15-lipoxygenase metabolites of docosahexaenoic acid inhibit prostate cancer cell proliferation and survival

Abstract

A 15-LOX, it is proposed, suppresses the growth of prostate cancer in part by converting arachidonic, eicosatrienoic, and/or eicosapentaenoic acids to n-6 hydroxy metabolites. These metabolites inhibit the proliferation of PC3, LNCaP, and DU145 prostate cancer cells but only at ≥1-10 µM. We show here that the 15-LOX metabolites of docosahexaenoic acid (DHA), 17-hydroperoxy-, 17-hydroxy-, 10,17-dihydroxy-, and 7,17-dihydroxy-DHA inhibit the proliferation of these cells at ≥0.001, 0.01, 1, and 1 µM, respectively. By comparison, the corresponding 15-hydroperoxy, 15-hydroxy, 8,15-dihydroxy, and 5,15-dihydroxy metabolites of arachidonic acid as well as DHA itself require ≥10-100 µM to do this. Like DHA, the DHA metabolites a) induce PC3 cells to activate a peroxisome proliferator-activated receptor-γ (PPARγ) reporter, express syndecan-1, and become apoptotic and b) are blocked from slowing cell proliferation by pharmacological inhibition or knockdown of PPARγ or syndecan-1. The DHA metabolites thus slow prostate cancer cell proliferation by engaging the PPARγ/syndecan-1 pathway of apoptosis and thereby may contribute to the prostate cancer-suppressing effects of not only 15-LOX but also dietary DHA.

Figure 1. The proliferation responses of PC3, LNCaP, and DU145 prostate cancer cells to selected DHA and AA metabolites.

The indicated cell types were incubated for 3 days with the indicated metabolite and their proliferation presented as the mean ± SEM (≥3 independent experiments) fractions of that found in cells treated with the vehicle (culture media) for the metabolites.

Figure 2. Stimulatory effects of DHA metabolites on caspase, PPARγ activity, and SDC-1 expression in PC3 cells.

A. Cells were incubated with the indicated concentration of 17-HDHA or 17-HpDHA for 24 h and caspase-3 activity was measured by Caspase-Glo® 3/7 assay. Results are presented as mean ± SD (N = 3) relative to control cells treated with the medium for the metabolites. Responses to all doses at and above 10⁻⁸ M for 17-HpDHA and at or above 10⁻⁷ M for 17-HDHA were significantly greater than that of control cells (two way ANOVA, P<0.05). B. Cells transfected with luciferase PPARγ reporter gene were stimulated for 24 h with 10 µM of the indicated metabolite (the lowest dose where all had a clear effect on cell growth) or 5 µM of troglitazone and assayed for luciferase. Values represent the mean ± SD (N = 3). Bars labeled with the same letters are not significantly different from each other; bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05) C. Cells were treated with medium (control) or 10 µM of the indicated metabolite for 8 or 24 h and their SDC-1 mRNA was measured. Values represent the mean, ± SD (N = 3). Within each time group, bars labeled with the same letters are not significantly different from each other; bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05). D. Cells were treated with medium (control) or 10 µM of the indicated metabolite for 72 h and their lysates were analyzed for SDC-1. The Western blot is representative of 3 independent experiments. Values in graphs represent the mean ± SEM (N = 3 independent experiments). Bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05).

Figure 3. Inhibition of the effects of DHA metabolites in PC3 cells.

A. Cells not transfected or transfected with pcDNA3 or d/nPPARγ were challenged with 10 µM of the indicated metabolite for 24 h before assaying syndecan-1 mRNA. Values represent the mean ± SD (N = 3). Within a transfection group, bars labeled with the same letters are not significantly different from each other; bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05). B. Cells untransfected or transfected with pcDNA3 or d/nPPARγ were challenged with 10 µM of the indicated metabolite for 3 days before assaying proliferation. Values represent the mean ± SD (N = 3). Within a metabolite group, bars labeled with the same letters are not significantly different from each other; bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05). C. Cells were incubated with 0–1 µM of PPARγ antagonist, GW6692, for 30 min and with 10 µM of the indicated metabolite for 3 days before assaying proliferation. Results are presented as mean ± SEM (N = 3 independent experiments). Within a metabolite group, bars labeled with the same letters are not significantly different from each other; bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05). D. Cells untransfected, transfected with control siRNA, or transfected with SDC-1 siRNA were challenged with 10 µM of the indicated metabolite for 3 days before assaying proliferation. Results are presented as mean ± SD (N = 4). Within a transfection group, bars labeled with the same letters are not significantly different from each other; bars labeled with different letters are significantly different from each other (one-way ANOVA, P<0.05).