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Regular Articles |
From the Department of Pathology,*
Division of Clinical
Pharmacology, Department of Pharmacology,
and
Department of Urology,
Vanderbilt University
School of Medicine, Nashville, Tennessee
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to structural differences, 15-LOX-2 exhibits differences in certain enzymatic characteristics. In contrast to the reticulocyte type of 15S-lipoxygenase (15-LOX-1), 15-LOX-2 converts AA exclusively to 15S-hydroperoxyeicosatetraenoic acid (15-HPETE); this hydroperoxide is reduced by cellular peroxidases to 15S-hydroxyeicosatetraenoic acid (15-HETE). Also unlike 15-LOX-1, this newly characterized enzyme metabolizes linoleic acid poorly compared with AA.8 The 15-LOX-2 cDNA was cloned from human hair roots.8 In addition, 15-LOX-2 mRNA was detected in a limited number of tissues, including prostate, lung, and cornea. In contrast to the distribution of 15-LOX-1, 15-LOX-2 was not detected in peripheral blood leukocytes, nor was it detected in liver, kidney, spleen, thymus, testis, ovary, skeletal muscle, heart, brain, or intestinal tissue.8
The limited tissue distribution of 15-LOX-2 suggests a possible role in regulation of organ-specific functions or differentiation or possible alterations in disease states. In continuing efforts to elucidate potentially important molecular alterations in prostate cancer development or progression, a limited number of recent investigations using both human tissues and prostate carcinoma cell lines have begun focusing on AA-metabolizing enzymes, including cyclooxygenase,9 12-LOX,10 5-LOX,11 and 15-LOX-1.12 In the current study, we have characterized the expression of 15-LOX-2 in benign prostate by immunohistochemistry, demonstrated the ability of benign prostate to form the enzymatic product of 15-LOX-2 (15-HETE) from AA, and begun exploring the possible altered expression of 15-LOX-2 and 15-HETE formation in prostate adenocarcinoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh tissue was procured from radical prostatectomy or cystoprostatectomy specimens accessioned in the Surgical Pathology Laboratory at the Vanderbilt University Medical Center. For prostatectomies, the external aspects (surgical margins) were inked according to standard protocols and whole cross sections (perpendicular to the apex-base axis) were made. Based on gross examination, possible benign and tumor regions were separately excised in such a manner as to not compromise surgical pathological evaluation of the specimen. Fresh tissues were placed in liquid nitrogen or immediately placed in a -80°C freezer. Before utilization in enzyme assays, thin sections parallel to the longest axis of the tissue were removed, and frozen or routine formalin-fixed paraffin-embedded H&E-stained sections were prepared. The samples were assessed as to percentage of benign glandular, stomal, and adenocarcinoma tissue present. Although numerous samples of benign tissue were thus generated, owing to the known difficulty of recognizing prostate carcinoma grossly, only a subset of presumably malignant tissues so procured turned out to be 100% tumor. For enzyme assays, frozen normal tissue samples and pairs of pure benign glands and pure carcinoma admixed with stroma were used according to availability.
Whole-mount sections were processed for histology and subjected to standard surgical pathology evaluation. Cases that were used for AA incubations were also used for 15-LOX-2 immunohistochemistry. Additional random cases were selected to give a representative mixture of tumor grades with various combinations of organ-confined disease and extra-capsular extension (ECE) with or without seminal vesicle (SV) and margin involvement: five cases of Gleason grade 5 or 6 without ECE or SV involvement, three cases of Gleason grade 6 with ECE or SV involvement, four cases of Gleason grade 7 without ECE or SV involvement, three cases of Gleason grade 7 with ECE or SV involvement, and three cases of Gleason grade 8 to 10 with ECE or SV involvement. Numerous foci of well differentiated carcinoma (Gleason patterns 1 and 2) were also included (generally incidental in transition zone).
Preparation of 15-LOX-2 for Antibody Production
cDNA encoding the open reading frame of 15-LOX-2 was cloned into the pET3a vector and expressed in phage-induced Escherichia coli (HMS174 cells). Ten milliliters of overnight culture of HMS174 cells transformed with pET3a/15-LOX-2 in LB medium containing 50 µg/ml ampicillin was used to inoculate a fresh 500-ml culture of LB/ampicillin. Incubation was continued at 37°C until the absorbance at 600 nm reached 0.9 A. One hundred milliliters of phage culture (CE6, expressing T7 RNA polymerase) was then added together with 5 ml of 1 mol/L MgSO4, and incubation was continued for 3 hours at 37°C. Under these incubation conditions, overexpression of the 15-LOX-2 protein gave a catalytically inactive lipoxygenase, comprising 25% to 50% of the cellular protein. The bacterial pellets were collected by centrifugation, washed with phosphate-buffered saline (PBS), and resuspended in 40 ml of PBS, and the cells were disrupted by sonication. The insoluble fraction containing the majority of the lipoxygenase protein was collected by centrifugation and washed once with 1.5 mol/L NaCl followed by a wash with 1% Triton X-100 in PBS. The pellet was again washed in PBS, dissolved in 8 ml of loading buffer, and subjected to semipreparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% polyacrylamide gels with 4% stacking gels. The clearly visible band of 15-LOX-2 protein was cut from Coomassie-blue-stained gels, placed in a dialysis bag, and electroeluted. The protein was lyophilized, and an aliquot was quantified by its appearance on SDS-PAGE using bovine serum albumin as standard.
Preparation and Characterization of Polyclonal Antibody to 15-LOX-2
Rabbits were injected at six sites of back skin using an initial 0.1 mg of 15-LOX-2 per animal in 1:1 PBS/Freund's complete adjuvant. A booster injection of 0.1 mg using PBS/Freund's incomplete adjuvant was given 19 days later, and blood serum was collected beginning 14 days later. High-titer bleeds from a single rabbit were used for the Western analyses and immunohistochemistry reported herein.
The specificity of the rabbit antibody for 15-LOX-2 and possible cross-reactivity to other human lipoxygenases was examined by Western blotting using five human lipoxygenases. 15-LOX-1, 15-LOX-2, and 12R-LOX13 were prepared by bacterial expression, each including an amino-terminal histidine tag. The proteins were recovered using a nickel affinity column (Qiagen, Chatsworth, CA) essentially according to the manufacturer's instructions. Purified human 5-lipoxygenase was a generous gift from Dr. Denis Reindeau (Merck-Frosst). Aliquots of these four lipoxygenase proteins were run on SDS-PAGE, and after staining with Ponceau S, the samples were quantified by visual inspection relative to dilutions of a standard of bovine serum albumin. Aliquots of 50 or 5 ng were used for Western analysis. A partially purified preparation of human platelet 12-lipoxygenase from Oxford Biomedical Research (Oxford, MI) was used as a source of human 12S-LOX (platelet 12-LOX). For Western blots employing 5 ng of purified lipoxygenases, 8 µg of protein of the partially purified 12S-LOX was used, which, assuming the 12-LOX comprises at least 0.1% of the extract, corresponds to at least 8 ng of 12S-LOX protein. Adequate protein loading of this sample was further confirmed by staining of gels and visual inspection. Proteins were separated by 12% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes using standard conditions.
The Western analyses were carried out using 15-LOX-2 polyclonal antibody (1:6000) and an alkaline-phosphatase-conjugated affinity-purified donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA; 1:7500 in bovine serum albumin/Tris-buffered saline/0.1% Tween-20) and Tris-buffered levamisole (Sigma Chemical Co., St. Louis, MO) followed by nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate for development.14
Immunohistochemistry and Quantitation of 15-LOX-2 Immunostaining
Whole-mount sections that contained adequate benign tissue and tumor typical of the overall specimen were selected. In general, both right and left halves of the respective whole-mount sections were recut onto separate immuno-slides for subsequent immunohistochemical staining. In some cases, multiple whole-mount blocks were used for immunostaining. Paraffin immunoperoxidase studies were performed using a 1:2500 dilution of either primary rabbit antisera for 15-LOX-2 or preimmune sera on an automated immunostainer (Ventana 320 automated immunohistochemistry system, Ventana Medical Systems, Tucson, AZ) with an avidin-biotin complex method without antigen retrieval techniques.15 15-LOX-2 immunostaining in benign prostate tissue was uniform and is expressed descriptively or semiquantitatively as 0 to 4+: 0, no staining; 1+, focal weak staining; 2+, weak to moderate intensity staining in up to one-half of the cells in most glands or most cells in up to one-half of the glands; 3+, moderate to intense staining in greater than one-half of the cells or one-half of the glands but less than strong, uniform staining; 4+, strong uniform staining in essentially all cells. 15-LOX-2 immunostaining was quantitated in tumor portions by carefully mapping out with ink on the glass slides the areas of tumor that were positive and negative and then by making enlarged photocopies of such outlines. Areas were determined using a digitized graphics tablet (WACOM) and NIH Image Analysis software. Results are expressed as the percentage of tumor negative for 15-LOX-2 immunostaining.
Tissue Incubations and HPLC Analysis
A 50- to 100-mg amount of benign or tumorous prostate tissue was
homogenized in 4 vol of buffer (50 mmol/L Tris with 100 mmol/L NaCl and
100 µmol/L CaCl2, pH 7.4). Incubations were begun by
addition of 50 µmol/L [1-14C]arachidonic acid (final
concentration; New England Nuclear, Boston, MA) in 
1% (final v/v)
ethanol. Samples were incubated for 1 hour at 37°C with continual
agitation and terminated by addition of 2.5 vol of cold methanol. After
addition of 1.25 vol of dichloromethane, the sample was centrifuged to
remove the protein precipitate, and the products were recovered in the
mixed phase of methanol/water/dichloromethane. The sample was
evaporated under a stream of nitrogen to remove most of the
dichloromethane and methanol, water was added, and the products were
recovered by C18 Sep-Pak extraction.8
The extracts were
analyzed by reversed-phase HPLC using a Beckman Ultrasphere 5-µm ODS
column (25 x 0.46 cm) with a solvent of methanol/water/glacial
acetic acid, either 90:10:0.01 (by volume) at a flow rate of 1.1
ml/minute (retention time of 15-HETE was approximately 5 minutes) or
80:20:0.01 at 1.1 ml/minute (retention time of 15-HETE approximately 15
minutes). Unlabeled HETEs (5-, 8-, 9-, 11-, 12-, and 15-HETEs) were
added to each sample before HPLC analysis; this permitted an exact
determination of the retention times of each HETE product within each
individual chromatographic run. Ultraviolet (UV) spectra and the
profiles at 205, 220, 235, and 270 nm were recorded using a
Hewlett-Packard 1040A diode array detector, and radioactivity was
monitored on-line using a Radiomatic Instruments Flo-One detector.
Product formation in some incubations of benign and malignant prostate
tissue pairs was also analyzed in more detail using reverse-phase HPLC
with a solvent system of methanol/water/glacial acetic acid
(75:25:0.01, by volume) at a flow rate of 1.01 ml/minute (retention
time of 15-HETE approximately 31.5 minutes), with collection of
0.5-minute fractions and 10-minute scintillation counting of all
fractions. In some samples, the main radiolabeled peaks were further
characterized by normal-phase HPLC using a Beckman Ultrasphere 5-µm
silica column (25 x 0.46 cm) and a solvent of hexane/isopropyl
alcohol/glacial acetic acid 100:1:0.1 (by volume) at a flow rate of 1.1
ml/minute with UV detection using the diode array detector and on-line
radioactive detection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubations of benign prostate tissue homogenates with
[14C]AA resulted in the formation of 15-HETE in all cases
examined (n = 8; Figure 1
). Identity of this product was
demonstrated by its precise co-elution on reverse-phase HPLC (and on
straight-phase HPLC in selected samples) with co-injected unlabeled
15-HETE (see Materials and Methods). The addition of a mixture of
unlabeled HETEs to each sample allowed an exact definition of the
retention times within each chromatographic run. The identity of the
radiolabeled product as 15-HETE was further confirmed by the
characteristic UV spectrum of the mixture of [14C]15-HETE
and the co-injected authentic standard16
; the UV
absorbance of the 15-HETE peak was distinctly elevated by the
contribution from the [14C]15-HETE. The amount of 15-HETE
produced under these incubation conditions was variable. However, in
all cases, 15-HETE was the major product detected, with variably sized
peaks in the prostaglandin region (Figures 1 and 3)
. From analysis of
these eight samples of benign prostate, minor amounts of other
hydroxyeicosatetraenoates were occasionally detected; the most
prominent of these additional HETE peaks, present in only one sample,
was the one, tentatively identified as 12-HETE, eluting at 36 minutes
in Figure 3B
.
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The polyclonal antibody prepared against purified 15-LOX-2 was demonstrated to be specific for 15-LOX-2 and to not cross-react with other pertinent lipoxygenases by Western blotting. Whereas a strong immunoreactive band was obtained with purified 15-LOX-2 protein (5 ng), no immunoreactivity was seen with purified 15-LOX-1 or 5-LOX (5 or 50 ng) or a sample of partially purified 12S-LOX (not shown). Weak immunostaining was seen with a purified preparation of a recently identified 12R-LOX,13 which may be expected, given the greater percentage of identity between 15-LOX-2 and 12R-LOX (approximately 50%) than between 15-LOX-2 and 5-LOX, 12S-LOX, and 15-LOX-1 (approximately 40%).8,13 However, unlike 12S-LOX, which has been reported in prostate (see Discussion), 12R-LOX has not been identified in prostate. Hence, minimal cross-reactivity of a 15-LOX-2 antibody with a 12R-LOX is not an issue in prostate immunostaining, as benign prostate makes large amounts of 15-HETE (as reported herein), and prostate has not been observed to make 12R-HETE.
The cellular location of 15-LOX-2 was identified as ductal and acinar
epithelium by immunohistochemistry. Benign prostatic epithelium
consistently stained strongly and uniformly with the polyclonal
antiserum against human 15-LOX-2 by paraffin immunoperoxidase (Figure 2A)
. Stromal, vascular, and inflammatory
cells were uniformly negative (Figure 2B)
. The distribution of
immunostaining was carefully examined in the benign portions of
prostate sections from radical prostatectomies performed for prostate
adenocarcinoma. There was strong uniform staining of apical (secretory)
cells in peripheral zone glands, with essentially all cells staining in
all glands (Figure 2, A and C)
. The pattern of staining was
predominantly cytoplasmic granular, with frequent nuclear staining also
evident (Figure 2C)
. Occasional detached cells in glandular and ductal
lumens showed immunostaining, but corpora amylacea were negative. Basal
cells did not stain (Figure 2C)
; this lack of staining was evident also
in areas of basilar hyperplasia (not shown). Strong uniform 15-LOX-2
immunostaining was present in columnar secretory cells of large
periurethral ducts and secondary prostatic ducts (Figure 2, D and E)
.
In contrast, transitional epithelium of the prostatic urethra,
periurethral ducts, and areas of transitional metaplasia was completely
negative (Figure 2E)
.
|
. This slightly reduced 15-LOX-2 immunostaining
was noted in variably prominent nodules of transition zone glandular
hyperplasia (the histological correlate of benign prostatic
hyperplasia, or BPH), including in cystically dilated hyperplastic
glands. In general, staining was stronger in glands in the periphery of
such nodules, with lesser staining, including negative glands, in the
center of hyperplastic nodules. Compared with the completely uniform
strong staining in essentially all peripheral zone glands, benign
glands in the central zone (surrounding the ejaculatory ducts) also
showed slightly reduced intensity and frequency of 15-LOX-2
immunostaining (Figure 2G)
. Ejaculatory ducts and seminal vesicles were
uniformly negative (Figure 2H)
. Reduced and even absent immunostaining
was also noted in atrophic glands in the peripheral zone (Figure 2I)
,
with intermediate staining intensity occasionally noted in partially
atrophic glands. No immunostaining was detected in any glandular
elements (or any other tissue compartment) with negative preimmune
serum controls (n = 5; data not shown). In
addition, staining of benign prostate glands was eliminated by
preincubation of antisera with purified 15-LOX-2 protein8
(not shown). A summary of the semiquantitative 15-LOX-2
immunohistochemical staining in benign prostate is shown in Table 1
.
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15-LOX-2 activity was assessed in benign prostate tissue and
prostate adenocarcinoma frozen from the same radical prostatectomy
specimens. In all benign samples, incubation with [14C]AA
resulted in formation of 15-HETE as detected by reverse-phase HPLC
analysis. In contrast, markedly reduced or no 15-HETE production was
detected in simultaneously incubated samples of prostate adenocarcinoma
(n = 3; Figure 3
).
Reduced 15-LOX-2 in Prostate Adenocarcinoma by Immunohistochemistry
In contrast to the strong, uniform 15-LOX-2 immunostaining in
benign prostate glands (particularly in the peripheral zone), 15-LOX-2
immunostaining was markedly reduced in prostate adenocarcinoma (Figure 4)
. 15-LOX-2 immunostaining was at least
focally absent in essentially all tumors, with the vast majority
showing substantial portions of tumor that were negative on
immunostaining. In 14 of 18 cases, 15-LOX-2 was absent in more than
25% of the tumor examined by immunohistochemistry. In these cases,
15-LOX-2 immunostaining was negative in 74.9 ± 24.4% of the
tumor. 15-LOX-2 was absent in >50% of the tumor in 10 of 18 cases and
essentially completely absent in 8 of 18 of the tumors.
|
. In two cases of
overall Gleason 7 adenocarcinoma (one 3 + 4 and one 4 + 3), areas of
Gleason pattern 2 and 3 were 15-LOX-2 immunopositive, whereas other
areas of Gleason patterns 3 and 4 were negative, including in one case
areas where the tumor was penetrating beyond the prostate capsule
(Figure 4, E–G)
. These two cases were among those assessed by HPLC,
the tumor tissue for which was taken from the posterior peripheral zone
(see also Figure 5, A and B
). Lack of
15-HETE formation from this tumor tissue likely correlates with the
reduced 15-LOX-2 immunostaining in more peripheral (and higher-grade)
portions of the tumor. Another example of possible tumor heterogeneity
with regard to 15-LOX-2 expression was seen in a case with a mixed
ductal and acinar pattern. Whereas the more peripherally located acinar
carcinoma was largely 15-LOX-2 immunopositive, the more centrally
located ductal carcinoma was negative (Figure 4, H and I)
. Examples of
the degree of heterogeneity of loss of 15-LOX-2 immunostaining within
individual tumors are shown in Figure 5
, in which parts A–C correspond
to the tumor cases analyzed by HPLC (as shown in Figure 3
). Figure 5D
shows an extreme example of the multifocal or heterogeneous
nature of 15-LOX-2 negativity seen in some cases with only partial loss
of 15-LOX-2 immunostaining.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Characterization of the 15-lipoxygenase metabolism and its designation
as 15-LOX-2 is based on the following lines of evidence. 1) Previous
multitissue Northern blots demonstrated 15-LOX-2 mRNA expression in
benign prostate,8
and we have recently detected 15-LOX-2
mRNA in individual benign prostates on Northern Blots (S. B.
Shappell, unpublished observations). 2) Analysis of lipoxygenase
product formation by HPLC identifies the specific formation of 15-HETE
in all of our incubations of benign prostate. Typically, the
reticulocyte type of 15-lipoxygenase, 15-LOX-1, forms a mixture of
15-HETE and 12-HETE,17
whereas 15-LOX-2 oxygenates AA
solely at C-15.8
In our experiments the characteristics of
15-HETE formation matched the catalytic activities of 15-LOX-2 (eg,
Figure 1
and Figure 3, A and C
). The exception, when both 12- and
15-HETE formation was evident (Figure 3B)
, might be accounted for by
co-expression of 12-LOX and a 15-lipoxygenase. 3) An additional feature
of the 15-lipoxygenase metabolism in prostate, again characteristic of
15-LOX-2, was the greater formation of 15-HETE from AA than of
13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid in
side-by-side incubations of the same benign prostate tissue (not
shown); this correlates with the substrate specificity of
15-LOX-28
and contrasts with the known preference of
15-LOX-1 for linoleic over arachidonic acid.18
4) The
antibody used in the current immunohistochemical studies
specifically detects 15-LOX-2 and does not cross-react with
15- LOX-1,19
12S-LOX, or 5-LOX (results
herein). Furthermore, there was complete suppression of the positive
reactions in the immunohistochemical staining by pre-absorption with
purified 15-LOX-2 protein. The specificity of our antibody for 15-LOX-2
versus 15-LOX-1 was also supported by the staining of the
prostatic epithelium with concomitant lack of staining of inflammatory
cells, including macrophages, which might be expected to stain for
15-LOX-1.20
Although inflammation was variable and
generally mild in the sections examined, one benign prostate examined
by both enzyme assay and immunostaining was a cystoprostatectomy
specimen containing granulomatous inflammation secondary to previous
BCG instillation therapy for bladder transitional cell
carcinoma. In addition to formation of large amounts of 15-HETE on
incubation of this prostate tissue with AA, by immunohistochemistry,
the anti-15-LOX-2 antibody uniformly stained benign prostate epithelium
but did not stain the large numbers of histiocytes (macrophages)
forming the granulomas (not shown).
Few studies have examined the possible significance of AA metabolism or the presence of cyclooxygenase or lipoxygenase enzymes in benign prostate and prostatic neoplasia. Chaudry et al9 reported prostaglandin E2 (PGE2) synthesis to be 10-fold higher in carcinoma tissue compared with benign prostatic hyperplasia (BPH) tissue. Whether the enzymes responsible for PGE2 synthesis were epithelial, stromal, or vascular was not elucidated. Increased formation of PGE2 has been demonstrated previously in human cancer cell lines, including human prostate cancer cell lines.21
More recently, multiple reports have described the presence of
lipoxygenase enzymes in prostate cancer and prostatic carcinoma cell
lines. Gao et al10
reported that 12-lipoxygenase (12-LOX)
mRNA was elevated in 46 of 122 (38%) prostate tumor samples compared
with normal tissue from the same patient. They found that 12-LOX mRNA
was more likely to be elevated in tumor versus control
tissue in patients with extracapsular extension (T3), positive lymph
nodes, higher-grade lesions (Gleason score 
8), and positive
surgical margins. By in situ hybridization they detected
12-LOX mRNA within epithelial cells of the prostate, including basal
cells of normal prostate tissue. Secretory cells were generally
negative in normal tissue. This basal cell distribution of
12S-LOX in benign prostate tissue contrasts with the apical
cell distribution of 15-LOX-2 in benign prostate demonstrated by
immunohistochemistry herein. In contrast to benign glands, Gao et al
reported that tumor epithelial cells demonstrated heterogeneous and
occasionally marked expression of 12-LOX mRNA.10
Biological effects of 12-HETE include promotion of tumor cell adhesion
and endothelial cell contraction, indicating a potential contribution
to tumor cell metastasis2,22,23
and possible modulation of
tumor growth by induction of angiogenesis.24
12-LOX enzyme
activity has not been reported in actual prostate tissues or prostate
cancers, however. We did not observe detectable 12-HETE accumulation in
most incubations of benign prostate tissue with exogenous arachidonic
acid. We also did not observe 12-HETE formation in similar incubations
with prostate adenocarcinoma. However, although the cases examined for
15-LOX-2 immunostaining included multiple examples of high-grade
(Gleason 8) and T3 carcinoma, only one of the cases examined by
HPLC was positive for extracapsular extension (T3), and none had
positive surgical margins or lymph node metastases. Hence, the
possibility of detecting 12-HETE formation and possible simultaneous
decreased 15-HETE and increased 12-HETE formation under the
experimental conditions used will require examination of higher-grade
and higher-stage lesions.
A possible role for 5-LOX in modulation of growth of the prostate cancer cell line PC-3 was reported by Ghosh and Myers.11 Arachidonic acid stimulated PC-3 cell growth, which was inhibited by selective 5-LOX inhibitors but not by 12-LOX or cyclooxygenase inhibitors. Furthermore, 5-HETE was produced from exogenous AA in PC-3 cells.11 More recently, these investigators and others have demonstrated induction of apoptosis in PC-3 and LNCaP cell lines by inhibitors of 5-LOX and 5-LOX-activating protein (FLAP).25,26 Again, we did not observe 5-HETE production from exogenous AA in benign prostate or a limited number of fresh-frozen prostate tumors. Given the known chromosomal and other possible genetic abnormalities of established prostate carcinoma cell lines, whether AA metabolic pathways in these cultured cells generated from individual tumors reflect similar alterations in actual cancers in vivo will require correlative studies, with analysis of high-grade and high-stage cancers, perhaps including metastatic tumors.
Spindler et al12 recently provided evidence for a possible 15-lipoxygenase in the human prostate cancer cell lines LNCaP and PC-3, which they indicated as the same as the rabbit reticulocyte 15-lipoxygenase (designated herein as 15-LOX-1). These findings may require re-evaluation in light of the recent discovery of 15-LOX-2. However, based on the evidence we have obtained in human prostate cancer samples, transformed prostate carcinoma cell lines, such as LNCaP and PC-3 cells, would not be expected to express 15-LOX-2. The reported detection of 13-HODE in a single specimen of human prostate carcinoma by frozen-section immunohistochemistry (and its absence in surrounding benign epithelium)12 does not correlate with the patterns of 15-LOX-2 expression observed in the series of samples we have examined, although 15-LOX-2 was still present to some degree by immunohistochemistry in a significant percentage of tumor cases. Certainly the origins of immunodetectable 13-HODE in tumor tissue might include its production through non-enzymatic mechanisms.
The biological significance of 15-LOX-2 expression in benign prostatic
epithelium is not clear. The uniform expression in prostate apical or
secretory cells suggests a possible role of this novel enzyme in
secretory function. Reduced expression in atrophic prostate glands and
prostate adenocarcinoma may thus parallel less secretory
differentiation. However, given our demonstration of the loss of this
protein and its associated enzymatic activity in prostate carcinoma, it
is possible that reduction or loss of 15-LOX-2 and 15-HETE formation is
a crucial event in the development or progression of prostate
adenocarcinoma. In this regard, it is noteworthy that the murine
homologue of 15-LOX-2, a recently identified 8S-lipoxygenase
(8-LOX), is up-regulated in benign squamous papilloma of mouse skin
induced by phorbol ester treatment and lost with progression to frank
squamous cell carcinoma with additional treatments.19,27
It is possible that the products of these related enzymes
(15S-HETE and 8S-HETE, respectively) regulate
aspects of cellular proliferation or differentiation and that the loss
of this function is associated with development or progression of a
malignant phenotype. The recent description of the activation of
transcription-regulating PPAR receptor subtypes (peroxisome
proliferator-activated receptors) by various AA-related products,
including HETEs, indicates one potential mechanism of
action.28-31
This is a particularly intriguing hypothesis
in light of the evidence that the synthetic activators of PPAR, the
aromatic fatty acids phenyl acetate and phenyl butyrate, inhibit
prostate cancer cell line growth in vitro and show activity
against hormone-insensitive prostate cancer in
vivo.32-34
More recently, Kubota et
al35
demonstrated expression of PPAR
in PC-3, DU-145,
and LNCaP cells and inhibition of PC-3 cell proliferation by PPAR
ligands, including the thiazolidinedione troglitazone and
15-deoxy-
12,14-prostaglandin J2. The recent
demonstration of activation of PPAR-dependent transcription by a
variety of oxidized lipids, including specifically
15-HETE,36
suggests a possible role of reduced 15-HETE
activation of PPAR in prostate carcinogenesis or progression.
Whether this signaling pathway or some other mechanism of cell
regulation is affected by alterations of prostate 15-LOX-2 expression
remains to be further clarified.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grant GM-53638 (A.R. Brash). S.B. Shappell is the recipient of a Department of Defense Prostate Cancer Research Program New Investigator Award (PC970260).
Accepted for publication March 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes. J Biol Chem 1985, 260:4508-4515
-leukotriene B4 pathway to inflammation control. Nature 1996, 384:39-43[Medline]
12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR. Cell 1995, 83:803-812[Medline]
and promotes adipocyte differentiation. Cell 1995, 83:813-819[Medline]
(Troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 1998, 58:3344-3352. Cell 1998, 93:229-240[Medline]
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