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

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: e.lalani@ic.ac.uk

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