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

Abstract

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.

Figure 1

RT-PCR of 15-LO-1 and β-actin expression in LAPC-4 and PC3-15LOS cells (control).

Figure 2

PUFA modulation of LAPC-4 proliferation. Cells were propagated on medium containing 10% FBS. Monolayers were harvested, and cells were plated (5 x 10³ cells/well and considered as 100% cells) in 96-well plates on a medium either containing different concentrations (0–300 µM) or lacking (control) EPA (△-△), DHA (●-●), EPA + DHA (◆-◆), and 15-HEPE (□-□). Viable cell content was determined by quantification of formazan production. For clarity, SD error bars from the mean of quadruplicate wells (SD ≤ 10% of the mean) have been omitted.

Figure 3

Effect of LA (100 µM; ■-■), AA (100 µM; △-△), LA + EPA (50 µM; ●-●), LA + EPA + DHA (50 µM; □-□), AA + EPA (○-○), and AA + EPA + DHA (◆-◆) on the growth of 5 x 10³ cells/well LAPC-4 in vitro. The untreated cells were used as control (▽-▽). Each point represents the mean ± SD of triplicate determinations from two separate experiments. For clarity, SD error bars from mean of triplicate wells (SD ≤ 10% of the mean) have been omitted. Values are half times for either population doubling (positive values) or viable cell loss (negative values). Half times were obtained by the solution of regression equations.

Figure 4

MSRP. Male athymic male BALB/C nude (nu/nu) mice (6–8 weeks old) fed for 9 weeks. LAPC-4 cells were injected subcutaneously on week 1, and tumor growth was monitored for 9 weeks. On week 9, tumors were surgically removed and mice were randomly assigned to high ω-6 LA, high ω-3 SDA, and no-fat diet groups and fed accordingly until the termination of the experiment on week 15. Serum PSA was measured on weeks 1, 7, 9, 12, and 15, respectively. Tumor volume, food consumption, and mouse weight were measured every week.

Figure 5

Average tumor volume per week in experimental mice. Average tumor volume (mm³; dotted line) per mouse was examined every week up to week 9 (before tumor resection) in the no-fat diet group (n = 72).

Figure 6

(A) Average PSA per tumor volume in the no-fat diet-fed mice on week 7 vs week 9 (n = 72). ***P = .2. (B) Number of mice showing tumor recurrence, average tumor volume, and PSA, after tumor resection, in mice fed no-fat, high ω-3 SDA, and high ω-6 LA diets on week 12 vs week 15. NO, not observed. *Estimated value.

Figure 7

Composition of LA, AA, EPA, and DHA in erythrocyte phospholipids (n = 3) from mice fed no-fat, high ω-3 SDA, and high ω-6 LA diets on (A) week 12 vs (B) week 15. Fatty acid methyl esters from erythrocytes were analyzed by gas liquid chromatography, as described in Materials and Methods section. The fatty acids are expressed as percentages of total phospholipids. *P < .01, **P < .05, ***P = .2, ****P = .3.

Figure 8

Representative immunostaining (A–D) and hematoxylin and eosin (H&E) staining (E and F) of tumors from mice fed high ω-3 SDA and high ω-6 LA diets on week 15 with polyclonal antibody for 15-LO-1 (brown), dual staining with Ki-67 (brown) as a proliferation marker, and caspase-3 (red; arrowheads) as an apoptosis marker (x40). ω-3 SDA (A, C, and E) and ω-3 SDA (B, D, and F). (A and B) 15-LO-1; (C and D) dual Ki-67/caspase-3; (E and F) H&E staining.

Figure 9

Assessment of (A) proliferation index and (B) apoptotic index in recurrent tumors on week 15 from mice fed high ω-3 SDA (n = 2) and high ω-6 LA diets (n = 2). Sections of formalin-fixed paraffin-embedded LAPC-4 tumor tissues were tested for the presence of 15-LO-1, Ki-67, and caspase-3. Proliferation and apoptotic indices (%) were estimated as described in Materials and Methods section.

Figure 10

Modulation of the enzymes of the LA and AA pathways by ω-3 and ω-6 fatty acids.