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Cytoplasmic induction and over-expression ofcyclooxygenase-2 in human prostate cancer: implications forprevention and treatmentS. MADAAN*, P.D. ABEL*, K.S. CHAUDHARY, R. HEWITT, M.A. STOTT, G.W.H. STAMP and
E.-N. LALANI
Departments of Histopathology and *Surgery, Imperial College School of Medicine, Hammersmith Hospital Campus, London and
Department of Urology, Royal Devon and Exeter Hospital, Exeter, UK
Objective To assess the level and morphological distribu-
tion of cyclooxygenase (COX)-1 and -2 in human
prostates and to determine any association with the
Gleason grade of prostate cancer.
Materials and methods The study comprised 30 samples
from patients with benign prostatic hyperplasia (BPH)
and 82 with prostate cancer. Immunohistochemistry
was used to assess the expression of COX-1 and -2, and
13 samples were also assessed using immunoblotting
(six BPH and seven cancers).
Results For both BPH and prostate cancer, COX-1
expression was primarily in the fibromuscular
stroma, with variable weak cytoplasmic expression
in glandular/neoplastic epithelial cells. In contrast,
COX-2 expression differed markedly between BPH and
cancer. In BPH there was membranous expression of
COX-2 in luminal glandular cells and no stromal
expression. In cancer the stromal expression of COX-2
was unaltered, but expression by tumour cells was
significantly greater (P=0.008), with a change in the
staining pattern from membranous to cytoplasmic
(P
-
homeostasis and water reabsorption by the renal
collecting tubules. In contrast, COX-2 is inducible and
thought to be involved in differentiative processes such as
inflammation and ovulation [7]. Of the two isoforms,
COX-2 is the most consistently up-regulated in many
cancers, including oesophagus [8], stomach [9], colon
[10], lung [11], pancreas [12] and head and neck [13].
Recent studies on rat mammary glands suggest that
hormonal influences on cancer development may also be
mediated by COX-2 gene expression and PG synthesis
[14,15]. COX-2 inhibitors are chemopreventive against
colon [16] and lung cancers [17] in mouse models.
Furthermore, COX-2/ApcD716 double-gene knockout
mice have fewer and smaller intestinal polyps than
ApcD716 knockout mice [18]. Evidence that increased
levels of COX-2 may be important in the development of
prostate cancer comes from preliminary results in human
[19,20] and canine prostates [21]. Thus the aim of the
present study was to assess the co-expression of COX-1
and -2 proteins in adult human benign and malignant
prostates, and to analyse their expression pattern.
Materials and methods
The study included 112 specimens (formalin-fixed and
fresh-frozen) of prostates obtained during TURP (30 of
BPH and 82 of adenocarcinoma), from the Department
of Histopathology and Human Biomaterials Resource
Centre, Imperial College School of Medicine,
Hammersmith Hospital Campus, London, and the
Department of Histopathology, West Middlesex
University Hospital, London. The median (range) age of
the patients was 70 (5192) years. The tumours were
graded according to the Gleason system by one author
(E-N.L.). Of the adenocarcinomas, 10 were well differ-
entiated (Gleason grade 24), 32 moderately differen-
tiated (Gleason grade 57) and 40 poorly differentiated
(Gleason grade 810).
For immunohistochemistry (IHC) all specimens were
fixed in 10% neutral buffered formalin, paraffin
embedded and processed routinely; 4 mm thick serialsections were taken onto poly-L-lysine-coated slides. A
three-step immunoperoxidase method (described pre-
viously [22]) was used to detect the expression of COX.
Briefly, sections were de-waxed in xylene, hydrated
through graded alcohols and water, and immersed in
0.3% v/v H2O2 in distilled water for 30 min to block
endogenous peroxidases. Antigens were retrieved by
microwaving at 750 W for 15 min in 0.01 mol/L
trisodium citrate buffer (pH 6.0). Sections were rinsed
well in standard PBS (pH 7.27.4) and nonspecific
binding sites blocked with 10% normal rabbit serum
(Dako, Denmark) for 30 min. Sections were incubated
with COX-1 (160110) or COX-2 (160112) mouse mAb
(Cayman Chemicals, Ann Arbor, MI) at a dilution of
1 : 300 and 1 : 200 in PBS, respectively, for 16 h at 4uC.After rinsing with PBS, sections were incubated with
biotinylated rabbit antimouse immunoglobulins (Dako)
at a dilution of 1 : 200 in PBS for 45 min. Sections were
rinsed with PBS and incubated with avidin-biotin
horseradish peroxidase complex solution (Dako) for
30 min, rinsed with PBS and immersed for 510 min
in a peroxidase substrate solution containing 0.05% w/v
3,3k-diaminobenzidine (Sigma Chemical Co., Poole, UK)and 0.02% v/v H2O2 in PBS. Sections were counter-
stained with Coles haematoxylin (Pioneer Research
Chemicals, UK), dehydrated, cleared, and mounted in
mounting medium. Normal colon sections known to
express COX-1 and COX-2 were used as positive controls,
while for negative controls the primary antibody was
replaced with PBS. The immunostaining was evaluated
independently by two authors (S.M. and E-N.L.) analys-
ing the intensity, distribution and pattern of immuno-
staining. If there was a discrepancy, a consensus was
reached after further evaluation. The intensity of
immunostaining was graded as 0 (negative), 1 (weak),
2 (moderate) and 3 (strong).
Western blotting was used on 13 fresh-frozen samples
(six BPH and seven prostate cancers); 30 sections (15 mmthick) were cut from each sample using a cryostat (at
x25uC) and placed in pre-chilled Eppendorf microfugetubes. The tubes were transferred to ice and 300 mL oflysis buffer (715 mol/L 2-mercaptoethanol, 10% glycerol,
2% SDS, 40 mmol/L Tris pH 6.8, 1 mmol/L EDTA)
containing a cocktail of protease inhibitors (Boehringer
Mannheim, UK) was added to each tube. After 20 min
the lysates were centrifuged at 13 000 rpm at 4uC for5 min. The supernatants were transferred to clean
microfuge tubes and the protein concentration deter-
mined using a protein assay reagent (Bio-Rad
Laboratories, Hercules, CA) and Ultraspec III spectro-
photometer (Pharmacia Biotech, UK). About 30 mg oftotal protein from each sample was subjected to SDS-
PAGE, the proteins transferred to nitrocellulose mem-
branes (Millipore, UK), and the membranes blocked with
5% non-fat milk (Marvel, Cadbury Schweppes, UK) in
Tris-buffered saline solution containing 0.5% Tween 20
(TBST) for 1 h at room temperature. The blots were then
probed overnight with COX-1 or COX-2 mouse mAb
(Cayman Chemicals) at a dilution of 1 : 1000, washed in
TBST for 1 h with a change of buffer every 10 min, and
probed with horseradish peroxidase-conjugated rabbit
antimouse immunoglobulins (Dako) at a dilution of
1 : 1000. After another wash in TBST for 2 h with a
change of buffer every 15 min, the blots were placed in
enhanced chemiluminescence solution (ECL, Amersham,
UK) and exposed to X-ray film (Hyperfilm, Amersham).
The relative expressions of the different proteins were
COX-1 AND -2 EXPRESSION IN PROSTATE 737
# 2000 BJU International 86, 736741
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measured using a densitometer (Molecular Dynamics,
UK).
Fishers exact test was used to compare the intensity
and pattern of expression of COX-1 and COX-2 between
benign and malignant samples, and among the various
grades of malignancy, with P
-
and prostate cancer. The results from six samples (three
BPH and three prostate cancers) are shown in Fig. 2.
Discussion
This study showed that COX-1 and COX-2 are differen-
tially expressed in benign prostates, and that the
expression of COX-2 differed significantly between BPH
and prostate cancer samples. COX-2 was expressed in
luminal glandular epithelial cells of BPH and in the
neoplastic epithelial cells of prostate cancers. However,
the level of expression in the epithelial cells of prostate
cancers was significantly higher than that in BPH. The
results of IHC were supported by immunoblotting. The
intracellular localization of COX-2 in the epithelial cells of
BPH and prostate cancer also differed. While in BPH it
was mainly localized at the basal and basolateral cell
membrane, in prostate cancers it was predominantly
cytoplasmic. In contrast, COX-1 protein expression was
not significantly different between BPH and prostate
cancer; in both tissues staining was primarily stromal,
with variable weak cytoplasmic expression in the
epithelial component. The expression of COX-1 on IHC
did not vary (P=0.72), while COX-2 expressionincreased with grade (P
-
cell adhesion [26], over-expression of matrix-metallo-
proteinase 2 with an associated increase in invasiveness
[28], and modulated production of angiogenic factors by
cancer cells [29]. COX-2 over-expression in cancer cells
has also been shown to inhibit immune surveillance [30]
and increase metastatic potential [28]. Furthermore,
COXs may play a role in the bioactivation of several
polycyclic aromatic hydrocarbons and aromatic amines,
two classes of carcinogens which induce extrahepatic
neoplasia [31].
To our knowledge the present is the first study
analysing the expression of COX-1 and COX-2 proteins
in benign and malignant human prostates. Given that
the actions of COX-2 induce features of the malignant
phenotype, there is a powerful argument that COX-2
should be evaluated further as a promising therapeutic
target, both in prostate cancer and in other cancers.
Acknowledgements
This work was partly funded by a grant from The Friends
of Hammersmith Hospital. We thank Dr R.W. Stirling,
Consultant Histopathologist, West Middlesex University
Hospital, London for providing us with some of the
prostate cancer samples. We also thank Prof. A. Wanji,
University of Hong Kong for reviewing the manuscript
and constructive suggestions.
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AuthorsS. Madaan, MS, FRCS, Clinical Research Fellow.
P.D. Abel, ChM, FRCS, Reader in Urology and Honorary
Consultant.
K.S. Chaudhary, PhD, Senior House Officer.
R. Hewitt, PhD, Clinical Scientist.
M.A. Stott, FRCS, Consultant Urologist.
G.W.H. Stamp, FRCPath, Chairman and Professor of
Histopathology.
E.N. Lalani, PhD, MRCPath, Reader in Molecular & Cellular
Pathology and Honorary Consultant Histopathologist.
Correspondence: Dr El-Nasir Lalani, Department of
Histopathology, Division of Investigative Sciences, Imperial
College School of Medicine, Hammersmith Hospital Campus,
Du Cane Road, London W12 0NN, UK.
e-mail: [email protected]
COX-1 AND -2 EXPRESSION IN PROSTATE 741
# 2000 BJU International 86, 736741