confirm that they were in the linear
range of amplification. Amplification of the house-
keeping gene b-actin served as a control for normal-

895
 HU et al.

TABLE 2. Omi/HtrA2 mRNA assay results in pros-

tate cancer and benign prostatic hyperplasia (X∫SD)
n ratio p
CaP 20 0.8736∫0.1096 0.000
BPH 7 0.4363∫0.1443

ization of the results. PCR products (10 ml) were run

on 1.5% agarose gel and visualized using the MAGI-
AS-1000 visualization system. The densitometric
analysis of PCR products was performed using
MAGIAS-1000 software and normalized relative to
the b-actin expression for each sample. The same ex-
periments were performed three times. Results are ex-
pressed as the ratio of Omi/HtrA2 and b-actin-nega-
tive phase absorbance¿area.

Statistical analysis
Statistical analysis was carried out using Chi-
Square and Mann-Whitney tests. Significance was as-
sumed for values p⬍0.05. Fig. 2. Omi/HtrA2 mRNA level in prostate cancer
and benign prostatic hyperplasia. DL2000 marker
(lane M); Omi/HtrA2 mRNA level of prostate cancer
(lane 1); Omi/HtrA2 mRNA level of benign prostatic
RESULTS hyperplasia (lane 2).

Omi/HtrA2 expression in CaP, BPH and NP

In the samples of prostate cancer analyzed,
immunopositivity for Omi/HtrA2 was observed
in 61 (93.84%) of the 65 cancers cases (Fig. 1A).
20 benign prostatic hyperplasia samples and 10
normal prostate tissue samples displayed weak
or no immunoreactivity for Omi/HtrA2 (Fig.
1B & C). The expression of Omi/HtrA2 was
correlated with prostate cancer differentiation
(p⬍0.005). According to pathological and clin-
ical data, the 65 prostate carcinoma samples
were classified as well, moderately, moderately–
poorly, and poorly differentiated groups. All of
eight poorly differentiated prostate cancer
samples (Gleason 9–10) showed strong Omi/
HtrA2 immunopositivity (πππ), 10 of 20
(50%) moderately-poorly differentiated prostate
cancer samples (Gleason 7–8) showed strong
Omi/HtrA2 immunopositivity (πππ), whereas
only 6 of 37 well- and moderately differentiated
(16.2%) samples showed strong immunopositiv-
ity. The cells immunostained for Omi/HtrA2
were evenly dispersed in the sections, and the
immunostaining of Omi/HtrA2––when pres-
ent––was cytoplasmic (Fig. 1A & B). Negative
controls using nonimmune serum as primary
antibody showed no signal.
896
Omi/HtrA2 mRNA assay results in prostate
cancer and benign prostatic hyperplasia
The semiquantitative RT-PCR assay shows the
expression of Omi/HtrA2 in the prostate cancer
group was much higher than that in benign pros-
tatic hyperplasia group (Table 2; Fig. 2).
DISCUSSION
The Omi/HtrA2 serine protease is a nuclear-en-
coded mitochondrial protein that can inhibit
multiple apoptosis proteins such as IAPs (12,
13) and ped/pea-15 (14, 15) identified in mam-
mals. Cellular stress, such as UV exposure, in-
duces cleavage of the Omi/HtrA2 mitochondrial
localization sequence, thereby generating a ma-
ture active molecule featuring a new apoptogen-
ic NH2 terminus, termed the IAP-binding motif
(16–18). This motif consists of a short stretch of
hydrophobic amino acids and can competitively
bind to the BIR domain of IAPs. This event
leads to the release and reactivation of the BIR-
bound caspases. Thus, Omi/HtrA2 binding dis-
places IAPs from caspases, releasing the sup-
pressive effect on caspase activity. Furthermore,
 OMI/HTRA2 EXPRESSION IN PROSTATE CANCER
Omi/HtrA2 can also trigger apoptosis in a cas-
pase-independent pathway, which entirely de-
pends on its serine protease function. Indeed,
IAPs are substrates for Omi/HtrA2 and their
degradation may contribute to caspase acti-
vation by Omi/HtrA2. Ped/pea-15 is also iden-
tified as a novel Omi/HtrA2 interactor that can
inhibit death receptors, such as TNF and
TRAIL, that induce apoptosis.
To date, several reports have described Omi/
HtrA2 mRNA expression in cancer cell lines
(19–21). Although Omi/HtrA2 mRNA is widely
expressed in cancer cell lines, the intensity of
Omi/HtrA2 expression is cell-type specific.
Whether Omi/HtrA2 expression plays a role in
the development or progression of prostate can-
cer is not known. Our study found Omi/HtrA2
overexpressed in prostate cancer and correlated
with prostate cancer differentiation. By con-
trast, normal prostate and benign prostatic hy-
perplasia shows no or only weak expression. As
benign prostatic hyperplasia and normal pros-
tate have the same degree of Omi/HtrA2 expres-
sion, we used in vivo benign prostatic hyper-
plasia tissue as control in the Omi/HtrA2
mRNA assay. The results, which coincided with
the outcome of immunohistochemistry, suggest
that Omi/HtrA2 expression in prostate cancers
might play a role in the development of prostate
cancer.
During the development and progression of
cancers, cancer cells become capable of evading
apoptosis, thereby protecting themselves from
various apoptotic stimuli. Failure of apoptosis
in the cancer cells with this capability could
allow their survival. Why Omi/HtrA2 overex-
pression does not lead to increased prostate
cancer cell apoptosis is not known. Three hypo-
theses could explain the discrepancy. One possi-
bility might be that the full-length form of Omi/
HtrA2 is located in the mitochondria and has
no proapoptosis function, mature Omi/HtrA2
transformation depending on the stimulation of
intracellular or extracellular stress. The second
possibility might involve IAPs as substrates of
Omi/HtrA2 are located in the cytosol. The third
possibility might be that overexpression of IAPs
and ped/pea-15 in prostate cancer can inhibit
the Omi/HtrA2 proapoptosis function (22).
These hypotheses would also explain why Omi/
HtrA2 makes tumor cells more sensitive to
chemotherapeutic agents.
In conclusion, the data provided in the cur-
rent study define for the first time the in vivo
patterns of Omi/HtrA2 expression in prostate
cancers. Different expression of Omi/HtrA2
among normal prostate, benign prostatic hyper-
plasia and prostate cancer cells suggests that
regulation of Omi/HtrA2 expression might play
a pivotal role in the development of prostate
cancers. As Omi/HtrA2 is highly expressed in
prostate cancer, it is relevant to study the effect
of Omi/HtrA2 on gene treatment of prostate
cancer.
This research was supported by the National Nature
Science Foundation of China (30070756).
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