Concordant induction of 15-lipoxygenase-1 and mutant p53 expression in human prostate adenocarcinoma: correlation with Gleason staging

Uddhav P.Kelavkar3, Cynthia Cohen1, Hideki Kamitani2, Thomas E.Eling2 and Kamal F.Badr

Center for Glomerulonephritis, Renal Division, Emory University and the VAMC, Atlanta, GA 30322,
1 Department of Pathology, Emory University, Atlanta, GA and
2 Laboratory of Molecular Carcinogenesis, NIEHS, Research Triangle Park, NC, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We recently reported that the mutant form of the tumor-suppressor gene p53 up-regulates 15-LO-1 gene expression in a murine cell line. Here, we examine the expression of 15-lipoxygenase (LO)-1 and mutant p53 (mtp53) in human prostatic tissues and 15-LO-1 in the human prostate adenocarcinoma cell line PC-3. Reverse transcription–PCR and western analyses conclusively demonstrated expression of 15-LO-1 in PC-3 cells. Western blotting for 15-LO-1 in freshly resected `normal' and prostate adenocarcinoma specimens showed 15-LO-1 expression in normal tissue, but significantly higher levels were detected in prostate adenocarcinomas. Prostate adenocarcinoma tissues generated chirally pure 13-S-hydroxyoctadecadienoic acid from exogenous linoleic acid, a preferred substrate of 15-LO-1. To study the correlation of 15-LO-1 expression with mtp53 in prostate cancer, we immunostained 48 prostatectomy specimens obtained by transurethral resection of the prostate and needle biopsy (median age 68 years, range 52–93) of different Gleason grades (n = 48), using antibodies specific for 15-LO-1, mtp53 and MIB-1 (a proliferation marker). We compared staining in cancerous foci with adjacent normal appearing prostate tissues. In only 5 of 48 patients did `normal' tissue adjacent to cancerous foci display staining for 15-LO-1. However, no staining for mtp53 was observed in any of the normal tissues. In cancer foci, robust staining was observed for both 15-LO-1 (36 of 48, 75%) and mtp53 (19 of 48, 39%). Furthermore, the intensities of expression of 15-LO-1 and mtp53 correlated positively with each other (P < 0.001) and with the degree of malignancy, as assessed by Gleason grading (P < 0.01). By immunohistochemistry, 15-LO-1 was located in secretory cells of peripheral zone glands, prostatic ducts and seminal vesicles, but not in the basal cell layer or stroma. Based on these and other studies, we propose a model describing a possible role for 15-LO-1 expression in influencing the malignant potential and pathobiological behavior of adenocarcinomas.

Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; DAB, diaminobenzidine; DAG, diacylglycerol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; IL, interleukin; LA, linoleic acid; LO, lipoxygenase; LXs, lipoxins; mtp53, mutant p53; NDGA, nordihydroguaiaretic acid.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cancer of the prostate is the most commonly diagnosed malignancy among men in the USA and Europe, killing thousands every year. Metastatic prostate cancer responds initially to androgen withdrawal therapy, but hormone resistance always develops. Chemotherapeutic agents currently available have little or no impact on the survival of patients with hormone-refractory prostate cancer. For this reason, metastatic prostate cancer almost always has a fatal outcome. Of note is that studies have shown that diets high in fat seem to be associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. There are a number of mutated genes, as well as several genes that are up- or down-regulated in prostate cancer (www.nci.nih.gov). The `anti-inflammatory' arachidonate 15-lipoxygenase (LO)-1 gene is also overexpressed in adenocarcinoma tissues, as well as in all prostate cancer cell lines, including an androgen-independent, p53-null PC-3 prostate cancer cell line. 15-LO-1 oxidatively metabolizes linoleic acid (LA) and arachidonic acid (AA). The primary metabolites from this pathway, 13-S-hydroxyoctadecadienoic acid (HODE), 15-S-hydroxyeicosatetraenoic acid (HETE) and lipoxins (LXs), are subject to a further cascade of transformations resulting in a rich spectrum of biologically active products. Evidence in the literature has suggested that cancer cells within the prostate tissue itself communicate via such lipid mediators and that tumor progression and/or metastasis depends, in part, on the synthesis and disposition of these classes of compounds (116). To date, the most consistently observed site of point mutations is the p53 gene and these mutations are common in advanced prostate disease (1720). While investigating the cellular actions of the 15-LO-1 gene and its product, we discovered and published evidence that 15-LO-1 expression is up-regulated by mutant p53 (mtp53) (21).

15-LO-1 oxidizes LA, AA and low density lipoprotein, reactions of potential relevance to inflammation, membrane remodeling and atherosclerosis (22). 15-LO-1 is found in reticulocytes, eosinophils, macrophages, prostate, liver, kidney, spleen, thymus, testis, ovary, skeletal muscle, heart, brain and intestinal tissue (Table IGo). Studies have examined the possible significance of LA and AA metabolism or the presence of cyclooxygenase or lipoxygenase enzymes in benign prostate and prostatic neoplasia. Also, several reports have described the presence of lipoxygenase enzymes in benign prostatic hyperplasia, prostate cancer and prostatic carcinoma cell lines (2325), suggesting an important role for lipoxygenases in promoting cancer development. Initial studies have suggested biological effects and the significance of 12-LO in promoting tumor cell adhesion and endothelial cell contraction, indicating a potential contribution to tumor cell metastasis and possible modulation of tumor growth by induction of angiogenesis (2629). More recently, induction of apoptosis in the prostate cancer cell lines PC-3 and LNCaP by inhibitors of 5-LO and 5-LO-activating protein (30,31) indicated a possible role of 5-LO in prostate cancer. Similarly, Spindler et al. (32) detected 13-HODE in a prostate adenocarcinoma tissue and 15-LO-1 expression in the prostate cancer cell lines LNCaP and PC-3. However, this issue was debated in recent studies with 15-LO-2 (25). Our present data with a large number of samples substantiates the presence of 15-LO-1 in prostate adenocarcinoma and correlated positively with mtp53 expression and the degree of malignancy. The biological significance of 15-LO-1 expression in the epithelium of prostatic adenocarcinoma is not entirely clear. The diverse patterns of p53 mutation among high grade and low grade prostatic intra-epithelial neoplasias and prostatic adenocarcinomas suggest multiclonal development of prostatic precancerous lesions (33), indicating that other molecular mechanisms may also play a role in the development of prostate cancer (34). Here, we characterize the expression of 15-LO-1 in prostate carcinoma by immunohistochemistry and biochemical analysis, correlate this expression with that of mtp53 and clinical indices of malignancy and begin to explore the possible implications of 15-LO-1 overexpression in this disease.


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Table I. Occurrence of 15-lipoxygenase-1 (15-LO-1: M23892) in different tissues. Source-NCBI/GenBank
 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Tween 20, acrylamide, SDS, non-fat dry milk, molecular weight protein markers and rabbit anti-sheep IgG–horseradish peroxidase conjugate were purchased from Bio-Rad. Recombinant human interleukin (IL)-13 and IL-4 were purchased from Upstate Biotechnology (Lake Placid, NY). The enhanced chemiluminescence western blotting detection system and PVDF membranes (0.45 µm) were purchased from Millipore (Bedford, MA). Phenylmethanesulfonyl fluoride, leupeptin and pepstatin were from Boehringer Mannheim. Penicillin, streptomycin, Ham's F-12 nutrient mixture medium and trypsin: EDTA were purchased from Life Technologies (Gaithersburg, MD). TRI reagent was from the Molecular Research Center (Cincinnati, OH). The RT–PCR kit was from Perkin Elmer (CA). Common buffer salts and chemicals were obtained from Fisher (Pittsburgh, PA). Fetal calf serum was from Sigma (St Louis, MO).

Cell culture
PC-3 prostate cancer cells (epithelial cells) were obtained from ATCC and cultured in Ham's F-12 nutrient mixture medium (Life Technologies) without phenol red, containing 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 25 µg/ml fungizone in 5% CO2 at 37°C. When cells were 70–80% confluent, IL-13 and IL-4 were added at ED50 concentrations of 5 and 0.2 ng/ml, respectively. Controls were not treated with either cytokine.

Antibodies
The isotypes and specificities of the monoclonal and polyclonal antibodies specific for human 15-LO-1, mtp53 and MIB-1 (Ki-67) are as follows: polyclonal CheY antibody IgG1 is specific for 15-LO-1 (obtained from Dr E. Sigal, CA), monoclonal antibody IgG1 is specific for MIB-1, cyclooxygenase (COX)-1 and COX-2 (Immunotech, FL) and antibody IgG1 is specific for mtp53 (Novocastra, CA).

SDS–PAGE and western blotting
For detection of 15-LO-1, COX-1 and COX-2, 25 µg protein whole cell extract or immunoprecipitated samples from control and experimentally grown PC-3 cells (2x106 cells) or frozen prostate tissues were mixed with loading buffer, boiled for 5 min and then loaded onto a 10% polyacrylamide gel containing 0.1% SDS. The gel was run at 200 V for 40 min. The separated proteins were either Coomassie blue and silver stained or transferred onto individual PVDF membranes by electroblotting. Ponceau S staining of the blots was conducted to ensure equivalent loading. After being blocked with 5% non-fat dry milk in phosphate-buffered saline, the nitrocellulose membranes were incubated with their respective antibodies (1:10 000 dilution) for 1 h at room temperature. Following incubation with either goat anti-rabbit (1:8000 dilution) or donkey anti-rabbit (1:2000 dilution) IgG–horseradish peroxidase second antibody, proteins were visualized using Luminol/Enhancer solutions as described by the manufacturer. Samples were compared with 15-LO-1, COX-1 and COX-2 standards.

Isolation and analysis of mRNAs
PC-3 cells were harvested and RNA was extracted for semi-quantitative RT–PCR. Briefly, RT–PCR of 15-LO-1, 15-LO-2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in PC-3 cells was performed using total RNA. One microgram was reverse transcribed in 45 µl of 50 mM Tris–HCl buffer, pH 8.2, containing 8 mM MgCl2, 30 mM KCl, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 30 U RNase inhibitor, 0.166 mM each dNTP, 150 pmol oligo(dT) primer and 15 U reverse transcriptase according to the manufacturer's instructions (Perkin-Elmer). Samples were heated to 95°C for 10 min. The PCR primers for 15-LO-1 (5'-GAGAGTTGACTTTGAGGTTTCGC-3' and 5'-GCCACGTCTGTCTTATAGTGG-3') and 15-LO-2 (5'-TGCCTCTCGCCATCCAGCT-3' and 5'-TGTTCCCCTGGGATTTAGATGGA-3') were selected for regions displaying minimal sequence similarity to the sequences of human 12- and 15-LOs and leukocyte 5-LO.

The primers for PCR of GAPDH were 5'-TCGGAGTCAACGGATTTGGTCGTA-3' and 5'-GGACTGTGGTCATGAGTCCTTC-3'. After initial denaturation for 3 min at 94°C, PCR was carried out for 23 cycles. Each cycle consisted of a denaturing period (40 s at 94°C), an annealing phase (30 s at 60°C for GAPDH and at 62°C for 15-LO) and an extension period (30 s at 72°C for all primer sets). The reaction mixture was 10 mM Tris–HCl buffer, pH 8.3, containing 50 mM KCl, 2 mM MgCl2, 0.1 mg/ml gelatine, 6 pmol primer sets, 100 µM each dNTP, 100 µg/ml bovine serum albumin and 2.5 U Taq DNA polymerase. PCR products were separated by 1.5% agarose gel electrophoresis. For semi-quantification, DNA was stained with ethidium bromide and analyzed densitometrically.

Immunohistochemical analysis of prostate samples
We examined for cross-reaction of 15-LO-2 protein by western analysis with both the polyclonal 15-LO-1 (Sigal) antibody and one from Parke-Davis. We did not observe any cross-reactivity between the 15-LO-1 and 15-LO-2 proteins (data not shown).

Sections of formalin-fixed, paraffin-embedded tissue (5 µm) were tested for the presence of 15-LO-1 (1:1600) (32,35,36), MIB-1 (1:50) (37) and mtp53 (1:20) (38), using an avidin–biotin complex technique and steam heat-induced antigen retrieval. An avidin–biotinylated enzyme complex kit (LSAB2; Dako Corp., Carpintera, CA) was used in combination with an automated Dako Autostainer. Sections of tonsil were used as a positive control. Negative controls had primary antibody adsorbed with 15-LO-1 antigen or replaced by buffer. Sections were deparaffinized and rehydrated, then steamed in citrate buffer (pH 6) for 20 min and cooled for 10 min before immunostaining. All tissues were then exposed to 3% hydrogen peroxide for 5 min, primary antibody for 25 min, biotinylated secondary linking antibody for 25 min, streptavidin–enzyme complex for 25 min, diaminobenzidine (DAB) as chromogen for 5 min and hematoxylin as counterstain for 1 min. These incubations were performed at room temperature; between incubations, sections were washed with sterile Tris-buffered saline.

Image cytometric nuclear quantitation
Quantitation was performed using a CAS 200 (Becton Dickinson Cellular Imaging Systems, San Jose, CA) according to the procedure described by Goel et al. (39). Briefly, this quantitation is based on differential staining of areas positive or negative for the antigen. Using the peroxidase/DAB staining procedure, positive nuclei stain brown, leaving the negative nuclear areas to be counterstained with hematoxylin. Using the two CAS 200 sensors, measurements are made at different wavelengths. At 620 nm, both the blue and brown color in the nuclei absorb. The threshold is set at 500 nm so that only the brown color absorbs, allowing the immunopositive nuclear areas to be measured independently. Comparison with the 620 nm mask gives the percentage of nuclear area stained positively. Densitometry measurements provide staining intensity data because the amount of light absorbed or optical density is proportional to the concentration of the chromogen.

The image cytometer is standardized by adjusting the light source of the microscope to a predetermined value on an empty field. Then, in control mode, the antibody threshold is adjusted to determined areas considered negative. The computer-generated mask is compared with the blue and brown stained images seen through the microscope and the slide is assayed in specimen mode. At least 15 consecutive fields in a predetermined area of interest are analyzed on each slide. The analysis starts in the upper left-hand corner, proceeds to the lower left-hand corner, then to the right from lower to upper, etc. Folded areas are excluded. Areas analyzed are isolated from adjacent areas of non-involvement by using the scene segmentation function, which allows the operator to precisely define portions of the image to be assessed. Histograms showing percentage nuclear area stained positively on the vertical and nuclear optical density on the horizontal axis were analyzed for nuclear area, percent stained nuclear area and average percent positive intensity of immunostain.

Case selection and histology
Fresh tissue procured from radical prostatectomy accessioned in the Surgical Pathology Laboratory at Emory University were analyzed for 15-LO-1 metabolic studies using [14C]LA and [14C]AA. Fresh tissues were placed in liquid nitrogen or immediately placed in a –80°C freezer. For enzyme assays, proteins extracted from these frozen cancer tissue samples were used.

Similarly, tissues procured from radical prostatectomy (n = 18), transurethral resection (n = 21) and needle biopsy specimens (n = 9) were fixed in 10% buffered formalin and paraffin embedded by a routine method, after dehydration. Five micron sections were prepared for hematoxylin and eosin staining and subjected to standard surgical pathology evaluation. Cases were selected to give a representative mixture of tumor grades with various Gleason grades and combinations of organ-confined disease and extra-capsular extension with or without seminal vesicle and margin involvement: these included 4 cases of Gleason grade scores 2–4, 13 cases of Gleason grade scores 5–6, 15 cases of Gleason grade score 7 and 16 cases of Gleason grade scores 8–10. The specificity of the 15-LO-1 antibody and possible cross-reactivity with other human lipoxygenases was not examined in the immunohistochemistry experiments as we saw no cross-reactivity in the western analysis (data not shown) and this has also been independently evaluated and reported by others (35,36).

Image cytometric 15-LO-1 quantitation
Sections immunostained for 15-LO-1, mtp53 and MIB-1 were semi-quantitatively evaluated either visually or using a CAS 200 analyzer (Becton Dickinson) as described earlier in the text. Briefly, for 15-LO-1 they were assigned a score descriptively or semi-quantitatively of 0 to 3+: 0, no staining; 1+, weak to moderate intensity staining in up to half of the cells in most glands or most cells in up to half of the glands; 2+, moderate to intense staining in greater than half of the cells but less than strong, uniform staining; 3+, strong, uniform staining in essentially all cells. p53 was semi-quantitatively evaluated as 0–3+, intensity and percentage of nuclei immunostaining >10% being regarded as positive. MIB-1 immunostaining was similarly quantitated in the tumor portions. Results are expressed as the percentage of samples positive for 15-LO-1 immunostaining.

Tissue incubations and HPLC analysis
A 50–100 mg amount of prostate adenocarcinoma tissue was individually homogenized in 4 vol buffer (50 mmol/l Tris with 100 mmol/l NaCl and 100 µmol/l CaCl2, pH 7.4). Cell homogenates (800 µg total protein) were incubated with 25 µM [14C]AA or [14C]LA (1x106 c.p.m., 15 nmol) in ethanol (2.5% of final volume) at 30°C for 15 min. The reaction mixture (600 µl) contained 50 mM Tris–HCl, pH 7.4, and 5 mM CaCl2. In all buffers, protease inhibitors were added just before use: phenylmethylsulfonyl fluoride and benzamidine at 1 mM each, aprotinin and leupeptin at 10 µg/ml, pepstatin A at 1 µg/ml. In some reaction samples, indomethacin (Sigma) was added at a final concentration of 10 µM or nordihydroguaiaretic acid (NDGA) (Sigma) to a final concentration of 20 µM. Fatty acids were isolated from the incubation buffer on a C18-PrepSep solid phase extraction column (Waters) after acidification to pH 3.5 with acetic acid. The column was then washed with acidified water, the metabolite eluted with methanol, evaporated to dryness and reconstituted with HPLC solvent.

Reverse phase HPLC analysis was performed using an Ultrasphere ODS column (5 mm, 4.6x250 mm; Beckman). The solvent system consisted of a methanol/water gradient at a flow rate of 1.1 ml/min. Radioactivity was monitored using a Flow Scintillation Analyzer (Packard) with EcoLume (ICN Biochemicals) as the liquid scintillation cocktail. UV analysis was performed by monitoring absorbance at 234 nm with a Waters 486 detector. Straight phase analyses were conducted with a µPorasil column (10 µm; Waters Associates) and eluted with hexane/isopropanol/acetic acid (100:1:0.1) at 2 ml/min. For chiral analysis, the samples were first converted to methyl esters by dissolving in 50 µl of methanol and adding 200 µl of diazomethane ether. After reaction for 2 min at room temperature, the samples were evaporated to dryness under argon. The methyl esters of LA metabolites were analyzed using a Pirkle-type dinitrobenzoyl phenylglycine column (5 µm, 4.6x250 mm; Regis Chemical, Morton Grove, IL) with a moble phase consisting of hexane/isopropanol (100:1) at a flow rate of 1.0 ml/min. Effluents were monitored with a Waters UV detector at 235 nm and the radioactivity with a Flow Scintillation Analyzer (Packard) with EcoLume (ICN Biochemicals) as the liquid scintillation cocktail. Authentic standards of 12-S-HETE, 15-S-HETE, 13-S- and 13-R-HODE, 9-S- and 9-R-HODE and prostaglandin B1 were obtained from Cayman Chemical. Radiolabeled [14C]AA and [14C]LA (40–60 mCi/mmol) were from DuPont-New England Nuclear (Boston, MA). All solvents were HPLC grade and were from Baker (Phillipsburg, NJ).

Statistical analysis
All experimental data are representative of triplicate or duplicate results from at least four different experiments. Correlations between 15-LO-1 and mtp53 expression and Gleason grade, mtp53 and 15-LO-1 expression were analyzed statistically by Kendall's {tau} and Fisher's exact tests.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of 15-LO-1 in prostate adenocarcinoma
Since northern analysis failed to detect the message for 15-LO in PC-3 cells (data not shown), RT–PCR was attempted using primers specific for the human 15-LO-1 and 15-LO-2 cDNAs. The results, as shown in Figure 1Go, indicate that PC-3 cells treated with IL-4 display a strong band (2- to 3-fold) whereas cells alone (control) and treated with IL-13 display a weaker, although readily detectable, band for 15-LO-1. 15-LO-1 expression was further substantiated by western blot analyses. Total proteins from three randomly picked prostate adenocarcinoma tissues (Figure 2AGo) and immunoprecipitated 15-LO-1 protein from the PC-3 cell line, a normal (Figure 2BGo, lane 4) and two prostate adenocarcinoma tissues with Gleason grades 6 and 7 (Figure 2BGo, lanes 5 and 6), respectively, were analyzed. Increased expression of 15-LO-1 has been reported earlier by Spindler et al. (32) in PC-3 cells. However, there was no detectable expression of COX-2 (data not shown), whereas COX-1 was detectable in the tissues analyzed (Figure 2AGo).



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Fig. 1. 15-lipoxygenase-1 (15-LO-1) and 15-lipoxygenase-2 (15-LO-2) (RT–PCR) expression in the PC-3 prostate cancer cell line without (Control) and supplemented with interleukin (IL)-4 and IL-13. Lane 1, molecular weight marker; lane 2, 15-LO-1 standard; lane 3, control; lane 4, IL-4; lane 5, IL-13; lane 6, blank; lane 7, 15-LO-2 standard; lane 8, control; lane 9, IL-4; lane 10, IL-13; lane 11, molecular weight marker.

 


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Fig. 2. Western blot analysis of (A) total proteins from three randomly picked prostate adenocarcinoma tissues for 15-LO-1, COX-1 and ß-actin and (B) immunoprecipitated proteins from PC-3 cells and prostate tissues. Lane 1, control PC-3; lane 2, IL4 + PC-3; lane 3, IL-13 + PC-3; lane 4, `normal' prostate; lane 5, prostate adenocarcinoma tissue 1; lane 6, prostate adenocarcinoma tissue 2; lane 7, 15-LO-1 standard; lane 8, negative control (A549 cells).

 
Enzymatic conversion of linoleic acid and arachidonic acid
Enzymatic activity present in the frozen prostate adenocarcinoma tissue with high Gleason grade scores was examined using AA and LA as substrates. AA was converted to one major metabolite which co-eluted with 15-HETE on reversed phase HPLC in only one of the four tissues examined (Figure 3IGoA). Further analysis by straight phase HPLC characterized it as 15-HETE (Figure 3IGoB). Exogenous LA appeared to be more extensively metabolized and converted to several metabolites, with the major metabolite eluting as a peak detected at ~66–68 min retention time. The metabolite at 68 min co-eluted with authentic standards of 13-HODE and 9-HODE. Figure 3GoIIA–D shows the metabolism of LA with four different preparations of adenocarcinona samples. Incubation of LA with the lipoxygenase inhibitor NDGA (20 µM) reduced the formation of 9/13-HODE metabolites, while addition of the COX inhibitor indomethacin (10 µM) did not inhibit LA metabolism (data not shown).




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Fig. 3. HPLC analysis of AA and LA metabolites from prostate adenocarcinoma tissues. (IA) Reverse phase HPLC profile of AA metabolite products. (IB) Straight phase HPLC profile of AA metabolite products. The arrow indicates 15-HETE. (II) Reverse phase HPLC profile of LA metabolite products from four (AD) randomly selected adenocarcinoma tissues.

 
Chiral analysis of linoleic acid metabolites
To further characterize the LA metabolites formed by prostate tissue, the material eluting as the HODE fraction on reverse phase HPLC was collected and analyzed in a straight phase HPLC system that resolves 13-HODE and 9-HODE. On straight phase HPLC, the prostate metabolites co-eluted with the 13-HODE standard (Figure 4AGo). To further characterize the chirality of the 13-HODE, the metabolite was converted to the methyl ester and analyzed by chiral phase HPLC, which resolves the S and R enantiomers. The prostate-derived 13-HODE eluted as a single peak, which co-eluted with 13-S-HODE (Figure 4BGo). Thus, LA is metabolized by prostate tissue to chirally pure 13-S-HODE. These results indicate that the formation of 13-S-HODE is mediated by 15-LO-1 and not by COX.



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Fig. 4. (A) Straight phase HPLC profile of LA metabolite products. Arrows indicate 13-HODE and 9-HODE. (B) Chiral analysis of LA metabolite 13-HODE from prostate adenocarcinoma tissues. Arrows indicate 13-S-HODE and 13-R-HODE, respectively.

 
15-LO-1 and p53 immunostaining in prostate adenocarcinoma
The distribution of immunostaining was carefully examined in the cancerous portions of prostate sections from 48 prostate carcinoma specimens compared with `normal' or benign prostate tissue. A summary of the semi-quantitative 15-LO-1 and mtp53 immunohistochemical staining in prostate carcinomas of different grades is shown in Table IIGo.


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Table II. Histologic analysis of 15-LO-1 and mt p53 immunostaining in prostate cancer tissues
 
Immunostaining of normal human prostate with a polyclonal antibody for 15-LO-1 showed uniform staining in the cytoplasm of secretory cells, prostatic ducts and seminal vesicles, but was negative in basal cells of the prostate glands and the prostatic urethral transitional mucosa (Figure 5AGo–D). More samples expressed 15-LO-1 (93%) and mtp53 (68%) in prostatic cancer with a high Gleason score than in samples from cancer with a lower score (50 and 0%, respectively, in well-differentiated carcinoma). The majority of prostate adenocarcinomas showed a significant increase in 15-LO-1 in >75% of the tumor. Thus, 15-LO-1 is strongly expressed in prostate adenocarcinoma tissues (Figure 2AGo) and its epithelium and cytoplasm (Figure 6Go). Mutant p53 is mainly nuclear and 15-LO-1 is cytoplasm associated, although in a few cases they were detectable in both nucleus and cytoplasm (data not shown). Though informative, we were not able to analyze serial sections to demonstrate 15-LO-1 and mtp53 expression in the same tumor area, partly due to experimental constraints and partly due to the quantity of tissue available for analysis.



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Fig. 5. Immunostaining of normal human prostate with polyclonal antibody for (15-LO-1) (200x). (A) Immunopositive (brown) in cytoplasm of secretory cells, but negative in basal cells. (B) Prostatic duct with immunopositivity (brown) in surface columnar cells, but negative in basal cells. (C) Positive (brown) in seminal vesicle. (D) Negative in prostatic urethral transitional mucosa.

 


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Fig. 6. Immunostaining of human prostate adenocarcinoma with polyclonal antibody for 15-LO-1 (100x). (A) Immunopositive (brown) for cytoplasmic 15-LO-1. (B) Negative control without primary 15-LO-1 antibody and only biotinylated secondary antibody. (C) Negative control, i.e. 15-LO-1 antigen adsorbed.

 
The polyclonal antibody prepared against purified 15-LO-1 was demonstrated to be specific for 15-LO-1 and does not cross-react with other pertinent lipoxygenases (36,37). Also, 15-LO-1 protein is not detected with an anti-15-LO-2 antibody (25). The immunoreactive band with anti-15-LO-1 antibody was thus specific for partially purified 15-LO-1 protein, with no immunoreactivity seen with purified 15-LO-2, 5-LO or 12-LO (data not shown). The cellular location of 15-LO-1 in benign prostate tissues was similar to that for 15-LO-2 in a previous study (25). However, in contrast to 15-LO-2, 15-LO-1 is uniformly detected in seminal vesicles (Figure 5CGo). Stromal, vascular and inflammatory cells were uniformly positive (data not shown). Though cells show only focal positive immunostaining in normal prostate (Figure 5Go), all high Gleason grade adenocarcinoma tissues show stronger and more diffuse positive immunostaining (Figures 9 and 10GoGo). We made the following observations: (i) in tissues with a Gleason score of 2–4, 50% of cases analyzed expressed 15-LO-1 only and not mtp53 (Figure 7IGo and Table IIGo) whereas in the remaining 50% both 15-LO-1 and mtp53 were undetectable (Figure 7GoII and Table IIGo); (ii) in tissues with a Gleason score of 7, 66% of the cases analyzed expressed 15-LO-1 and 46% expressed mtp53 (Figure 9A and BGo and Table IIGo); (iii) in tissues with a Gleason score of 8–10, 93% of carcinomas analyzed expressed 15-LO-1 and 68% expressed mtp53 (Figure 10A and BGo and Table IIGo). Thus the data suggest that as 15-LO-1 and mtp53 correlate with increasing Gleason score, proliferation increases, as assessed by MIB-1 immunostaining (antibody to Ki-67, a proliferation marker) (Figures 8C, 9C and 10CGoGoGo). The prostatic epithelium consistently stained strongly and uniformly with the polyclonal antiserum against human 15-LO-1 by paraffin immunohistochemistry. There was strong uniform staining of apical (secretory) cells in peripheral zone glands, with essentially all cells staining in all glands (Figures 3A, 5A, 6A and 7AGoGoGoGo) and seminal vesicles (Figure 5CGo). The pattern of staining was predominantly cytoplasmic granular, with occasional nuclear staining also evident. The prostatic duct was immunopositive for 15-LO-1 in surface columnar cells (Figure 5BGo), but basal cells (Figure 5AGo) and the prostatic urethral transitional mucosa did not stain (Figure 5DGo). These are similar results to findings with 15-LO-2 in benign tumors, however, in contrast to 15-LO-2, ejaculatory ducts and seminal vesicles were uniformly positive (Figure 5CGo). No immunostaining was detected in negative controls, i.e. without primary 15-LO-1 antibody or with the 15-LO-1 antigen adsorbed (Figures 6C, 7D, 8D, 9D and 10DGoGoGoGoGo).



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Fig. 9. Immunostaining of human prostate adenocarcinoma (different Gleason grades) with polyclonal antibody for 15-LO-1, mtp53 and MIB-1 as a proliferation marker for Ki-67 (100x). Gleason grade 3 + 4= 7. (A) Cytoplasmic immunostain for 15-LO-1 in a malignant gland (brown); (B) carcinoma positive for nuclear mtp53 (brown); (C) nuclear MIB-1 (Ki-67), proliferation index (brown) = 15.5%; (D) negative control, i.e. 15-LO-1 antigen adsorbed.

 


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Fig. 10. Immunostaining of human prostate adenocarcinoma (different Gleason grades) with polyclonal antibody for 15-LO-1, mtp53 and MIB-1 as a proliferation marker for Ki-67 (100x). Gleason grade 5 + 4= 9. (A) Robust cytoplasmic immunostain for 15-LO-1 in a malignant gland (brown); (B) carcinoma positive for nuclear mtp53 (brown); (C) nuclear MIB-1 (Ki-67), proliferation index (brown) = 25.6%; (D) negative control, i.e. 15-LO-1 antigen adsorbed.

 


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Fig. 7. Immunostaining of human prostate adenocarcinoma (different Gleason grades) with polyclonal antibodies for 15-LO-1, mtp53 and MIB-1 as a proliferation marker for Ki-67 (100x). (I) Gleason grade 1 + 3= 4. (A) Cytoplasmic immunostain for 15-LO-1 in a malignant gland (brown); (B) carcinoma negative for nuclear mtp53; (C) nuclear MIB-1 (Ki-67), proliferation index = 0.9%; (D) negative control, i.e. 15-LO-1 antigen adsorbed. (II) Gleason grade 2 + 2= 4. (A) Negative for cytoplasmic immunostain for 15-LO-1; (B) negative for nuclear mtp53; (C) nuclear MIB-1 (Ki-67), proliferation index (brown) = 0.7%; (D) negative control, i.e. 15-LO-1 antigen adsorbed.

 


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Fig. 8. Immunostaining of human prostate adenocarcinoma (different Gleason grades) with polyclonal antibody for 15-LO-1, mtp53 and MIB-1 as a proliferation marker for Ki-67 (100x). (I) Gleason grade 3 + 2= 5. (A) Cytoplasmic immunostain for 15-LO-1 in a malignant gland (brown); (B) carcinoma positive for nuclear mtp53 (brown); (C) nuclear MIB-1 (Ki-67), proliferation index = 3.3%; (D) negative control, i.e. 15-LO-1 antigen adsorbed. (II) Gleason grade 3 + 2= 5. (A) Negative for cytoplasmic immunostain for 15-LO-1; (B) negative for nuclear mtp53; (C) nuclear MIB-1 (Ki-67), proliferation index (brown) = 2.5%; (D) negative control, i.e. 15-LO-1 antigen adsorbed.

 
Increased 15-LO-1 in prostate adenocarcinoma by immunohistochemistry
We analyzed the pattern of 15-LO-1 and mtp53 expression in tissues with Gleason scores of 2–4, 5–6, 7 and 8–10. The expression pattern of 15-LO-1 and mtp53 in tissues with Gleason scores of 2–4 and 5–6 was suggestive of tissue heterogeneity. One case showed the presence of 15-LO-1 and an absence of mtp53 (Gleason grade 1 + 3), while another with a Gleason grade 2 + 2 adenocarcinoma showed the opposite results. Similar patterns were also seen in two cases with a Gleason score 5–6 adenocarcinoma (3 + 2). These examples were among those that show possible tumor heterogeneity with regard to 15-LO-1 expression. Examples of the degree of heterogeneity of 15-LO-1 immunostaining within individual tumors are shown in Figures 5 and 6GoGo. However, with Gleason scores of 7 and 8–10, the tissues showed 15-LO-1 immunoreactivity in >90% of cancer foci (data not shown). Of particular significance was that in both Gleason grades the 15-LO-1 and mtp53 immunostaining increased in intensity from 2+ in tissues with a Gleason score of 7 to 3+ in tissues with a Gleason score of 8–10 for 15-LO-1 and from +1 to +3 for mtp53.

The following observations were made after analyzing these tissues: (i) in contrast to the strong, uniform 15-LO-1 immunostaining in carcinomatous prostate glands, 15-LO-1 immunostaining was markedly reduced and focal in `normal' or benign prostatic carcinoma; (ii) 15-LO-1 immunostaining was focally present in essentially all tumors; (iii) in 12 of 48 cases 15-LO-1 was absent from >25% of the tumor examined by immunohistochemistry, in which cases 15-LO-1 immunostaining was negative in 60% of the tumor; (iv) 15-LO-1 was present in >75% of the tumor in 25 of 31 cases; (v) the correlation of increased 15-LO-1 with known prognostic factors such as mtp53 in prostate cancer indicate a possible correlation of increased 15-LO-1 immunostaining with increased tumor grade or biological aggressiveness (Table IIGo).

The correlation of the data for 15-LO-1 and mtp53 expression and of Gleason score compared with mtp53 and 15-LO-1 expression analyzed statistically by Kendall's {tau} and Fisher's exact tests are highly significant (P < 0.001). So is the data for age and Gleason score compared with mtp53 and 15-LO-1 expression (P < 0.01).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human tumors, both primary and metastatic, continue to proliferate in spite of the presence of T and B lymphocytes, plasma cells, natural killer cells and macrophages within and around the tumor. Tumors interact with their environment, reprogramming host cells to induce responses such as angiogenesis, inflammation, immunity and immune suppression. To understand these processes, it is important to identify and isolate new genes whose expression is induced in host tissues in response to tumors. It seems that inflammatory cells and cells within the cancer tissue itself communicate via lipid mediators from the metabolism of LA and AA and that the progression and outcome of inflammation depend, in part, on the synthesis and disposition of these classes of compounds. This raises an intriguing question as to the role of these mediators, especially products of 15-LO-1 reactions. There are no studies relating an association of 15-LO-1 with prostate cancer, although it is expressed in the prostate and several other cancer tissues (Table IGo).

IL-4 and the closely related cytokine IL-13 share many biological and immunoregulatory functions on B lymphocytes, monocytes, dendritic cells and fibroblasts. We have used IL-4 and IL-13 in our study with PC-3 cells to study whether these cytokines induce 15-LO-1, as shown previously by us in the lung cancer cell line A549 (40). The IL-4 and IL-13 receptors are multimeric and share at least one common chain, called IL-4R{alpha} (41). Deleuran et al. (42), while studying functional aspects of IL-4 and IL-13 receptors, found that compared with IL-4, IL-13 induced a similar alteration of the cytokine cascade and AA metabolism, suggesting a common receptor complex or signal pathway. Maini et al. (43) have previously reported that human prostate cancer cell lines also express high affinity functional IL-13 receptors. IL-4 and IL-13 are also known to induce 15-LO-1 in human monocytes/macrophages (44).

Recently, we demonstrated regulation of 15-LO-1 gene expression by the mutant form of p53 tumor suppressor protein (21). Though the majority of studies have revealed a low percentage of p53 abnormalities in early stage (clinically organ-confined) cancer of the prostate, the overwhelming bulk of evidence suggests that the frequency of p53 abnormalities does increase with disease progression and is highest in tissues from patients with hormone-refractory prostate cancer (17).

One study has suggested that analysis of biological changes (including p53 alterations in regional lymph node metastases) could be of value in assessment of the biological aggressiveness of prostate carcinoma (18). p53 overexpression is associated with the known proliferative capacity of basal cells in benign hyperplastic prostate glands and mutations of p53 play a role in the pathogenesis of a subset of high grade prostate adenocarcinomas (19). However, p53 expression in the primary tumor does not appear to influence patient outcome (20). Thus mtp53 quantitations by immunohistochemical analysis need to be further confirmed by p53 gene mutation analysis studies, though there are various problems of interpretation in making detailed comparisons between them. However, in view of the importance of p53, particularly in prostate cancer progression and metastasis, gene therapy studies have been initiated (45).

15-LO-1 has a wide tissue distribution, including peripheral blood leukocytes (Table IGo). In the current study, we have characterized the distribution of 15-LO-1 and mtp53 in 48 prostectomy transurethral resection of the prostate and needle biopsy specimens (median age 68 years, range 52–93) with adenocarcinoma at different stages of cancer progression by immunohistochemistry. We have further demonstrated the ability of prostate adenocarcinoma tissues to form 13-S-HODE from exogenous LA. Immunostaining with anti-15-LO-1, anti-p53 and anti-MIB-1 antibodies showed proportional increases in 15-LO-1 and mtp53 expression with the degree of proliferation as assessed by MIB-1, suggesting an association between prostatic tumor grade and increasing expression of 15-LO-1 and mtp53.

The wide tissue distribution of 15-LO-1 suggests a possible role in regulation of organ-specific functions, differentiation or possible alterations in disease states. Recent investigations using both human tissues and prostate carcinoma cell lines to study potentially important molecular mechanisms in prostate cancer development or progression have shed some light on the possible aspects, including cyclooxygenase (16), 12-LO (23), 5-LO (24), 15-LO-1 (32) and 15-LO-2 (25). In particular, the recent discovery that 15-LO-2 is absent from prostate adenocarcinomas and is found only in benign prostate cancer suggests individual roles for 15-LO-1 in adenocarcinoma and 15-LO-2 in benign prostate hypertrophy, respectively.

The role of the oxidation products of LA as regulators of cellular activity is a rapidly expanding area of investigation (18). Numerous biological activities have been associated with production of these compounds and a variety of organs and tissues are affected. Various activities, from vascular homeostasis to cell growth and differentiation, have been shown to utilize LA oxidation products as part of the signaling system (6,11).

Two major metabolic pathways of lipid peroxidation by 15-LO-1 conspire to lend biological significance to its inappropriate expression in prostate cancer.

Oxidation of LA yielding 13-S-HODE. Though a study has shown that 13-S-HODE may inhibit cell proliferation (46), our present data, supported by other observations in the literature, strongly indicate that 13-S-HODE in fact mediates the effect of LA in enhancing cellular proliferation (116). Sauer et al. (47) have recently shown that 13-S-HODE enhances epidermal growth factor-dependent mitogenesis and is the mitogenic signal which is responsible for LA-dependent growth in hepatoma 7288CTC in vivo. Diets rich in LA, an n-6 fatty acid, stimulate the progression of human breast cancer solid tumors in athymic nude mice (48,49), suggesting that 13-S-HODE is potentially capable of promoting survival of prostate cancer cells following androgen withdrawal, thereby supporting tumor growth in the absence of androgen. Studies have also shown that LA supports the growth of the androgen-unresponsive human prostate cancer cell line PC-3 in vitro (50) and that of the androgen-responsive cell line DU145 in vivo (51,52). These and other similar studies in the literature (32,53) strongly suggest that dietary fatty acids are involved in the carcinogenic process within the prostate gland. The mechanism(s) by which the products of 15-LO-1 reactions control the survival of prostate cancer cells is an intriguing problem. In our present metabolism studies, incubation of LA with NDGA (a lipoxygenase inhibitor) inhibited the formation of 9/13-HODE metabolites, while addition of indomethacin (a COX inhibitor) did not inhibit LA metabolism (data not shown). These observations indicate that the HODE metabolite is a product of 15-LO-1 and not due to cyclooxygenases.

There are also reports on the presence of other LA and AA metabolizing enzymes in benign prostate tumors and prostatic neoplasia (2325). In this study we have unequivocally demonstrated, by RT–PCR followed by sequencing of the product, western blotting of the protein and HPLC analysis of metabolic products as well as immunohistochemistry, that 15-LO-1 is overexpressed in prostate adenocarcinoma. Incubation of prostate adenocarcinoma tissues with [14C]LA resulted in formation of 13-S-HODE, as determined by reverse and straight phase HPLC and chiral analysis. Incubation of LA in the presence of NDGA inhibited the formation of 13-S-HODE, while addition of indomethacin did not inhibit LA metabolism (data not shown). The antibody used in the current immunohistochemical studies specifically detects 15-LO-1 and does not cross-react with 15-LO-2, 12-LO or 5-LO. Furthermore, there was complete suppression of positive reactions in the immunohistochemical staining by pre-absorption of tissue with purified 15-LO-1 protein. Thus, these data also conclusively demonstrate that the metabolism of LA in human prostate tissues is mediated by 15-LO-1 and not due to cyclooxygenases or 15-LO-2.

Oxidation of AA yielding 15-S-HETE and LXs. In 1990, Brezinski and Serhan (54) reported 15-S-HETE esterification into inositol-containing lipids. Moreover, the esterified phosphoinositol membrane lipid pool containing 15-S-HETE could serve as a `primed' pool for subsequent generation of LXs and inhibition of leukotriene B4 synthesis from endogenous sources in human neutrophils. This novel membrane priming event was shown to have an impact on signal transduction. Subsequently, our own group (55,56) and others (5760) have presented evidence supporting the specific capacity of 15-S-HETE and LXA4 to effectively paralyze polymorphonuclear cell and macrophage activation, chemotaxis and trans-endothelial migration. Data from these studies suggest that substitution of 15-S-HETE/LXA4 for esterified AA in the diacylglycerol (DAG) component of phosphatidylinositol bisphosphate leads to remodeling of the membrane topology, thereby thwarting recognition of inflammatory agonists and adhesion molecules by leukocyte surface receptors. Legrand et al. (58) have suggested that should cellular activation still occur in 15-S-HETE preincubated cells, a significant fraction of the `second messenger' DAG will be substituted by 15-S-HETE (or LXA4) (`false' DAG), incapable of activating protein kinases. Although the precise mechanism for potential 15-HETE/DAG–kinase interactions has yet to be elucidatied, this two-step `tripping' of macrophage/polymorphonuclear cell activation potentially endows 15-lipoxygenated eicosanoids with `anti-inflammatory' properties.

Recently, in vivo evidence for the anti-inflammatory impact of the 15-LO-1 pathway has been reported. Stable analogs of LXs have been shown to be topically active and anti-inflammatory in mouse models of inflammation (61,62). Finally, IL-13, an anti-inflammatory lymphokine, is known to up-regulate 15-LO-1 gene expression in human monocytes (27) and to induce LXA4 receptor expression in human enterocytes (63).

The biological significance of 15-LO-1 expression in the epithelium of prostatic adenocarcinoma is not entirely clear. The uniform expression in prostate apical or secretory cells suggests a possible role for this enzyme in secretory functions. Its presence in seminal vesicles may suggest involvement of eicosanoids in regulation of the mitogenic effect of testosterone on these vesicles found in seminal vesicular carcinomas (64). mtp53 expression is concomitant and further supports our earlier observation (21) that mtp53 up-regulates 15-LO-1 gene expression. Based on our studies and those reported by others, we have proposed a model (Figure 11Go) describing a possible role of 15-LO-1 expression in influencing the malignant potential and patho-biological behavior of adenocarcinomas. (i) Low or `normal' levels of 15-LO-1 serve normal physiological functions, such as tissue repair through membrane remodeling. (ii) Proliferating cancerous tissues accumulate mtp53. As an innate response, the cells favor 15-LO-1 overexpression (likely due to mtp53 or chemokines such as IL-4 and/or Il-13 secreted by lymphocytes infiltrating the tumor), whose metabolic products, such as 13-S-HODE, may potentiate growth or proliferation. The levels of LA- and AA-derived products from 15-LO-1, 5-LO or 12-LO may then play a crucial role, whether the cells differentiate and undergo apoptosis or continue to proliferate by up-regulating the levels of 15-LO-1 to levels higher than `normal'. Recent observations in our laboratory (U.P.Kelavkar and K.F.Badr, in preparation) and those of others (65) suggest that the LA- and AA-derived products 13-S-HODE and 15-S-HETE may have a role in cell proliferation regulation acting either through bcl2/bax-regulated cell growth or through an insulin-like growth factor 1 receptor-mediated cell signaling pathway(s). Similarly, Rao et al. (66) have shown in their studies that LA and its product HPODE induce DNA synthesis, c-fos, c-jun and c-myc mRNA expression and MAPK activation in vascular smooth muscle cells, leading to their proliferation. (iii) The anti-inflammatory potential of 15-LO-1 (67) reaction products (such as 15-HETE and LXs) provide a zone of localized hypo-immunity, protecting the developing carcinoma from effective removal by activated leukocytes and/or natural killer cells (22).



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Fig. 11. Proposed hypothesis for a role of the anti-inflammatory actions of 15-LO-1 in epithelial cancers. Under `normal' circumstances, transient expression of 15-LO-1 in epithelial cells (as in reticulocytes) aids in cellular turnover and repair through lipid peroxidation/membrane remodeling. During `cancer', mtp53 and possibly other stimulants lead to `inappropriately sustained' expression of 15-LO-1 in malignant cells. Through its actions on LA, 15-LO-1 generates 13-S-HODE, which is a known stimulator of cellular proliferation. Through its actions on AA, 15-LO-1 leads to the formation of 15-S-HETE and LXs, thereby providing a milieu in which killer T cells, macrophage (M{phi}) and polymorphonuclear neutrophil (PMN) functions are suppressed (hypo-immunity). Thus, cancer-restricted expression of 15-LO-1 affords developing tumors with protection against immune destruction and increased potential for proliferation.

 
This study highlights important facets of 15-LO-1 gene expression during prostate cancer development. Further detailed study based on the proposed model may provide an opportunity for designing novel interventional strategies. Thus biological and chemical agents inhibiting 15-LO-1 are attractive new tools for arresting tumor growth and/or metastasis through preferential inhibition of 15-LO-1.


    Notes
 
3 To whom correspondence should be addressed at: 3304 WMB, Renal Division, School of Medicine, Emory University, 1639 Pierce Drive, GA 30322, USA Email: kelavkar{at}emory.edu Back


    Acknowledgments
 
We thank the histopathology laboratory for tissue sectioning, Diane Lawson and Debbie Sexton (Emory University) for immunohistochemistry and Dr Joe Haseman of the Biometry Branch for analyzing the data and Mr Mark Geller (National Institutes of Environmental Health) for HPLC analysis. This work was supported in part by National Institutes of Health (NIH/NIDDK) grant no. 2RO1DK43883 (to K.F.B.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Bertomeu,M.C., Gallo,S., Lauri,D., Haas,T.A., Orr,F.W., Bastida,E. and Buchanan,M.R. (1993) Interleukin 1-induced cancer cell/endothelial cell adhesion in vitro and its relationship to metastasis in vivo: role of vessel wall 13-HODE synthesis and integrin expression. Clin. Exp. Metastasis, 11, 243–250.[ISI][Medline]
  2. Buchanan,M.R., Horsewood,P. and Brister,S.J. (1998) Regulation of endothelial cell and platelet receptor-ligand binding by the 12- and 15-lipoxygenase monohydroxides, 12-, 15-HETE and 13-HODE. Prostaglandins Leukot. Essent. Fatty Acids., 58, 339–346.[ISI][Medline]
  3. Ikawa,H., Kamitani,H., Calvo,B.F., Foley,J.F. and Eling,T.E. (1999) Expression of 15-lipoxygenase-1 in human colorectal cancer. Cancer Res., 59, 360–366.[Abstract/Free Full Text]
  4. Kamitani,H., Geller,M. and Eling,T. (1998) Expression of 15-lipoxygenase by human colorectal carcinoma Caco-2 cells during apoptosis and cell differentiation. J. Biol. Chem., 273, 21569–21577.[Abstract/Free Full Text]
  5. Natarajan,R. and Nadler,J. (1998) Role of lipoxygenases in breast cancer. Front. Biosci., 3, E81–E88.[Medline]
  6. Reddy,N., Everhart,A., Eling,T. and Glasgow,W. (1997) Characterization of a 15-lipoxygenase in human breast carcinoma BT-20 cells: stimulation of 13-HODE formation by TGF alpha/EGF. Biochem. Biophys Res. Commun., 231, 111–116.[ISI][Medline]
  7. Cesano,A., Visonneau,S., Scimeca,J.A., Kritchevsky,D. and Santoli,D. (1998) Opposite effects of linoleic acid and conjugated linoleic acid on human prostatic cancer in SCID mice. Anticancer Res., 18, 1429–1434.[ISI][Medline]
  8. Zock,P.L. and Katan,M.B. (1998) Linoleic acid intake and cancer risk: a review and meta-analysis. Am. J. Clin. Nutr., 68, 142–153.[Abstract]
  9. Bairati,I., Meyer,F., Fradet,Y. and Moore,L. (1998) Dietary fat and advanced prostate cancer. J. Urol., 159, 1271–1275.[ISI][Medline]
  10. Willett,W.C. (1997) Specific fatty acids and risks of breast and prostate cancer: dietary intake. Am. J. Clin. Nutr., 66, 1557S–1563S.[Abstract]
  11. Glasgow,W.C. and Everhart,A.L. (1997) The role of linoleic acid metabolism in the proliferative response of cells overexpressing the erbB-2/HER2 oncogene. Adv. Exp. Med. Biol., 407, 393–397.[ISI][Medline]
  12. Blask,D.E., Sauer,L.A., Dauchy,R., Holowachuk,E.W. and Ruhoff,M.S. (1999) New actions of melatonin on tumor metabolism and growth. Biol. Signals Recept., 8, 49–55.[ISI][Medline]
  13. Banni,S., Angioni,E., Casu,V., Melis,M.P., Carta,G., Corongiu,F.P., Thompson,H. and Ip,C. (1999) Decrease in linoleic acid metabolites as a potential mechanism in cancer risk reduction by conjugated linoleic acid. Carcinogenesis, 20, 1019–1024.[Abstract/Free Full Text]
  14. Rose,D.P. and Connolly,J.M. (1998) Influence of dietary linoleic acid on experimental human breast cancer cell metastasis in athymic nude mice. Int. J. Oncol., 13, 1179–1183.[ISI][Medline]
  15. Marnett,U. (1992) Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res., 52, 5575–5589.[ISI][Medline]
  16. Smith,W.L., Garavito,R.M. and DeWitt,D.L. (1996) Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem., 271, 33157–33160.[Free Full Text]
  17. Heidenberg,H.B., Sesterhenn,I.A., Gaddipati,J.P., Weghorst,C.M., Buzard,G.S., Moul,J.W. and Srivastava,S. (1995) Alteration of the tumor suppressor gene p53 in a high fraction of hormone refractory prostate cancer. J. Urol., 154, 414–421.[ISI][Medline]
  18. Cheng,L., Leibovich,B.C., Bergstralh,E.J., Scherer,B.G., Pacelli,A., Ramnani,D.M., Zincke,H. and Bostwick,D.G. (1999) p53 alteration in regional lymph node metastases from prostate carcinoma: a marker for progression? Cancer, 85, 2455–2459.[ISI][Medline]
  19. Kallakury,B.V., Figge,J., Ross,J.S., Fisher,H.A., Figge,H.L. and Jennings,T.A. (1994) Association of p53 immunoreactivity with high Gleason tumor grade in prostatic adenocarcinoma. Hum. Pathol., 25, 92–97.[ISI][Medline]
  20. Schlechte,H., Lenk,S.V., Loning,T., Schnorr,D., Rudolph,B.D., Ditscherlein,G. and Loening,S.A. (1998) p53 tumor suppressor gene mutations in benign prostatic hyperplasia and prostate cancer. Eur. Urol., 34, 433–440.[ISI][Medline]
  21. Kelavkar,U.P. and Badr,K.F. (1999) Effects of mutant p53 expression on human 15-lipoxygenase-promoter activity and murine 12/15-lipoxygenase gene expression: evidence that 15-lipoxygenase is a mutator gene. Proc. Natl Acad. Sci. USA, 96, 4378–4383.[Abstract/Free Full Text]
  22. Samuelsson,B., Dehien,S.E., Lindgren,J.A., Rouzer,C.A. and Serhan,C.N (1987) Leukotrienes and lipoxins: structures, biosynthesis and biological effects. Science, 237, 1171–1176.[ISI][Medline]
  23. Gao,X., Grignon,D.J., Chbihi,T., Zacharek,A., Chen,Y.O., Sakr,W., Porter,A.T., Crissman,J.D., Pontes,J.E., Powell,I.J. and Honn,K.V. (1995) Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer. Urology, 46, 227–237.[ISI][Medline]
  24. Ghosh,J. and Myers,C.E. (1997) Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase. Biochem. Biophys. Res. Commun., 235, 418–423.[ISI][Medline]
  25. Shappell,S.B., Boeglin,W.E., Olson,S.J., Kasper,S. and Brash,A.R. (1999) 15-Lipoxygenase-2 (15-LOX-2) is expressed in benign prostatic epithelium and reduced in prostate adenocarcinoma. Am. J. Pathol., 155, 235–245.[Abstract/Free Full Text]
  26. Tang,D.G. and Honn,K.V. (1994) 12-Lipoxygenase, 12(S)-HETE and cancer metastasis. Ann. N. Y. Acad. Sci., 744, 199–215.[ISI][Medline]
  27. Honn,K.V., Tang,D.G., Grossi,I., Duniec,Z.M., Timar,J., Renaud,C., Leithauser,M., Blair,I.A., Johnson,C.R., Digllo,C.A., Kimler,V.A., Taylor,J.D. and Marnett,U. (1994) Tumor derived 12(S)-hydroxyeicosatetraenoic acid induces microvascular endothelial cell retraction. Cancer Res., 54, 565–574.[Abstract]
  28. Chen,Y.O., Duniec,Z.M., Liu,B., Hagmann,W., Gao,X., Shimoji,K., Marnett,U., Johnson,C.R. and Honn,K.V. (1994) Endogenous 12-S-HETE production by tumor cells and its role in metastasis. Cancer Res., 54, 1574–1579.[Abstract]
  29. Nie,O., Hillman,G.G., Geddes,T., Tang,K., Pierson,C., Grignon,D.J. and Honn,K.V. (1998) Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth. Cancer Res., 58, 4047–4051.[Abstract]
  30. Ghosh,J. and Myers,C.E. (1998) Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells. Proc. Natl Acad. Sci. USA, 95, 13182–13187.[Abstract/Free Full Text]
  31. Anderson,K.M., Seed,T., Vos,M., Mulshine,J., Meng,J., Alrefal,W., Ou,O. and Harris,J.E. (1998) 5-Lipoxygenase inhibitors reduce PC-3 cell proliferation and initiate nonnecrotic cell death. Prostate, 37, 161–173.[ISI][Medline]
  32. Spindler,S.A., Sarkar,F.H., Sakr,W.A., Blackburn,M.L., Bull,A.W., LaGattuta,M. and Reddy,R.G (1997) Production of 13-hydroxyoctadecadienoic acid (13-HODE) by prostate tumors and cell lines. Biochem. Biophys. Res. Commun., 239, 775–781.[ISI][Medline]
  33. Yasunaga,Y., Shin,M., Fujita,M.Q., Nonomura,N., Miki,T., Okuyama,A. and Aozasa,K. (1998) Different patterns of p53 mutations in prostatic intraepithelial neoplasia and concurrent carcinoma: analysis of microdissected specimens. Lab. Invest., 78, 1275–1279.[ISI][Medline]
  34. Stattin,P., Bergh,A., Karlberg,L., Nordgren,H. and Damber,J.E. (1996) P53 immunoreactivity as prognostic marker for cancer-specific survival in prostate cancer. Eur. Urol., 30, 65–72.[ISI][Medline]
  35. Nadel,J.A., Conrad,D.J., Ueki,I.F., Schuster,A. and Sigal,E. (1991) Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes and airway cells. J. Clin. Invest., 87, 1139–1145.[ISI][Medline]
  36. Lei,Z.M. and Rao,C.V. (1992) The expression of 15-lipoxygenase gene and the presence of functional enzyme in cytoplasm and nuclei of pregnancy human myometria. Endocrinology, 130, 861–870.[Abstract]
  37. Kirkegaard,L.J., DeRose,P.B., Yao,B. and Cohen,C. (1998) Image cytometric measurement of nuclear proliferation markers (MIB-1, PCNA) in astrocytomas. Prognostic significance. Am. J. Clin. Pathol., 109, 69–74.
  38. Cohen,C. and DeRose,P.B. (1994) Immunohistochemical p53 in hepatocellular carcinoma and liver cell dysplasia. Mod. Pathol., 7, 536–539.[ISI][Medline]
  39. Goel,A., Abou-Ella,A., DeRose,P. and Cohen,C. (1996). The prognostic significance of proliferation in prostate cancer. J. Urol. Pathol., 4, 213–225.
  40. Kelavkar,U.P., Wang,S. and Badr,K.F. (2000) Ku autoantigen (DNA helicase) is required for interleukins-13/-4-induction of 15-lipoxygenase-1 gene expression in human epithelial cells. Genes Immun., 1, 237–250.[ISI][Medline]
  41. Chomarat,P. and Banchereau,J. (1998) Interleukin-4 and interleukin-13: their similarities and discrepancies. Int. Rev. Immunol., 17, 1–52.[Medline]
  42. Deleuran,B., Iversen,L., Deleuran,M., Yssel,H., Kragballe,K., Stengaard-Pedersen,K. and Thestrup-Pedersen,K. (1995) Interleukin-13 suppresses cytokine production and stimulates the production of 15-HETE in PBMC. A comparison between IL-4 and IL-13. Cytokine, 7, 319–324.
  43. Maini,A., Hillman,G., Haas,G.P., Wang,C.Y., Montecillo,E., Hamzavi,F., Pontes,J.E., Leland,P., Pastan,I., Debinski,W. and Puri,R.K. (1997) Interleukin-13 receptors on human prostate carcinoma cell lines represent a novel target for a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin. J. Urol., 158, 948–953.[ISI][Medline]
  44. Nassar,G.M., Morrow,J.D., Roberts,L.J., Lakkis,F.G. and Badr,K.F. (1994) Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes. J. Biol. Chem., 269, 27631–27634.[Abstract/Free Full Text]
  45. Gurnani,M., Lipari,P., Dell,J., Shi,B. and Nielsen,L.L. (1999) Adenovirus-mediated p53 gene therapy has greater efficacy when combined with chemotherapy against human head and neck, ovarian, prostate and breast cancer. Cancer Chemother. Pharmacol., 44, 143–151.[ISI][Medline]
  46. Liu,B., Khan,W.A., Hannun,Y.A., Timar,J., Taylor,J.D., Lundy,S., Butovich,I. and Honn,K.V. (1995) 12(S)-Hydroxyeicostetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids. Proc. Natl Acad. Sci. USA, 92, 9323–9327.[Abstract]
  47. Sauer,L.A., Dauchy,R.T., Blask,D.E., Armstrong,B.J. and Scalici,S. (1999) 13-Hydroxyoctadecadienoic acid is the mitogenic signal for linoleic acid-dependent growth in rat hepatoma 7288CTC in vivo. Cancer Res., 59, 4688–4692.[Abstract/Free Full Text]
  48. Connolly,J.M., Gilhooly,E.M. and Rose,D.P. (1999) Effects of reduced dietary linoleic acid intake, alone or combined with an algal source of docosahexaenoic acid, on MDA-MB-231 breast cancer cell growth and apoptosis in nude mice. Nutr. Cancer., 35, 44–49.[ISI][Medline]
  49. Liu,X.H., Connolly,J.M. and Rose,D.P. (1996) Eicosanoids as mediators of linoleic acid-stimulated invasion and type IV collagenase production by a metastatic human breast cancer cell line. Clin. Exp. Metastasis, 14, 145–152.[ISI][Medline]
  50. Rose,D.P. and Connolly,J.M. (1991) Effects of fatty acids and eicosanoid synthesis inhibitors on the growth of two human prostate cancer cell lines. Prostate, 18, 243–254.[ISI][Medline]
  51. Connolly,J.M., Coleman,M. and Rose,D.P. (1997) Effects of dietary fatty acids on DU145 human prostate cancer cell growth in athymic nude mice. Nutr. Cancer., 29, 114–119.[ISI][Medline]
  52. Cesano,A., Visonneau,S., Scimeca,J.A., Kritchevsky,D. and Santoli,D. (1998) Opposite effects of linoleic acid and conjugated linoleic acid on human prostatic cancer in SCID mice. Anticancer Res., 18, 1429–1434.[ISI][Medline]
  53. Giovannucci,E., Rimm,E.B., Colditz,G.A., Stampfer,M.J., Ascherio,A., Chute,C.C. and Willett,W.C. (1993) A prospective study of dietary fat and risk of prostate cancer. J. Natl. Cancer Inst., 85, 1571–1579.[Abstract]
  54. Brezinski,M.E. and Serhan,C.N. (1990) Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: agonist-induced deacylation and transformation of stored hydroxyeicosanoids. Proc. Natl Acad. Sci. USA, 87, 6248–6252.[Abstract]
  55. Badr,K.F., DeBoer,D.K., Schwartzberg,M. and Serhan,C.N. (1989) Lipoxin A4 antagonizes cellular and in vivo actions of leukotriene D4 in rat glomerular mesangial cells: evidence for competition at a common receptor. Proc. Natl Acad. Sci.USA, 86, 3438–3442.[Abstract]
  56. Fischer,D.B., Christman,J.W. and Badr,K.F. (1992) Fifteen-S-hydroxyeicosatetraenoic acid (15-S-HETE) specifically antagonizes the chemotactic action and glomerular synthesis of leukotriene B4 in the rat. Kidney Int., 41, 1155–1160.[ISI][Medline]
  57. Brady,H.R., Lamas,S., Papayianni,A., Takata,S., Matsubara,M. and Marsden,P.A. (1995) Lipoxygenase product formation and cell adhesion during neutrophil-glomerular endothelial cell interaction. Am. J. Physiol., 268, F1–F12.[Abstract/Free Full Text]
  58. Legrand,A.B., Lawson,L., Meyrick,B.O., Blair,I.A. and Oates,J.A. (1991) Substitution of 15-hydroxyeicosatetraenoic acid in the phosphoinositide signaling pathway. J. Biol. Chem., 266, 7570–7577.[Abstract/Free Full Text]
  59. Takata,S., Matsubara,M., Allen,P.G., Janmey,P.A., Serhan,C.N. and Brady,H.R. (1994) Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium. J. Clin. Invest., 93, 499–508.[ISI][Medline]
  60. Takata,S., Papayianni,A., Matsubara,M., Jimenez,W., Pronovost,P.H. and Brady,H.R. (1994) 15-Hydroxyeicosatetraenoic acid inhibits neutrophil migration across cytokine-activated endothelium. Am. J. Pathol., 145, 541–549.[Abstract]
  61. Takano,T., Clish,C., Gronert,K., Petasis,N. and Serhan,C. (1998) Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues. J. Clin. Invest., 101, 819–826.[Abstract/Free Full Text]
  62. Takano,T., Fiore,S., Maddox,J., Brady,H., Petasis,N. and Serhan,C. (1997) Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors. J. Exp. Med., 185, 1693–1704.[Abstract/Free Full Text]
  63. Gronert,K., Gewirtz,A., Madara,J. and Serhan,C. (1998) Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon and inhibits tumor necrosis factor-induced IL-8 release. J. Exp. Med., 187, 1285–1294.[Abstract/Free Full Text]
  64. Klus,G.T., Nakamura,J., Li,J.S., Ling,Y.Z., Son,C., Kemppainen,J.A., Wilson,E.M. and Brodie,A.M. (1996) Growth inhibition of human prostate cells in vitro by novel inhibitors of androgen synthesis. Cancer Res., 56, 4956–4964.[Abstract]
  65. Du,J., Peng,T., Scheidegger,K.J. and Delafontaine,P. (1999) Angiotensin II activation of insulin-like growth factor 1 receptor transcription is mediated by a tyrosine kinase-dependent redox-sensitive mechanism. Arterioscler. Thromb. Vasc. Biol., 9, 2119–2126.
  66. Rao,G.N., Alexander,R.W. and Runge,M.S. (1995) Linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, stimulate c-Fos, c-Jun and c-Myc mRNA expression, mitogen-activated protein kinase activation and growth in rat aortic smooth muscle cells. J. Clin. Invest., 96, 842–847.[ISI][Medline]
  67. Munger,K.A., Montero,A., Fukunaga,M., Uda,S., Yura,T., Imai,E., Kaneda,Y., Valdivielso,J.M. and Badr,K.F. (1999) Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis. Proc. Natl Acad. Sci. USA, 96, 13375–13380.[Abstract/Free Full Text]
Received April 27, 2000; revised June 20, 2000; accepted June 28, 2000.