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.

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