Flaxseed, rich in α-linolenic acid (ALA), is a complementary breast cancer (BC) therapy; however ALA effectiveness and mechanism are unclear. Variation in cellular expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and estrogen (E2) levels may alter ALA effectiveness. This research determined the effect of ALA on growth, apoptosis, and phospholipid fatty acids of 4 BC cell lines with varying receptor expression ± E2. MCF-7 (ER+/PR+/HER2-), BT-474 (ER+/PR+/HER2+), MDA-MB-231 (ER-/PR-/HER2-) and MDA-MB-468 (ER-/PR-/HER2-) cells were incubated with ALA (50-200 μM) ± 1 nM E2 for 48-72 h. ALA dose-dependently reduced growth, measured by trypan blue exclusion, of all cells (55-80% with 75 μM), and this effect was not altered by E2. ALA (75 μM)+E2 induced apoptosis, measured by flow cytometry (up to 111.2%). Decreased growth and increased apoptosis is related to increased cell phospholipid % ALA (up to 25.1%), measured by gas chromatography. ALA is shown for the first time to reduce cell growth and induce apoptosis regardless of receptor expression and E2 environment, by incorporating into BC phospholipids, supporting the use of ALA and ALA-rich foods as a safe, inexpensive complementary therapy for a wide range of BC.

BackgroundDiets rich in the n-3 fatty acid alpha-linolenic acid (ALA) have been shown to reduce breast tumor growth, enhance the effectiveness of the HER2-targeted drug trastuzumab (TRAS) and reduce HER2 signaling in mouse models. It is unclear whether this is due to direct effects of ALA or due to its long-chain n-3 fatty acids metabolites including docosahexaenoic acid (DHA).MethodsThe ability of HER2-overexpressing BT-474 human breast cancer cells to convert ALA to long-chain n-3 fatty acids was determined by measurement of phospholipid fatty acids by gas chromatography following treatment with 100 μM ALA. The effects of 96 h treatment with ALA or DHA, at serum levels seen in mice (50–100 μM), alone and combined with TRAS (10 μg/ml), on BT-474 cell growth measured by trypan blue exclusion, apoptosis measured by flow cytometric analysis of Annexin-V/7-AAD stained cells (ALA and TRAS treatment only) and protein biomarkers HER2 signaling measured by western blot were determined.ResultsALA-treated BT-474 cells had higher phospholipid ALA but no increase in downstream n-3 metabolites including DHA. Both ALA and DHA reduced cell growth with and without TRAS. ALA had no effect on apoptosis. ALA and DHA showed opposite effects on Akt and MAPK phosphorylation; ALA increased and DHA decreased phosphorylation.ConclusionsTogether these data suggest that, while both ALA and its DHA metabolite can reduce HER2-overexpressing breast cancer growth with and without TRAS, they demonstrate for the first time that DHA is responsible for the effects of ALA-rich diets on HER2 signaling pathways.

Docosahexaenoic acid (DHA) is considered to be important for cardiac and brain function, and 17β-estradiol (E2) appears to increase the conversion of α-linolenic acid (ALA) into DHA. However, the effect of varying ALA intake on the positive effect of E2 on DHA synthesis is not known. Therefore, the objective of this study was to investigate the effects of E2 supplementation on tissue and serum fatty acids in mice fed a low-ALA corn oil-based diet (CO, providing 0.6 % fatty acids as ALA) or a high ALA flaxseed meal-based diet (FS, providing 11.2 % ALA). Ovariectomized mice were implanted with a slow-release E2 pellet at 3 weeks of age and half the mice had the pellet removed at 7 weeks of age. Mice were then randomized onto either the CO or FS diet. After 4 weeks, the DHA concentration was measured in serum, liver and brain. A significant main effect of E2 was found for liver and serum DHA, corresponding to 25 and 15 % higher DHA in livers of CO and FS rats, respectively, and 19 and 13 % in serum of CO and FS rats, respectively, compared to unsupplemented mice. There was no effect of E2 on brain DHA. E2 results in higher DHA in serum and liver, at both levels of dietary ALA investigated presently, suggesting that higher ALA intake may result in higher DHA in individuals with higher E2 status.

Breast cancer differs in cell receptors which then determines effective treatments. Alpha‐linolenic acid (ALA)‐rich flaxseed oil reduced estrogen receptor (ER)+ cell growth at low and high estrogen (E2) but its effect is unclear in cells with varying ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Our objective was to determine in vitro the ALA and E2 effect on growth and membrane lipids of four breast cancer cell lines with varying ER, PR and HER2 status. Cells were treated with 0 – 200uM ALA ± 1nM E2 and tested for growth by trypan blue exclusion method and fatty acids by gas chromatography. Receptor status confirmed by Western Blot: MCF7‐ER+PR+low HER2; BT474‐ER+PR+HER2+, MDA MB 231‐ER‐PR‐low HER2; MDA MB 468‐ER‐PR‐HER2‐. Compared to –E2 control, MCF7 had a significant E2, ALA and interaction effect on growth; other cell lines only had an ALA effect. 50uM and 75uM ALA significantly decreased growth by 14, 48, 31 and 63% and 38, 77, 80 and 78% in MCF7, BT 474, MDA MB231 and MDA MB468, respectively. E2 significantly increased (91.8%) MCF7 growth. Cells showed significant increases (9–25 fold) in % ALA which related to growth. Thus ALA reduces breast cancer cell growth ± E2 at doses as low as 50uM, likely through alteration of lipid composition. It is effective regardless of ER, PR and HER2 status which may be of importance in difficult to treat cancers such as triple negative. Funded by NSERC.Grant Funding Source: NSERC

n‐3 polyunsaturated fatty acids (PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial health effects. The conversion of α‐linolenic acid (ALA) to EPA and DHA is low, especially in males compared to females and varies in the literature. Estrogen (E2) is suggested to affect ALA conversion but few studies have confirmed the E2 effect. Our objective was to determine the independent effect of E2 on n‐3 PUFA levels in serum and various tissues in female ovariectomized mice fed diets with low and high ALA. After 7 weeks exposure to an E2 pellet, mice were randomized to a corn oil‐based basal diet or an ALA‐rich flaxseed diet (FS) with or without removal of the E2 pellet for 4 weeks. E2 responsiveness was indicated by the 269% higher (P<0.001) uterus weight in E2‐ treated mice compared to those with E2 pellet removed. FS‐fed mice had higher proportions of ALA, EPA and DHA in total lipids extracted from serum, liver and brain. E2‐treated mice had higher serum (15.1%; P<0.001) and liver (18.2%; P<0.001) DHA but not brain DHA. EPA levels were not affected by E2. There was a diet × E2 interaction for ALA in the liver where the FS+E2 group had lower ALA compared to FS‐E2. In conclusion, E2 results in higher serum and liver DHA, does not affect brain DHA and does not affect EPA in any of the tissues measured. This finding may help explain the variability in ALA effect in studies involving males and females.Grant Funding Source: NSERC