Long term consumption of flaxseed enriched diet decreased ovarian cancer incidence and prostaglandin E₂in hens

Abstract

Objective: Ovarian cancer is the most lethal gynecological malignancy. Prevention may be the best approach to reduce ovarian cancer. Flaxseed is the richest vegetable source of omega-3 fatty acids which may be effective in the prevention of ovarian cancer. Prostaglandin E₂ (PGE₂) is the most pro-inflammatory ecoisanoid and one of the downstream products of two isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Our objective was to determine if long-term consumption of a flaxseed enriched diet decreased ovarian cancer severity and incidence in the laying hen and to investigate its potential correlation with the expression of COX enzymes and PGE₂ concentration.

Methods: White Leghorn hens were fed 10% flaxseed-enriched or standard diet for 4years. The severity and incidence of ovarian cancer were determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE₂ concentrations in ovaries were measured by Western blot, quantitative real-time PCR and ELISA, respectively.

Results: The results demonstrated that there was a reduction in ovarian cancer severity and incidence in hens fed flaxseed diet. In correlation with decreased ovarian cancer severity and incidence, concentration of PGE₂ and expression of COX-2 were diminished in ovaries of hens fed flaxseed.

Conclusions: Our findings suggest that the lower levels of COX-2 and PGE₂ are the main contributing factors in the chemo-suppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Keywords: Cyclooxygenase; Flaxseed; Inflammation; Laying hen; Ovarian cancer; Prostaglandin E(2).

Fig. 1

(A) Gross anatomy of normal ovary with a hierarchy of developing follicles; (B) Ovary classified as a suspected; (C) Ovary taken from a hen with metastatic late stage ovarian cancer; (D) H&E stain of normal ovary with developing small follicles; (E) H&E stain of suspected ovary showed a disassociated stoma with unusually high numbers of blood vessels and formation of focal lesions in the stroma below the ovarian surface was apparent (arrow); (F) H&E stain of metastatic late stage ovarian cancer with solid areas composed of slit-like sheets containing cells with high-grade nuclear atypia and a few tiny glands were also seen without any papillae. Calibration Bar, 20µm (G) Percentage of ovarian cancer incidence in hens fed flaxseed enriched and control diet. Necropsy of hens was performed at different time-points (n=20) and ovaries were classified as normal, cancerous or questionable. Questionable ovarian tissues were analyzed by histology to confirm the cancerous ovarian tissue. No ovarian malignancy was detected in 1 (n=0/20), 1.5 (n=0/20) and 2 (n=0/20) year old hens in both flaxseed and control groups; however, 10% of 2.5 year old (n=2/20) hens fed control diet had ovarian cancer whereas 5% (n=1/20) of hens fed flaxseed had ovarian cancer. There was a difference in ovarian cancer incidence in 3 years old hens fed control diet (n=13/20) (65%) compared to hens fed a flaxseed enriched diet aged the same (n=9/20) (45%), but it was not statistically significant. Flaxseed enriched diet significantly reduced the incidence of ovarian cancer in 3.5 year old hens compared to hens fed control diet (P<0.05). (H) Comparison of early and late stage ovarian cancer between hens fed a flaxseed enriched diet and hens fed control diet. The cancerous ovary of 2.5 year old hen fed a flaxseed enriched diet was at the late stage but one early and one late stage ovarian cancer were found in hens aged the same on control diet. At the age of 3 year old, 8 early stage and only 1 late stage ovarian cancer were detected in hens fed flaxseed diet; similarly, 8 early stage ovarian cancer were observed in 3 year old hens on control diet but there was more late stage ovarian cancer (n=5). One early and 3 late stage ovarian cancer were observed in 3.5 year old hens fed a flaxseed enriched diet but interestingly, all of 10 ovarian cancers detected in 3.5 year old hens fed control diet were at the late stage. *, P<0.05.

Fig. 2

Expression of COX-1 protein and mRNA was measured in normal ovaries of hens fed a flaxseed enriched and normal ovaries of hens fed control diet at approximately 6 month intervals. (A& B) No significant differences in COX-1 protein expression in normal ovaries of hens fed either flaxseed enriched diet or control diet were observed at all ages (P>0.05). (C) Similar to the protein expression, flaxseed diet did not change the COX-1 mRNA expression in normal ovaries of hens compared to ovaries of hens fed control diet (P>0.05). Bars indicate standard error.

Fig. 3

Expression of COX-1 protein and mRNA was measured in cancerous ovaries of hens fed a flaxseed enriched diet and cancerous ovaries of hens fed control diet at approximately 6 month intervals. There was no significant difference in COX-1 protein (A&B) and mRNA (C) expression in cancerous ovaries of 3 and 3.5 year old hens fed a flaxseed enriched diet versus age matched hens fed control diet (P>0.05). Bars indicate standard error.

Fig. 4

Expression of COX-2 protein and mRNA was measured in normal ovaries of hens in both flaxseed and control groups at different ages. Normal ovaries of 2.5 (n=19), 3 (n=11) and 3.5 (n=16) year old hens fed a flaxseed enriched diet had significantly lower expression of COX-2 protein (A&C) and mRNA (C) compared to normal ovaries of 2.5 (n=18), 3 (n=7) and 3.5 (n=10) year old hens fed control diet. *, P<0.05; **, P<0.01; Bars indicate standard error.

Fig. 5

COX-2 protein and mRNA expression in cancerous ovaries of hens fed flaxseed enriched diet versus cancerous ovaries of hens fed control diet. The 3 (n=9) and 3.5 (n=4) year old hens fed a flaxseed enriched diet had lower COX-2 protein (A&B) and mRNA (C) expression than 3 (n=13) and 3.5 (n=10) year old hens with ovarian cancer fed control diet, respectively; *, P<0.05; Bars indicate standard error.

Fig. 6

Comparison of prostaglandin E₂ concentrations in ovaries of hens fed a flaxseed enriched diet and hens fed control diet. (A) There was a significant reduction in concentration of PGE₂ in normal ovaries of hens fed a flaxseed enriched diet compared to normal ovaries of hens fed control diet regardless of age. (B) PGE₂ concentration in cancerous ovaries of 3 (n=8) and 3.5 (n=4) year old hens fed a flaxseed enriched diet was significantly lower than PGE₂ concentration in cancerous ovaries 3 (n=8) and 3.5 (n=8) year old hens fed control diet. *, P<0.05; **, P<0.01; ***, P<0.001; Bars indicate standard error.