Opposing Effects of 15-Lipoxygenase-1 and -2 Metabolites on MAPK Signaling in Prostate

Human prostate tumors have elevated levels of 15-lipoxygenase-1 (15-LOX-1) and data suggest that 15-LOX-1 may play a role in the development of prostate cancer. In contrast, 15-LOX-2 expression is higher in normal rather than in tumor prostate tissue and appears to suppress cancer development. We recently reported that 13-(S)-HODE, the 15-LOX-1 metabolite, up-regulates the MAP kinase signaling pathway and subsequently down-regulates PPARγ in human colorectal carcinoma cells. To determine whether this mechanism is applicable to prostate cancer and what the effects of 15-LOX-2 are, we investigated the effect of 15-LOX-1, 15-LOX-2, and their metabolites on epidermal growth factor (EGF)- and insulin-like growth factor (IGF)-1 signaling in prostate carcinoma cells. In PC3 cells, 13-(S)-HODE, a 15-LOX-1 metabolite, up-regulated MAP kinase while in contrast 15-(S)-HETE, a 15-LOX-2 metabolite, down-regulated MAP kinase. As a result, 13-(S)-HODE increased PPARγ phosphorylation while a subsequent decrease in PPARγ phosphorylation was observed with 15-(S)-HETE. Thus, 15-LOX metabolites have opposing effects on the regulation of the MAP kinase signaling pathway and a downstream target of MAP kinase signaling like PPARγ. In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.

In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.
Human prostate tumors have higher expression of 15-LOX-1 compared with normal adjacent tissue and this expression correlates with the Gleason score of the cancer (5). 13-(S)-HODE, the 15-LOX-1 metabolite, is detected in adenocarcinoma tissue (5). Furthermore, nude mice injected with 15-LOX-1-overexpressing prostate cells have increased frequency and size of tumors compared with nude mice injected with control cells (6). These data taken together suggest a possible pro-tumorigenic role for 15-LOX-1 in prostate tumor development. In contrast, 15-LOX-2 is expressed in normal prostate tissue, but poorly expressed in prostate tumors (3). The 15-LOX-2 expression is inversely correlated with the Gleason score of the tumor (3). Furthermore, 15-(S)-HETE is detected in benign prostate tissue samples, and 15-LOX-2 is a negative cell cycle regulator in normal prostate epithelial cells (3,7). These results suggest there may be different, if not opposing, biological functions for 15-LOX-1 and 15-LOX-2 in the prostate.
Metabolites of 15-LOX have recently been reported to serve as ligands for PPAR␥. 13-(S)-HODE, 13-(S)-HpODE, and 15-(S)-HETE all show binding activity for PPAR␥ but at relatively high concentrations (19,20). Also, recently, it was shown that exogenous 15-(S)-HETE could activate PPAR␥ using luciferase reporter assays and inhibit proliferation in prostate cells (8). Thus, based on activation of PPAR␥, one could conclude that 15-LOX-1 may have antitumorigenic activity. However, this hypothesis is in conflict with the elevated 15-LOX-1 expression in tumors versus normal tissue and with observed increased * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recently, we reported that 15-LOX-1 linoleic acid metabolites up-regulate the MAP kinase signaling pathway (22). MAP kinase can subsequently phosphorylate PPAR␥, one potential downstream target of MAP kinase, and thus down-regulate PPAR␥ activity in human colorectal carcinoma cells (22). The up-regulation of the MAP kinase pathway provides a rationale to explain how 15-LOX-1 may play a protumorigenic role and may provide a clue to the difference between the biological function of 15-LOX-1 and 15-LOX-2. The aim of the present study is: 1) to compare the effects of 13-(S)-HODE, a 15-LOX-1 metabolite, to the effects of 15-(S)-HETE, a 15-LOX-2 metabolite, on MAP kinase signaling in prostate, 2) to determine whether changes in MAP kinase alter PPAR␥ in prostate, and 3) to determine whether any phenomenon observed is unique to EGF signaling.  (30 M) was added to the cells as described above in RPMI without phenol red prior to treatment with EGF. After stimulation with EGF or IGF-1, cells were harvested at the various time points indicated. Normal human tracheobronchial epithelial (NHTBE) cells, as previously described (23), were used as a positive control for the expression of 15-LOX. Differentiated 3T3-L1 cells, as previously described (24), were used as a positive control for the expression of PPAR␥.
Analysis of Arachidonic Acid and Linoleic Acid Metabolites in Intact cells-PC3 or 15-LOX-1 clone cells cultured in 150-cm 2 dishes at each condition were washed with serum-free medium twice. 10 ml of phosphate-buffered saline supplemented with 10 M CaCl 2 was then added to each plate, the appropriate treatments were added, and each plate was incubated for 15 min at 37°C. Nordihydroguaiaretic acid (NDGA) was used at a concentration of 10 M. Each plate was reacted with [ 3 H]arachidonic acid (3 Ci, 30 M) (PerkinElmer Life Sciences) or [ 14 C]linoleic acid (3 Ci, 30 M) (PerkinElmer Life Sciences) for 1 h. at 37°C. The media were collected, and each plate was washed with 2 ml of MeOH and 2 ml of 1% acetic acid. The cells were scraped into this wash and added to the appropriate tube containing the media previously collected. The total collected media was acidified with acetic acid to pH 3 and applied to a C 18 -PrepSep solid phase extraction column (Waters) pretreated with methanol. The samples were washed with acidified water, eluted with methanol, evaporated to dryness, and reconstituted with high pressure liquid chromatography (HPLC) solvent.
High Pressure Liquid Chromatography-Reverse-phase HPLC analysis was performed using an Ultrasphere ODS column (5 m; 4.6 ϫ 250 mm; Beckman). The solvent system consisted of a methanol/water gradient at a flow rate of 1.1 ml/min as previously described (26). Radioactivity was monitored using a Flow Scintillation Analyzer (Packard) with EcoLume (ICN Biochemicals) as the liquid scintillation mixture. Authentic standards of 13-(S)-HODE and 15-(S)-HETE (Cayman Chemical) were used.
Analysis of Densitometry Measurements-Autoradiograms from Western blots were scanned using a Umax™ Powerlook III™ scanner equipped with transparency adapter and scanning software. Bands were quantitated using Scion Image™ beta version 4.0.2. Western blot values were first corrected using their corresponding actin levels. Values shown are fold increases versus vehicle or zero hour as illustrated in the figure legends. Statistical analysis using Student's t test was also performed.

Endogenous 15-LOX-1, 15-LOX-2, and PPAR␥ Expression in
PC3 Cells-PC3 cells, a human prostate cancer cell line, were used for the experimental studies to follow. Basal levels of 15-LOX-1, 15-LOX-2, and PPAR␥ expression were first confirmed by Western analysis (data not shown). PC3 cells lack endogenous 15-LOX-1 and 15-LOX-2 expression, while PPAR␥1 is detectable. Differentiated 3T3-L1 cells, which express both PPAR␥1 and PPAR␥2 isoforms, were used as a positive control for PPAR␥ (27). No functional difference has been found between the two isotypes. PC3 cells express only the PPAR␥1 isoform. From this point on, we will refer to PPAR␥1 as PPAR␥ since all the subsequent experiments are done in PC3 cells.

Effect of 13-(S)-HODE or 15-(S)-HETE on MAPK
Phosphorylation-Following serum deprivation, PC3 cells were pretreated with 5 M 13-(S)-HODE or 10 M 15-(S)-HETE for 45 min prior to treatment with EGF (10 ng/ml). The effect of 13-(S)-HODE or 15-(S)-HETE on MAP kinase after EGF stimulation was examined by Western analysis at the indicated time points (Fig. 1). Using a phosphospecific MAPK antibody, an increase in MAPK phosphorylation was observed in cells treated with 13-(S)-HODE compared with cells treated with EGF alone (Fig. 1A). A 2.2 Ϯ 0.2-fold increase (n ϭ 3) (p Ͻ 0.001) in MAPK phosphorylation could be detected after treatment. In contrast to this, a 2.8 Ϯ 0.3-fold decrease (n ϭ 3) (p Ͻ 0.001) in MAPK phosphorylation was observed in cells treated with 15-(S)-HETE compared with cells treated with EGF alone (Fig. 1B). Total MAP kinase expression levels were also examined by antibodies to ERK-1 and ERK-2 for this experiment and all subsequent experiments measuring phosphorylated MAPK. In all cases, total MAP kinase levels did not change, and thus only the levels of phosphorylated MAPK were altered by treatment with 13-(S)-HODE, 15-(S)-HETE, or the treatment indicated for each experiment (data not shown). The density of the phosphorylated proteins (ERK1/2) was measured, normalized to actin, and are reported in the parentheses below the blots.  (Fig. 2A). An increase in MAPK phosphorylation was observed at concentrations as low as 0.1 M, but the strongest increase in MAPK phosphorylation was detected at 5 M 13-(S)-HODE. Conversely, a decrease in MAPK phosphorylation was observed in cells treated with 15-(S)-HETE compared with the cells treated with EGF alone (Fig. 2B). A decrease in MAPK phosphorylation was observed at 1 M, but 10 M was an optimal concentration. Total MAP kinase expression levels were also examined using antibodies to ERK-1 and ERK-2. No changes in total MAP kinase expression were noted. Thus, the response to lipid metabolites is to alter phosphorylation of MAP kinase. linoleic acid or 30 M arachidonic acid for 45 min prior to treatment with EGF (10 ng/ml). MAP kinase phosphorylation after EGF stimulation was examined by Western analysis at the indicated time points (Fig. 3). Actin expression was measured and used to normalize the data. The normalized density measurements are reported in the brackets. A 1.9 Ϯ 0.1-fold increase (n ϭ 3) (p Ͻ 0.002) in MAPK phosphorylation was observed in cells treated with linoleic acid plus EGF over the only EGF-treated cells (Fig. 3A). In contrast, the addition of arachidonic acid plus EGF to 15-LOX-1 PC3 cells caused a 2.5 Ϯ 0.1-fold decrease (n ϭ 3) (p Ͻ 0.002) in MAPK phosphorylation compared with only EGF-treated cells (Fig. 3B). Thus, endogenously produced 15-LOX metabolites had a similar effect on MAPK as exogenously added metabolites.
To determine if endogenously formed 13-(S)-HODE or 15-(S)-HETE also alters PPAR␥ phosphorylation, the 15-LOX-1-overexpressing PC3 cells were incubated with linoleic acid or arachidonic acid. PPAR␥ phosphorylation was increased by 1.7 Ϯ 0.2-fold (n ϭ 3) (p Ͻ 0.009) with linoleic acid (Fig. 5A). In contrast, incubation of the overexpressing cells with arachi-   (Fig. 6). This is consistent with inhibition of MEK activity. Likewise, PPAR␥ phosphorylation was ablated (Fig. 7). Total MAP kinase levels did not change, only the levels of phosphorylated MAPK were altered by treatment with 13-(S)-HODE or 15-(S)-HETE (data not shown). Normalization of the phosphorylation density measurements was performed, and the values are reported in parentheses. These results are consistent with the hypothesis that phosphorylation of PPAR␥ observed upon treatment with 15-LOX metabolites in EGF-stimulated cells is dependent on MAP kinase activity in prostate cells.

Effect of 13-(S)-HODE or 15-(S)-HETE on Akt in IGF-1treated
Cells-In addition to activating the MAPK signaling pathway, IGF-1 also can activate the PI3 kinase/Akt pathway. To determine whether this pathway is also affected by the 15-LOX metabolites, phosphorylated Akt was examined using an Akt-phosphospecific antibody. An increase in Akt phosphorylation was observed with 13-(S)-HODE plus IGF-1 (1.5 Ϯ 0.1-fold, n ϭ 3) (p Ͻ 0.004) while a decrease in Akt phosphorylation was observed with 15-(S)-HETE plus IGF-1 (1.4 Ϯ 0.05-fold, n ϭ 3) (p Ͻ 0.008) as compared with only IGF-1stimulated cells (Fig. 9). Total Akt protein levels were not altered by the treatments (data not shown). Interestingly, the changes in phosphorylation of Akt by the 15-LOX metabolites was not as great as that observed on MAPK phosphorylation. A 2-fold or greater change in MAPK phosphorylation was observed upon addition of the metabolites in the presence of IGF-1. However, a less than 2-fold change in Akt phosphorylation was observed.

DISCUSSION
Previously, we have shown that the 15-LOX-1 metabolite, 13-HODE, increases EGF-dependent MAPK activity, which in turn can cause a decrease in PPAR␥ activity in colorectal cancer cells (22). In the current study, we have furthered our studies to include 15-LOX-2 and prostate cells since 15-LOX-1 and 15-LOX-2 differ in their expression in tumor and normal prostate tissue. The difference between 15-HETE, the major metabolite of 15-LOX-2, and 13-HODE, the major metabolite of 15-LOX-1, effects on MAPK activity was investigated. Both exogenous and endogenous 13-HODE increases MAPK activity and thus decreases PPAR␥ activity. In contrast, both exogenous and endogenous 15-HETE decreases MAPK activity, which in turn results in an increase in PPAR␥ activity. In addition, the effects of the 15-LOX metabolites on MAPK are not unique to EGF signaling, as the IGF signaling pathway and Akt phosphorylation are also affected in prostate cells. These findings are of particular interest given the fact that 15-LOX-1 expression is higher in tumors whereas 15-LOX-2 expression is higher in normal prostate tissue. Also, given the importance of the IGF/Akt pathway in prostate tissue, showing the metabolites alter this signaling pathway is particularly relevant to tumor development. Thus, the findings presented here advance our understanding of the two human 15-LOX enzymes, 15-LOX-1 and 15-LOX-2, which differ in their expression and their apparent biological activity in human prostate.
In prostate, higher expression of 15-LOX-1 in tumors is linked to Gleason score of the tumor whereas 15-LOX-2 is expressed in normal prostate tissue and inversely correlates with Gleason score (3,5). Based upon several experimental findings, 15-LOX-1 appears to have a pro-tumorigenic activity while 15-LOX-2 appears to have anti-tumorigenic activity (6,7). These results thus suggest opposing biological functions for 15-LOX-1 and 15-LOX-2 in the prostate. The results presented here provide a rationale for understanding the different biological activity between 15-LOX-1 and 15-LOX-2 in human prostate and is based, in part, on differences in substrate preference for the two enzymes. 13-HODE and 15-HETE bind and activate PPAR␥ in vitro suggesting that the metabolites may function as endogenous ligands for PPAR␥ (19). However, these findings are observed with high concentrations of the metabolites (30 -100 M) and most of these studies employ only exogenous metabolites (19,20). The physiological relevance of these lipid substances as regulators of PPAR␥ in vivo is not fully established. One potential downstream target of MAPK signaling is PPAR␥. Phosphorylation of PPAR␥ results in a decrease in transcriptional activity (28 -31). MAP kinase, a central regulator of cell growth, phosphorylates a key residue, Ser-82 on PPAR␥1, which results in a decrease in PPAR␥ transcriptional activity (28,29,32). In PC3 cells, endogenous 15-LOX metabolites have opposing effects on the regulation of MAP kinase activity and have opposing effects on PPAR␥ activity. Based on data from this and previous studies (8,22), it appears that down-regulation of PPAR␥ activity is specific for linoleic acid metabolites as 13-(S)-HODE, 13-(R)-HODE, and 13-(S)-HpODE all increase MAPK activity and hence PPAR␥ phosphorylation whereas 15-(S)-HETE, an arachidonic acid metabolite, has the opposite effect. These effects on MAP kinase are observed with endogenously generated metabolites and the addition of the 15-LOX metabolites alters MAP kinase at lower concentrations than required to observe PPAR␥ ligand binding in vitro. Thus, these findings indicate that endogenous 15-LOX metabolites alter PPAR␥ transcriptional activity via MAPK phosphorylation rather than acting as a ligand for this receptor. PPAR␥ agonists inhibit proliferation and potentially induce differentiation in many carcinoma cell lines (8, 10 -18), suggesting that PPAR␥ could act as a tumor suppressor. The 15-LOX metabolites appear to modulate this activity via MAPK phosphorylation.
In addition to the EGF signaling, IGF-1 signaling appears to play an important role in prostate cancer. Epidemiologic studies suggest an association between increased serum levels of IGF-1 and an increased risk of prostate cancer (33). Furthermore, transgenic mice expressing human IGF-1 in basal epithelial cells of prostate led to activation of the IGF-1R and spontaneous tumorigenesis in prostate epithelium (34,35). In response to IGF-1, the Raf-MEK-ERK and PI3K-Akt signaling pathways are often simultaneously activated and play important roles in IGF-1R-induced cellular proliferation and the inhibition of apoptosis. Traditionally, the Ras/Raf/MAP kinase pathway was thought to primarily mediate the cell proliferative response to IGF-1, whereas the PI3 kinase pathway, which activates Akt/PKB, was primarily implicated in mediating the anti-apoptotic effects of IGF-1 (36 -38). However, recent studies have demonstrated a role for both pathways in mediating both responses. Coordination of the two pathways in a single cellular response may depend on cell type or the stage of differentiation (39 -41).
In prostate PC3 cells, 13-(S)-HODE and 15-(S)-HETE upregulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. However, the magnitude of the response on MAPK is greater than on Akt. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. Exactly where in the signaling pathway the 15-LOX metabolites are regulating MAP kinase in the prostate carcinoma cells is not known and remains to be elucidated. Nonetheless, it appears that regulation of MAP kinase by 15-LOX metabolites may have important implications relevant to tumorigenesis, particularly in the prostate.
Opposing biological actions by eicosanoids is frequently observed. For example, the conversion of arachidonic acid to thromboxane A 2 promotes platelet aggregation and vasoconstriction, whereas the formation of prostacyclin inhibits platelet aggregation and promotes vasodilation (42). The two lipoxygenases, 15-LOX-1 and 15-LOX-2, are other examples where the balance between two enzymes that metabolize cis-unsatur- Growth factors (such as EGF or IGF) interact with their respective receptor that activates MAP kinase signaling. Endogenous 13-HODE up-regulates growth factor initiated MAP kinase activity and as a result, MAP kinase-dependent PPAR␥ phosphorylation is increased. This causes a down-regulation or loss of PPAR␥ transcriptional activity. In contrast, 15-HETE has the opposite effect, causing a down-regulation of MAPK activity. 13-HODE and 15-HETE are also thought to be endogenous ligands for PPAR␥ and cause activation of PPAR␥ receptor. However, a more likely scenario may be to regulate PPAR␥ through the MAPK signaling pathway. Thus a balance between these two opposing effects of 15-LOX metabolites may determine the role they play in the development of prostate cancer. Perhaps there is a shift in expression of 15-LOX-2 to 15-LOX-1 as a cell undergoes neoplastic progression from a normal cell to a tumor cell. ated fatty acids appear to antithetically modulate the activity of a key pathway regulating biological events. In this particular case, a balance between two opposing effects could determine the role 15-LOX plays in the development of prostate cancer. Perhaps there is a shift in expression of 15-LOX-2 to 15-LOX-1, and presumably a shift from 15-(S)-HETE to 13-(S)-HODE, as a cell undergoes neoplastic progression from a normal cell to a tumor cell (Fig. 10). These metabolites exert a dramatically opposite effect on EGF and IGF-1 signaling pathways leading to an increase or decrease in MAP kinase activity. MAP kinase may be a key player in determining whether a pro-tumorigenic activity or an anti-tumorigenic response is observed. Loss of 15-LOX-2 expression and gain of 15-LOX-1 may contribute to prostate cancer development and progression. Thus, a shift from 15-LOX-2 expression to 15-LOX-1 expression may serve as a useful marker of prostate cancer development. Further investigation of 15-LOXs in prostate will need to be done to clarify their function in cancer development.