Pro-apoptotic and anti-angiogenic actions of 2-methoxyestradiol and docosahexaenoic acid, the biologically derived active compounds from flaxseed diet, in preventing ovarian cancer

Abstract

Background: We have previously shown that a whole flaxseed supplemented diet decreased the onset and severity of ovarian cancer in the laying hen, the only known animal model of spontaneous ovarian cancer. Flaxseed is rich in omega-3 fatty acids (OM3FA), mostly α-Linoleic acid (ALA), which gets converted to Docosahexaenoic acid (DHA) by the action of delta-6 desaturase enzyme. Ingestion of flaxseed also causes an increase in production of 2-methoxyestradiol (2MeOE₂) via the induction of the CYP1A1 pathway of estrogen metabolism. We have previously reported that the flaxseed diet induces apoptosis via p38-MAPK pathway in chicken tumors. The objective of this study was to investigate the effect of the flaxseed diet on ovarian cancer in chickens, focusing on two hallmarks of cancer, apoptosis and angiogenesis.

Results: The anti-cancer effects of two active biologically derived compounds of flax diet, 2MeOE₂ and DHA, were individually tested on human ovarian cancer cells and in vivo by the Chick Chorioallantoic Membrane (CAM) assay. Our results indicate that a flaxseed-supplemented diet promotes apoptosis and inhibits angiogenesis in chicken tumors but not in normal ovaries. 2MeOE₂ promotes apoptosis in human ovarian cancer cells, inhibits angiogenesis on CAM and its actions are dependent on the p38-MAPK pathway. DHA does not have any pro-apoptotic effect on human ovarian cancer cells but has strong anti-angiogenic effects as seen on CAM, but not dependent on the p38-MAPK pathway.

Conclusions: Dietary flaxseed supplementation promotes a pro-apoptotic and anti-angiogenic effect in ovarian tumors, not in normal ovaries. The biologically derived active compounds from flaxseed diet act through different pathways to elicit their respective anti-cancer effects. A flaxseed-supplemented diet is a promising approach for prevention of ovarian cancer as well as having a significant potential as an adjuvant treatment to supplement chemotherapeutic agents for treatment of advanced stages of ovarian cancer.

Keywords: 2-methoxyestradiol; Angiogenesis; Apoptosis; Docosahexaenoic acid; Flaxseed; Ovarian cancer; p38-MAPK.

Fig. 1

TUNEL staining on normal and cancerous chicken ovarian tissue TUNEL staining on normal and cancerous chicken ovarian tissue from control, whole flax, defatted flax meal and flax oil diet groups. Two-way ANOVA, error bars: SD, p < 0.05

Fig. 2

Immmunohistochemical staining on normal and cancerous chicken ovarian tissue for angiogenic markers a Normal and cancerous chicken ovaries from control, whole flax, defatted flax meal and flax oil groups were stained with anti-CD31, anti-⍺ smooth muscle actin, anti-vascular endothelial growth factor, anti-vascular endothelial growth factor receptor type 2 antibodies and NG2 co-stained with CD31. b Quantified expression of the angiogenic markers, number of positive cells over total number of cells per field of view. Two-way ANOVA, error bars: SD, p < 0.05

Fig. 3

TUNEL staining on BG1, HeyC2 and TOV112D cells after 2MeOE₂ treatment BG1, HeyC2 and TOV112D cells treated with 10 μM 2MeOE₂ and after an incubation of 26 h TUNEL staining was performed

Fig. 4

2MeOE₂ treatment of human ovarian cancer cells a BG1, HeyC2 and TOV112D cells were treated with 10 μM 2MeOE₂, 10 μM SB203580 and 10 μM 2MeOE₂ and SB203580 together. Cells were photographed after 24 h from treatment. 10 μM 2MeOE₂ plates had significant reduction in cell numbers from the control plate. b Western blot analysis performed on protein lysates from control and 10 μM 2MeOE₂ against p38 and phospho-p38 on BG1, HeyC2 and TOV112D cells (n = at least 3 for each dataset). One-way ANOVA, error bars: SEM, p < 0.05. c Western blot analysis performed on protein lysates from control, 10 μM 2MeOE₂, 10 μM SB203580 and 10 μM 2MeOE₂ + 10 μM 2MeOE₂ against caspase 3 and cleaved caspase 3 on BG1, HeyC2 and TOV112D cells (n = 3). Amount of caspase-3 cleaved into the active cleaved caspase-3 was quantified to estimate the amount of apoptosis induced by each treatment. Two-way ANOVA, error bars: SEM, p < 0.05

Fig. 5

DHA treatment of human ovarian cancer cells a BG1, HeyC2 and TOV112D cells were treated with 10 μM DHA. Cells were photographed after 24 h of treatment. b Western blot from whole cell lysates following 10 μM DHA treatment for p38, phospho-p38, caspase-3 and cleaved caspase-3 (n = at least 3 for each experiment)

Fig. 6

Anti-angiogenic effect of 2MeOE₂ and DHA on CAM a 2MeOE₂ was added to a filter paper in increasing concentrations of 1 μM (n = 9), 10 μM (n = 5) and 100 μM (n = 6), and placed on the CAM. Control group (n = 5) was treated with only the solvent, 100% ethanol. After an incubation of 48 h, CAMs were harvested and viewed under the microscope and number of sprouting vessels around the filter paper was counted. One-way ANOVA, error bars: SEM, p < 0.05. b DHA was added to a filter paper in increasing concentration – 1 μM (n = 9), 10 μM (n = 5), 100 μM (n = 6) and 1 mM (n = 8); and placed on the CAM. Control group (n = 10) treated with only the solvent, 100% ethanol. Following an incubation of 48 h, CAMs were harvested and viewed under the microscope and number of sprouting vessels around the filter paper was counted. One-way ANOVA, error bars: SEM, p < 0.05

Fig. 7

Involvement of p38-MAPK pathway in anti-angiogenic action of 2MeOE₂ and DHA on CAM a 10 μM 2MeOE₂ (n = 10), 10 μM SB203580 (n = 5) or both 2MeOE₂ and SB203580 were added to filter papers and placed on CAMs. Control group (n = 6) was treated with only the solvent, 100% ethanol. Two-way ANOVA, error bars: SEM, p < 0.05. b 10 μM DHA (n = 8), 10 μM SB203580 (n = 8) or both DHA and SB203580 together (n = 11) were added to filter papers and placed on CAMs. Control group (n = 9) was treated with only the solvent, 100% ethanol. Two-way ANOVA, error bars: SEM, p < 0.05