5-Lipoxygenase expression in benign and malignant canine prostate tissues ∗ Original Article

Original Article
DOI: 10.1111/j.1476-5829.2010.00245.x
5-Lipoxygenase expression in benign
and malignant canine prostate tissues∗
L. A. Goodman1 , C. L. Jarrett2 , T. M. Krunkosky2 , S. C. Budsberg1 ,
N. C. Northrup1 , C. F. Saba1 and B. E. LeRoy3
1 Department
of Small Animal Medicine and Surgery, University of Georgia College of Veterinary Medicine,
Athens, GA, USA
2 Department of Anatomy and Radiology, University of Georgia College of Veterinary Medicine, Athens,
GA, USA
3
Department of Pathology, University of Georgia College of Veterinary Medicine, Athens, GA, USA
Abstract
Keywords
carcinoma, dog,
hyperplasia,
immunohistochemistry,
leukotriene, prostatitis
5-Lipoxygenase (5-LO) is overexpressed in human prostate carcinomas (PCs), and its inhibition
decreases proliferation and induces apoptosis in prostate cancer cell lines. We hypothesized that
5-LO would be overexpressed in canine PC compared with benign prostate tissue and may be
important in the pathogenesis of the disease. Immunoblot analysis of canine PC and benign prostatic
hyperplasia (BPH) tissues demonstrated 5-LO expression in both. 5-LO immunohistochemical
staining was not significantly different within the stromal or epithelial components of canine primary
PC, BPH or suppurative prostatitis, suggesting that differential expression of this enzyme does not
occur in these conditions. The percentage of tumour cells expressing 5-LO was significantly lower in
metastatic PC lesions compared with primary PC (P < 0.0001). This decreased expression may
indicate down-regulation or altered expression of the enzyme with progression of canine PC to a
metastatic phenotype.
Introduction
Correspondence address:
N. C. Northrup
Department of Small
Animal Medicine and
Surgery
University of Georgia
College of Veterinary
Medicine
501 D.W. Brooks Dr.
Athens
GA 30602, USA
e-mail: [email protected]
Leukotrienes are pro-inflammatory lipid mediators
derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism. Produced
primarily by inflammatory cells such as neutrophils, macrophages and mast cells, leukotrienes
have been implicated in the pathogenesis of several inflammatory conditions in people including osteoarthritis,1 atopic dermatitis2 and allergic
asthma.3,4 In addition, they are thought to play
a role in atherosclerosis5 and brain ageing.6 Currently, the most recognized clinical indication for
leukotriene inhibition in human medicine is for
treatment of allergic asthma.7
∗ An
abstract was presented at the 29th Annual Conference
of the Veterinary Cancer Society, Austin, TX, USA, 16–19
October 2009.
© 2010 Blackwell Publishing Ltd
Recently, the 5-LO pathway has been recognized
as a potential new pharmacologic target in cancer
therapy as research indicates that it may play an
important role in the pathogenesis of cancer. A
comprehensive review of the cyclooxygenase and
lipoxygenase pathways and their roles in cancer is
available elsewhere.8 5-LO overexpression has been
identified in a number of human cancers, including colonic,9,10 breast,11 pancreatic,12 bladder,13
oesophageal,14 oral,15 renal16 and brain.17 Human
prostate carcinoma (PC) has been shown to overexpress 5-LO compared with benign prostatic
hyperplasia (BPH) and normal prostatic tissue.18,19
In addition, when compared with benign prostate
tissues, human PC overexpresses leukotriene receptors and has an increased concentration of 5-LO
metabolic byproducts.19 – 21 Cell culture studies
involving a variety of cancer types, including PC,
1
2
L. A. Goodman et al.
demonstrate that leukotriene inhibition through
receptor antagonism or 5-LO enzyme inhibition
suppresses cancer cell proliferation and induces
apoptosis.13,14,18,22 – 25 Postulated mechanisms for
the anticancer activity of leukotriene inhibition
and induction of apoptosis include inhibition of
angiogenesis by decreasing vascular endothelial
growth factor expression,23 abrogation of 5-LOmediated resistance to anoikis26 and stimulation of
apoptosis through cytochrome c release, caspase9 activation22 and alterations in the Bcl-2/Bax
ratio.25 Taken together, current studies suggest
that the 5-LO pathway plays a role in malignant
transformation of cells and that inhibition of this
pathway represents a potential target for anticancer
therapy.
Canine PC may serve as a spontaneous animal
model for studying androgen refractory, poorly
differentiated PC in men.27 – 29 Dogs are the only
large mammals other than humans to develop a
significant number of spontaneous PC, and as
in humans, PC in dogs are highly metastatic
with a propensity to metastasize to the lumbar
vertebrae and pelvis.28 Both human and canine PC
overexpress cyclooxygenase-2 (COX-2).30 Based on
observed similarities between human and canine PC
and experimental data describing 5-LO in human
PC tissues and cell lines, we speculated that 5-LO
would play a role in the pathogenesis of canine PC.
At the present time there are no reports describing
5-LO in canine neoplasms. Therefore, the primary
aim of this study was to compare 5-LO expression
in neoplastic and benign canine prostate tissue.
A secondary aim was the comparison of 5-LO
expression in canine PC metastases and primary PC.
We hypothesized that canine PC would overexpress
5-LO compared with benign tissue samples and that
expression of 5-LO by metastases would be similar
to primary tumours.
Materials and methods
Western blot analysis
Frozen prostate tissue samples stored at −80 ◦ C
were evaluated from three dogs with PC and
from seven intact, young adult dogs with BPH.
Samples were verified as PC or BPH following
histopathologic evaluation by a single pathologist
(B. L.). A buffy coat preparation stored at −80 ◦ C
from a clinically normal dog was used as a positive
control. A human recombinant hexahistidinetagged protein (Cayman Chemical, Ann Arbor,
MI, USA) was used as a second positive control.
Four hundred micrograms of each tissue sample
was minced using a razor blade and added to 1.0 mL
of a Western lysis buffer (50 mM Tris–HCl pH 7.4,
150 mM NaCl, 1% Triton X-100, 1 mM EGTA,
6 mM sodium deoxycholate, 1 mM Na3 VO4 , 1 mM
NaF, 20 μg mL−1 aprotinin, 20 μg mL−1 leupeptin
and 1 mM PMSF). To serve as the positive control,
a 250 μL aliquot of concentrated canine leukocytes
was thawed and added to 250 μL of a Western
lysis buffer. All samples were homogenized at 4 ◦ C
with a pellet pestle, ultrasonicated up to four times
and centrifuged at 20 000 g for 5 min at 4 ◦ C. The
supernatant was removed and was frozen in aliquots
at −80 ◦ C to avoid multiple freeze–thaw cycles.
Total protein concentrations were determined
utilizing a commercially available Bradford total
protein assay (Bio-Rad, Hercules, CA, USA). Active
colour changes of a dye reagent were measured
by spectrophotometry at a 595-nm wavelength
and analysed by a computer program (Softmax
Pro, Bio-Rad, Richmond, CA, USA). Sample total
protein concentrations were determined as μg μL−1
concentrations.
Ten micrograms of the canine leukocyte sample and 40 μg of the BPH or PC sample were
equilibrated with a Western lysis buffer containing protease inhibitors and 2× sample buffer. Each
prepared sample or the human recombinant 5-LO
Western blot positive control was loaded onto a
10% SDS–polyacrylamide gel. The gel was electrophoresed and proteins were transferred to a
nitrocellulose membrane. After blocking with a
5% dry non-fat milk solution, the membrane was
incubated overnight at 4 ◦ C with either a rabbit
polyclonal 5-LO primary antibody (1:250 concentration; Abcam, Cambridge, MA, USA) or the
primary antibody (1:250 concentration) after it
had been incubated with a 5-LO blocking peptide
(Cayman Chemical) in a 1:1 ratio to test for binding
specificity. Although the human recombinant 5-LO
primary antibody, 5-LO Western positive control
and 5-LO blocking peptide were obtained from
different manufacturers, they were all generated
© 2010 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, doi: 10.1111/j.1476-5829.2010.00245.x
5-LO expression in benign and malignant canine prostate tissues 3
using an identical 5-LO protein sequence. Following
primary antibody incubations, blots were then
incubated with horseradish-peroxidase-conjugated
secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and treated with SuperSignal West
Dura extended duration chemiluminescence substrate solution (Pierce Biotechnology). Blots were
analysed using a Fluor-S Max2 (Bio-Rad, Richmond, CA) MultiImager system and associated
software.
Immunohistochemical analysis
Formalin-fixed paraffin-embedded samples of
canine primary PC, metastatic PC, BPH and
suppurative prostatitis lesions were identified by
searching the University of Georgia College of
Veterinary Medicine Athens Diagnostic Laboratory
database from January 2000 to December 2008
or were obtained from the University of Georgia
College of Veterinary Medicine Animal Cancer
Tissue Repository. Samples were included in the
study if they were obtained by surgical biopsy
or necropsy. Tru-cut biopsy samples and small
tissue samples were excluded as a lesion could have
been missed because of the sampling technique.
Cases were included as primary PC only if the
prostate was the primary organ affected. Cases in
which the primary tumour location could not be
distinguished as a result of both prostate and urinary
bladder invasions were excluded. The samples were
histologically classified as PC, BPH or suppurative
prostatitis by one veterinary pathologist (B. L.).
Tissue sections (3 μm) were mounted onto
Superfrost Plus slides (Fisher Scientific, Pittsburgh,
PA, USA). Additionally, positive controls (canine
leukocyte cell pellet) were sectioned and mounted
onto the slides. Sections were deparaffinized in
xylene and rehydrated in a graded alcohol series,
ending in tap water. Heat-induced epitope retrieval
was performed by submerging the slides in 0.01 M
sodium citrate buffer, pH 6.0 in a polypropylene
coplin jar and heating in a tabletop autoclave
to 121 ◦ C. Endogenous peroxidase was blocked
by submerging the slides for 20 min in 3%
hydrogen peroxide in tap water. Slides were dried
and sections were outlined with an ImmunoEdge
pen (Vector Laboratories, Burlingame, CA, USA).
The sections were rehydrated in tap water, and
blocking was performed for a minimum of 1 h
at room temperature, using 10% goat serum in
phosphate-buffered saline (PBS) pH 7.4 (Sigma, St
Louis, MO, USA). Primary antibody (rabbit anti-5LO, polyclonal, 0.004 μg μL−1 ; Abcam) diluted in
blocking serum was applied overnight at 4 ◦ C. The
following morning, sections were washed 3× in PBS
with 0.05% Tween 20 (Sigma) (PBST). Secondary
antibody (biotinylated goat anti-rabbit; Vector
Laboratories) was diluted in blocking serum and
applied for 30 min at room temperature. Sections
were washed 3× with PBST. Avidin-conjugated
peroxidase (Neutra-Avidin; Pierce Biotechnology)
was diluted to 5 μg mL−1 in PBS and applied for
1 h at room temperature. Sections were washed 3×
with PBST. A DAB peroxidase substrate kit was
applied as directed by the manufacturer (Vector
Laboratories). Nuclei were stained with Mayer’s
haematoxylin (Sigma). Slides were dehydrated in a
graded alcohol series ending in xylene. Slides were
permanently coverglassed. A purified rabbit IgG
(Santa Cruz Biotechnology, Santa Cruz, CA, USA),
applied at the same protein concentration as the
primary antibody, was utilized as an isotype control
to confirm that antibody binding was not because
of non-specific interactions.
5-LO staining was evaluated semi-quantitatively
by light microscopy and all immunohistochemical
grading was performed by one investigator (B. L.).
Epithelial cells and stromal cells within each sample
were graded separately for stain distribution and
stain intensity using a modification of a previously
used grading scheme.31 Stain distribution grades
were based on the percentage of cells that stained
positive for 5-LO within the sample (Table 1). Stain
intensity grades were determined by estimating the
average amount of stain uptake of the positive cells
within a sample (Table 1, Fig. 1).
Statistical analysis for immunohistochemical
grading
The Shapiro–Wilk W test was used to evaluate immunohistochemical grade distributions for
departures from normality for each of the four
sample groups. Histograms were also examined.
Because most distributions were found to be
© 2010 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, doi: 10.1111/j.1476-5829.2010.00245.x
4
L. A. Goodman et al.
Figure 1. Examples of 5-LO immunohistochemical staining intensity grades 1 (A, mild), 2 (B, moderate) and 3 (C, marked)
in canine primary PC. All photomicrographs are at ×400 magnification.
Table 1. Semi-quantitative grading schemes used to score
5-LO immunohistochemical staining in canine prostate
tissues
Stain distribution grades
Stain intensity grades
0 = No cells stain positive
1 = 1–33% of cells stain
positive
2 = 34–66% of cells stain
positive
3 = 67–100% of cells
stain positive
0 = No cells stain positive
1 = Mild intensity
2 = Moderate intensity
3 = Marked intensity
significantly non-normal, a Kruskal–Wallis test
was used to test for differences in staining distribution and intensity levels between each of
the four sample groups. Multiple comparisons
were adjusted for using Dunn’s test. All hypothesis tests were two-sided and the significance level
was α = 0.05. All statistical analyses except for the
Dunn’s test were performed using SAS V 9.2 (Cary,
NC, USA). The Kruskal–Wallis test was performed
using PROC NPAR1WAY in SAS. Dunn’s test
© 2010 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, doi: 10.1111/j.1476-5829.2010.00245.x
5-LO expression in benign and malignant canine prostate tissues 5
was calculated manually using Microsoft Excel
(Redmond, WA, USA).
Results
Western blot
Western blotting identified 5-LO in canine leukocytes and prostate tissues. The histone-tagged 5-LO
human recombinant control formed a band at the
78 kDa molecular weight (Fig. 2). Although the
native protein is 74 kDa, the histone tag increased
the molecular weight of the control peptide in
the expected manner. Similar bands at the slightly
decreased molecular weight of 74 kDa, appropriate for 5-LO, were found in cell lysate samples
from canine leukocytes as well as canine BPH tissue. Pre-incubation of the 5-LO antibody with the
peptide blocker abrogated staining demonstrating
specificity for 5-LO.
Results from the Western blots demonstrated
that both canine PC tissue and canine BPH
tissue express 5-LO. A band was detected at the
appropriate molecular weight for all three PC
samples and six BPH samples (Fig. 3).
PC metastases 9.4 years (range 7–12; age known for
5/6 cases), BPH 10.1 years (range 6–16; age known
for 23/39 cases) and suppurative prostatitis 8.3 years
(range 5–11; age known for 4/5 cases). Each of the
6 metastatic lesions was from a different dog and
corresponding primary PC samples were available
for three of the six dogs. The sites of metastasis
included lung (3), lymph node (1), bone (1) and
brain (1).
Significant differences in 5-LO stain distribution
and intensity grades in epithelial and stromal cells
of BPH, prostatitis and primary PC (Figs 4 and 5)
were not identified. However, significant differences
were noted when comparing PC metastases to other
groups. 5-LO stain distribution grades in epithelial
cells were significantly lower in PC metastases
compared with primary PC and BPH (P < 0.0001),
and stain intensity grades were significantly lower in
PC metastases compared with BPH (P = 0.0095).
Immunohistochemistry
Samples evaluated included 19 primary PC, 6 PC
metastases, 39 BPH and 5 suppurative prostatitis
samples. The age was known for 57% of the
dogs from which samples were obtained. Mean
ages of dogs in each category follow: primary PC
8.2 years (range 6–10; age known for 7/19 cases);
Figure 2. Western blot validating the use of the 5-LO
antibody in canine tissues. Lane 1 = His-tagged positive
control; Lane 2 = Canine leukocytes; Lane 3 = BPH tissue;
Lane 4 = His-tagged positive control with peptide blocker;
Lane 5 = Canine leukocytes with peptide blocker and
Lane 6 = BPH tissue with peptide blocker.
Figure 3. Western blot results demonstrating 5-LO
expression in canine PC tissue and canine BPH tissue.
Lane 1 = His-tagged positive control; Lanes 2–4 = PC and
Lanes 5–10 = BPH tissue.
Figure 4. Mean grades (±SE) for 5-LO immuno-
histochemical stain distribution (A) and intensity (B) for the
epithelial component of 63 canine prostate tissues and 6 PC
metastatic lesions.
© 2010 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, doi: 10.1111/j.1476-5829.2010.00245.x
6
L. A. Goodman et al.
Figure 5. Mean grades (±SE) for 5-LO
immunohistochemical stain distribution (A) and intensity
(B) for the stromal component of 63 canine prostate tissues
and 6 PC metastatic lesions.
No differences were noted in stain distribution
or intensity grades of stromal cells in any of the
samples evaluated.
Discussion
Contrary to our hypothesis, our results suggest
that 5-LO is not overexpressed in canine PC compared with benign prostate tissue. The immunohistochemical staining intensity and distribution
patterns in the epithelial and stromal components
were not significantly different between primary
PC, BPH and prostatitis samples. This indicates that
there is no differential expression of the enzyme in
these disease processes as assessed by our study
methodology.
Our findings in canine tissue are dissimilar
to those of human immunohistochemical studies
which show overexpression of 5-LO in PC compared with benign tissue.18,19 In humans, PC has
been shown to exhibit moderate to strong 5-LO
staining, similar to the canine PC staining results
reported here. The primary difference appears to be
the degree of 5-LO expression in human and canine
BPH tissue. Studies of BPH in men show weak 5-LO
staining whereas our study shows strong staining in
canine BPH tissue. The reason for this discrepancy
is unknown but may indicate a species-specific difference between the canine and human hyperplastic
prostate gland. Currently, no immunohistochemical studies have evaluated 5-LO expression in
human patients with suppurative prostatitis and
therefore a comparison between species could not
be made with regard to this disease process.
Another finding in this study was that 5-LO
was expressed in a lower percentage of epithelial
cells in PC metastases compared with primary PC
and benign prostatic tissues. This could be due
to loss of tissue differentiation in the metastases
resulting in decreased or loss of expression. Another
possibility is that in the development of a metastatic
phenotype, mutations occurred that altered the
amino acid sequence of the expressed protein,
decreasing the ability of the primary antibody
to recognize the epitope. Discrepancies in posttranscriptional enzyme modification may also have
occurred in the metastases that affected epitope
recognition by the antibody. It is also possible
that 5-LO expression down-regulation conferred an
adaptive advantage to a clone of transformed cells.
While the current study was not designed to
explore the association between inflammation and
5-LO expression, it is interesting to note that suppurative prostatitis 5-LO expression grades were not
significantly different from those of BPH or primary
PC, despite a higher degree of tissue inflammation.
The finding that 5-LO expression may not be related
to degree of inflammation is similar to results from
a recent study on COX-2 expression.32 In that
study, although prostate tumours without inflammation had a significantly higher COX-2 expression
than tumours with inflammation, the degree of PC
inflammation was not significantly associated with
the degree of COX-2 expression.
The results presented here must be interpreted
with caution given the small sample sizes and
© 2010 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, doi: 10.1111/j.1476-5829.2010.00245.x
5-LO expression in benign and malignant canine prostate tissues 7
the potential for type-II error. Also, competing
variables may have influenced 5-LO expression.
For example, although not yet evaluated in the
prostate, expression of 5-LO has been shown
to increase with age in CNS and cardiovascular
tissues.33 Because the ages of many dogs in this study
were unknown, an evaluation of the association
between 5-LO expression and age was not valid.
Therefore, it is possible that age-related differences
in 5-LO expression existed between the groups
and this could confound our results. In addition,
direct comparison between 5-LO expression in
primary PC versus PC metastasis within the same
individual could be made in only three cases. In
all three cases, the 5-LO stain distribution and
intensity in epithelial cells were lower in the
metastasis compared with the primary tumour.
Further comparison of 5-LO expression in a
larger number of primary versus metastatic PC
within the same individual would be necessary
to determine whether 5-LO expression is truly
decreased in metastatic lesions. Finally, although
multiple histomorphological types of canine PC
have been defined and may have been included in
our study, comparison of 5-LO expression between
types was beyond the scope of this study.27
To better assess the role of 5-LO in canine PC,
enzyme activity could be evaluated and compared
between benign and transformed tissues. The
location of the enzyme within the cell may have
a profound impact on its leukotriene synthetic
capacity, as has been shown in inflammatory cells.34
In non-activated leukocytes, 5-LO is a soluble
enzyme within the cytoplasm or nucleoplasm
that translocates to a membrane upon leukocyte
stimulation. At the membrane, 5-LO comes in close
contact with its arachidonic acid substrate as well
as other proteins that greatly enhance its activity.35
Given this, the leukotriene synthetic capacity of
a cell is determined by not only the amount of
enzyme expressed but also by its location within
the cell. Therefore, despite similar expression,
enzyme activity may have been markedly different
between groups. Assessment of enzyme activity
was not a goal of this study but measurement
and comparison of 5-LO metabolic byproducts
within sample groups would be a future study
direction.
In conclusion, this study suggests that 5-LO is
expressed similarly in neoplastic, hyperplastic and
inflamed canine prostate tissue. Interestingly, 5-LO
was expressed in a lower percentage of epithelial
cells in PC metastases compared with primary PC.
Although this finding suggests a possible alteration
in 5-LO transcription or translation associated
with the development of a metastatic phenotype,
it must be interpreted with caution given our
small sample size. Further characterization of the
metabolic activity of 5-LO in canine PC would
be helpful to fully assess its relevance or lack
thereof in the pathogenesis of this disease. Given
the overexpression of 5-LO in a variety of human
cancers, as well as the decreased proliferation
and increased apoptosis noted with leukotriene
inhibition in human cancer cell culture studies,
evaluation of the role of 5-LO in other canine and
feline cancers remains of interest.
Acknowledgments
This work was conducted at the University of
Georgia College of Veterinary Medicine and was
funded internally and through Georgia CaRES
(Cancer Research, Education and Service) for Pets
Fund. We would like to acknowledge Deborah Keys,
PhD, for her assistance with the statistical analysis.
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