Prostate tumor growth can be modulated by dietarily targeting the 15-lipoxygenase-1 and cyclooxygenase-2 enzymes.

The main objectives of our study were to determine the bioavailability of omega-3 (omega-3) to the tumor, to understand its mechanisms, and to determine the feasibility of targeting the omega-6 polyunsaturated fatty acids (PUFAs) metabolizing 15-lipoxygenase-1 (15-LO-1) and cyclooxygenase-2 (COX-2) pathways. Nude mice injected subcutaneously with LAPC-4 prostate cancer cells were randomly divided into three different isocaloric (and same percent [%] of total fat) diet groups: high omega-6 linoleic acid (LA), high omega-3 stearidonic acid (SDA) PUFAs, and normal (control) diets. Tumor growth and apoptosis were examined as end points after administration of short-term (5 weeks) omega-3 and omega-6 fatty acid diets. Tumor tissue membranes were examined for growth, lipids, enzyme activities, apoptosis, and proliferation. Tumors from the LA diet-fed mice exhibited the most rapid growth compared with tumors from the control and SDA diet-fed mice. Moreover, a diet switch from LA to SDA caused a dramatic decrease in the growth of tumors in 5 weeks, whereas tumors grew more aggressively when mice were switched from an SDA to an LA diet. Evaluating tumor proliferation (Ki-67) and apoptosis (caspase-3) in mice fed the LA and SDA diets suggested increased percentage proliferation index from the omega-6 diet-fed mice compared with the tumors from the omega-3 SDA-fed mice. Further, increased apoptosis was observed in tumors from omega-3 SDA diet-fed mice versus tumors from omega-6 diet-fed mice. Levels of membrane phospholipids of red blood cells reflected dietary changes and correlated with the levels observed in tumors. Linoleic or arachidonic acid and metabolites (eicosanoid/prostaglandins) were analyzed for 15-LO-1 and COX-2 activities by high-performance liquid chromatography. We also examined the percent unsaturated or saturated fatty acids in the total phospholipids, PUFA omega-6/omega-3 ratios, and other major enzymes (elongase, Delta [Delta]-5-desaturase, and Delta-6-desaturase) of omega-6 catabolic pathways from the tumors. We observed a 2.7-fold increase in the omega-6/omega-3 ratio in tumors from LA diet-fed mice and a 4.2-fold decrease in the ratio in tumors from the SDA diet-fed mice. There was an increased Delta-6-desaturase and Delta-9 desaturase enzyme activities and reduced estimated Delta-5-desaturase activity in tumors from mice fed the SDA diet. Opposite effects were observed in tumors from mice fed the LA diet. Together, these observations provide mechanistic roles of omega-3 fatty acids in slowing prostate cancer growth by altering omega-6/omega-3 ratios through diet and by promoting apoptosis and inhibiting proliferation in tumors by directly competing with omega-6 fatty acids for 15-LO-1 and COX-2 activities.

Polyunsaturated fatty acid metabolizing 15-Lipoxygenase-1 (15-LO-1) expression in normal and tumorigenic human bladder tissues.

Diets high in fat seem to correspond with an increased risk of certain forms of cancer, including bladder BlCa. This preliminary study examined the expression and enzyme activity profile of the polyunsaturated fatty acid metabolizing enzyme 15-Lipoxygenase-1 (15-LO-1) in human tissues from normal bladder and bladder tumors (stages CIS-T3/T4). Human tissue samples from normal (donor) bladder and bladder tumors (stages CIS-T3/T4; non-Bacillus Calmette-Guerin-treated) were grossly microdissected and analyzed for 15-LO-1 protein expression [immunohistochemistry (IHC)/Western blot], mRNA expression (quantitative real-time polymerase chain reaction) and enzyme activity profiles. Our results demonstrated that 15-LO-1 expression (protein/mRNA) and enzyme activity varied with BlCa progression. Specifically, IHC analyses of 15-LO-1 protein levels revealed decreased expression with increased bladder tumor stage. In particular, a statistically significant decrease in 15-LO-1 expression in stage T3/T4 bladder tumors compared with normal tissues (P<0.001) was observed. In agreement with IHC results, Western blot, quantitative real-time polymerase chain reaction, and enzymatic activity analyses demonstrated increased 15-LO-1 protein, mRNA, and enzyme activity, respectively, in normal human bladder tissues in comparison with stage T3/T4 human bladder tumors. Our finding of variable 15-LO-1 expression and enzyme activity in bladder tissues suggests a role for 15-LO-1 in bladder carcinogenesis.

Orthotopic expression of human 15-lipoxygenase (LO)-1 in the dorsolateral prostate of normal wild-type C57BL/6 mouse causes PIN-like lesions.

The lipid-peroxidating enzyme, 15-lipoxygenase (LO)-1 and its metabolite, 13-S-hydroxyoctadecadienoic acid (13-S-HODE), likely contribute to prostate tumorigenesis. Thus, this study evaluated adenovirus-mediated overexpression of 15-LO-1 on normal mouse prostate. Adenovirus expressing either human 15-LO-1 tagged with green fluorescent protein (GFP) or GFP alone was orthotopically injected into the dorsolateral prostates of C57BL/6 mice, three times over the course of 60 days. On day 90, pathological changes in prostate tissue were assessed by hematoxylin and eosin (H&E) staining. Expression of the proliferation marker Ki-67 was evaluated by immunohistochemistry and expression of angiogenesis markers were analyzed by an antibody array. Based on the latter study, immunoprecipitation analysis was used to measure the effect of 13-S-HODE, with or without conditioned media, on fibroblast growth factor-a and b (FGF-a and FGF-b) expression in human PrEC (normal prostate epithelial), PrSMC (normal prostate smooth muscle) and PrSC (normal prostate stromal) lines. Expression of viral 15-LO-1-GFP, but not GFP alone, resulted in the development of a prostate intraepithelial neoplasia (PIN)-like phenotype with increased expression of Ki-67. Aberrant 15-LO-1 expression also induced the angiogenic markers FGF-a and FGF-b. Human PrEC, PrSMC and PrSC lines demonstrated an increase in FGF-b expression upon stimulation with 13-S-HODE, which was further increased by the addition of conditioned media from the epithelial or smooth muscle cells. Using adenoviral mediated 15-LO-1 gene delivery, this study suggests that aberrant 15-LO-1 overexpression in normal prostate can trigger events leading to prostate epithelial and stromal cell proliferation. Thus, our findings demonstrate the effectiveness of this viral system for 15-LO-1 expression studies in tissues.

Conditional expression of human 15-lipoxygenase-1 in mouse prostate induces prostatic intraepithelial neoplasia: the FLiMP mouse model.

The incidence and mortality of prostate cancer (PCa) vary greatly in different geographic regions, for which lifestyle factors, such as dietary fat intake, have been implicated. Human 15-lipoxygenase-1 (h15-LO-1), which metabolizes polyunsaturated fatty acids, is a highly regulated, tissue-specific, lipid-peroxidating enzyme that functions in physiological membrane remodeling and in the pathogenesis of atherosclerosis, inflammation, and carcinogenesis. We have shown that aberrant overexpression of 15-LO-1 occurs in human PCa, particularly high-grade PCa, and in high-grade prostatic intraepithelial neoplasia (HGPIN), and that the murine orthologue is increased in SV40-based genetically engineered mouse (GEM) models of PCa, such as LADY and TRansgenic Adenocarcinoma of Mouse Prostate. To further define the role of 15-LO-1 in prostate carcinogenesis, we established a novel GEM model with targeted overexpression of h15-LO-1 in the prostate [human fifteen lipoxygenase-1 in mouse prostate (FLiMP)]. We used a Cre- mediated and a loxP-mediated recombination strategy to target h15-LO-1 specifically to the prostate of C57BL/6 mice. Wild-type (wt), FLiMP+/-, and FLiMP+/+ mice aged 7 to 21, 24 to 28, and 35 weeks were characterized by histopathology, immunohistochemistry (IHC), and DNA/RNA and enzyme analyses. Compared to wt mice, h15-LO-1 enzyme activity was increased similarly in both homozygous FLiMP+/+ and hemizygous FLiMP+/- prostates. Dorsolateral and ventral prostates of FLiMP mice showed focal and progressive epithelial hyperplasia with nuclear atypia, indicative of the definition of mouse prostatic intraepithelial neoplasia (mPIN) according to the National Cancer Institute. These foci showed increased proliferation by Ki-67 IHC. No progression to invasive PCa was noted up to 35 weeks. By IHC, h15-LO-1 expression was limited to luminal epithelial cells, with increased expression in mPIN foci (similar to human HGPIN). In summary, targeted overexpression of h15-LO-1 (a gene overexpressed in human PCa and HGPIN) to mouse prostate is sufficient to promote epithelial proliferation and mPIN development. These results support 15-LO-1 as having a role in prostate tumor initiation and as an early target for dietary or other prevention strategies. The FLiMP mouse model should also be useful in crosses with other GEM models to further define the combinations of molecular alterations necessary for PCa progression.

Prostate tumor growth and recurrence can be modulated by the omega-6:omega-3 ratio in diet: athymic mouse xenograft model simulating radical prostatectomy.

Evidence indicates that a diet rich in omega (omega)-6 polyunsaturated fatty acids (PUFAs) [e.g., linoleic acid (LA)] increases prostate cancer (PCa) risk, whereas a diet rich in omega-3 decreases risk. Precisely how these PUFAs affect disease development remains unclear. So we examined the roles that PUFAs play in PCa, and we determined if increased omega-3 consumption can impede tumor growth. We previously demonstrated an increased expression of an omega-6 LA-metabolizing enzyme, 15-lipoxygenase-1 (15-LO-1, ALOX15), in prostate tumor tissue compared with normal adjacent prostate tissue, and that elevated 15-LO-1 activity in PCa cells has a protumorigenic effect. A PCa cell line, Los Angeles Prostate Cancer-4 (LAPC-4), expresses prostate-specific antigen (PSA) as well an active 15-LO-1 enzyme. Therefore, to study whether or not the protumorigenic role of 15-LO-1 and dietary omega-6 LA can be modulated by altering omega-3 levels through diet, we surgically removed tumors caused by LAPC-4 cells (mouse model to simulate radical prostatectomy). Mice were then randomly divided into three different diet groups-namely, high omega-6 LA, high omega-3 stearidonic acid (SDA), and no fat-and examined the effects of omega-6 and omega-3 fatty acids in diet on LAPC-4 tumor recurrence by monitoring for PSA. Mice in these diet groups were monitored for food consumption, body weight, and serum PSA indicative of the presence of LAPC-4 cells. Fatty acid methyl esters from erythrocyte membranes were examined for omega-6 and omega-3 levels to reflect long-term dietary intake. Our results provide evidence that prostate tumors can be modulated by the manipulation of omega-6:omega-3 ratios through diet and that the omega-3 fatty acid SDA [precursor of eicosapentaenoic acid (EPA)] promotes apoptosis and decreases proliferation in cancer cells, causing decreased PSA doubling time, compared to omega-6 LA fatty acid, likely by competing with the enzymes of LA and AA pathways, namely, 15-LO-1 and cyclooxygenases (COXs). Thus, EPA and DHA (major components of fish oil) could potentially be promising dietary intervention agents in PCa prevention aimed at 15-LO-1 and COX-2 as molecular targets. These observations also provide clues as to its mechanisms of action.

15-lipoxygenase-1 expression upregulates and activates insulin-like growth factor-1 receptor in prostate cancer cells.

We previously discovered that a fat-metabolizing enzyme, 15-lipoxygenase-1 (15-LO-1), is high in human prostate cancer (PCa) and correlates with disease progression. The biologic link between the aberrant 15-LO-1/linoleic acid (LA) metabolism and fat (which is a rich source of growth factors) in PCa is unknown. Therefore, we tested the hypothesis that the metabolic product of the polyunsaturated fatty acid LA (i.e., 13-S-hydroxyoctadecadienoic acid or 13-(S)-HODE) affects the proliferation status of PCa cells through one or more growth factors. We used parental prostate cancer cell line-3 (PC-3) and engineered PC-3 cell lines [PC3-Zeo (mock-transfected), PC3-15LOS (15-LO-1-overexpressing), and PC3-15LOAS (15-LO-1-blocked)] to test our hypothesis. Of the growth factors examined, only insulin-like growth factor-1 (IGF-1) exhibited a two-fold to three-fold increase in growth response on PC3-15LOS cells compared to PC3-Zeo (control) cell line (P <.01). Insulin-like growth factor-1 receptor (IGF-1R) immunohistochemical analyses of human normal and adenocarcinoma prostate tissues, as well as levels in tumors derived from nude mice injected with PC-3 cells, demonstrated that elevated IGF-1R expression correlated with 15-LO-1 levels. Radioligand binding assays demonstrated two-fold higher IGF-1 binding sites in PC3-15LOS cells (P <.05 vs PC3-Zeo cells). IGF-1R promoter reporter assay and affinity-purified IGF-1R receptor levels demonstrated a four-fold higher activity in PC3-15LOS cells (P <.01 vs PC3-Zeo cells). IGF-1R promoter activation is 13-(S)-HODE-dependent. IGF-1R blockade with a dominant-negative adenovirus caused significant growth inhibition in PC-3 cells (P <.0001; PC3-15LOAS versus PC3-15LOS cells), as well as affected the IGF-1-stimulated mitogen-activated protein (MAP) kinase (Erk1/2) and Akt activation levels. Our study suggests that overexpression of 15-LO-1 in PCa contributes to the cancer progression by regulating IGF-1R expression and activation.

Overexpression of 12/15-lipoxygenase, an ortholog of human 15-lipoxygenase-1, in the prostate tumors of TRAMP mice.

Changes in the expression and activity of lipid-metabolizing enzymes, including the linoleic acid (LA)-metabolizing enzyme 15-lipoxygenase-1 (15-LO-1), may play a role in the development and progression of human prostate carcinoma (PCa). We reported that human 15-LO-1 (designated as leukocyte type 12-LO or 12/15-LO in mouse) is expressed in human prostate and increased in PCa, particularly high-grade PCa. Genetically engineered mouse (GEM) models of PCa could facilitate the study of this gene and its regulation and function in PCa progression. In this study, we examine the protein expression and enzyme activity levels of 12/15-LO associated with PCa progression in the TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) model of PCa. This GEM model develops prostatic intraepithelial neoplasia (PIN), followed by invasive gland-forming PCa and invasive and metastatic less differentiated PCa, with neuroendocrine (NE) differentiation (NE Ca). In the wild-type and TRAMP prostates, the most prominent LA metabolite was 13-hydroxyoctadecadienoic acid (13-HODE). Lesser amounts of 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid (HETE) were made from arachidonic acid (AA). In TRAMP prostates, 12/15-LO activity was increased compared to wild type at 20, 29, 39, and 49 weeks, as assessed by LA conversion to 13-HODE, and by AA conversion to 12/15-HETE, respectively. Immunostaining demonstrated that the increased capacity to generate 13-HODE was paralleled by an increase in neoplastic epithelial expression of 12/15-LO in PIN and invasive carcinomas. In conclusion, although there is a basal 12/15-LO activity in the wild-type mouse prostate, there is a marked increase in the expression of 12/15-LO with TRAMP PCa progression, paralleling our previously reported increased expression of the ortholog 15-LO-1 in high-grade human PCa. Thus, 12/15-LO and LA metabolism in the TRAMP model shares similarities to human PCa, and may allow to confirm a role for LA metabolism and other biologic functions of 15-LO-1 in human PCa. In addition, the TRAMP model will serve as a tool for testing the suitability of 12/15-LO-and ultimately human 15-LO--as a therapeutic target during PCa progression.