Julie K. Mason

Flaxseed oil-trastuzumab interaction in breast cancer.

Flaxseed oil (FO), which is rich in n-3 fatty acid, is commonly consumed by breast cancer patients because of its potential anti-cancer effects. Trastuzumab (TRAS) is the primary drug for epidermal growth factor receptor 2 (HER2) positive breast cancer. We investigated in athymic mice whether combining dietary FO (8%) with TRAS treatment (2.5 or 5mg/kg body weight) can cause better or adverse effect on established human breast tumors overexpressing HER2 (BT-474). Control tumors significantly grew 187%, TRAS2.5 treated tumors did not change, while TRAS5, FO+TRAS2.5 and FO+TRAS5 treated tumors significantly regressed 75%, 89% and 84%, respectively, after 4weeks treatment. Two weeks after stopping TRAS treatment while continuing on same diet, tumor size in FO+TRAS2.5 group was 87% lower than in TRAS2.5 group and was not different from TRAS5 group with or without FO. Combined TRAS2.5 treatment with FO caused a significantly lower tumor cell proliferation and higher apoptosis compared to TRAS2.5 treatment alone and showed similar effect to TRAS5 treatment with or without FO. Hence, FO did not interfere with TRAS but rather enhanced its tumor-reducing effects and combined FO and low dose TRAS was as effective as high dose TRAS treatment.

Flaxseed oil enhances the effectiveness of trastuzumab in reducing the growth of HER2-overexpressing human breast tumors (BT-474)

Flaxseed oil (FSO) reduces breast tumorigenesis and HER2 expression in animal models of luminal breast cancer. The primary treatment for HER2-overexpressing tumors is trastuzumab (TRAS). We aimed to determine the effect of 4% FSO alone and combined with TRAS on HER2-overexpressing tumor (BT-474) growth and to explore potential mechanisms with a specific focus on HER2, mitogen-activated protein kinase (MAPK) and Akt signaling and fatty acid profile. Athymic mice with established tumors were fed the basal diet (control) or 4% FSO diet, with or without TRAS (1 or 2.5 mg/kg) treatment for 4 weeks. Tumor growth, HER2 signaling biomarkers (mRNA and protein) and fatty acid profile were measured. Tumors treated with FSO alone showed no difference in tumor growth compared to control; however, compared to TRAS2.5 and other groups, FSO+TRAS2.5 caused significantly lower tumor growth and cell proliferation and higher apoptosis and the greatest lowering of signaling biomarker expressions (MAPK2, HER2 mRNA; pHER2 protein). Both TRAS and FSO had main effects of reducing the phosphorylated/total expression of Akt and MAPK protein expression. Dietary FSO altered the tumor fatty acid profile. In conclusion, 4% dietary FSO alone does not affect BT-474 tumor growth but enhances the tumor-reducing effect of TRAS (2.5 mg/kg). FSO×TRAS interactive effect may be modulated by their combined reductions of HER2 signaling through the Akt and MAPK pathways leading to reduced cell proliferation and increased apoptosis. FSO alters tumor fatty acid profile that likely contributes to effects on signaling pathways. This supports FSO as a complementary treatment for HER2+ breast cancer treated with TRAS.

Dietary Flaxseed–Trastuzumab Interactive Effects on the Growth of HER2-Overexpressing Human Breast Tumors (BT-474)

Flaxseed (FS) reduces breast tumorigenesis and human epidermal growth factor receptor 2 (HER2) expression in postmenopausal patients and animal models. The primary treatment for HER2-overexpressing tumors is trastuzumab (TRAS). FS oil enhances TRAS effectiveness in athymic mice but the FS effect is unknown and was therefore determined. Athymic mice with established BT-474 tumors were fed the basal diet (control), or 10% FS diet, with or without TRAS (2.5mg/kg) treatment for 5 wk. After 2 wk, TRAS and FS reduced tumor size with a trend for an FS × TRAS interaction; however, after 5 wk, only TRAS reduced tumor size and increased tumor apoptosis. FS did not further improve TRAS effect but increased overall survival. TRAS reduced signaling biomarkers [phosphorylated HER2 and mitogen-activated protein kinase (MAPK) proteins; Akt1, Akt2, MAPK, and estrogen receptor-α mRNA], FS reduced phosphorylated-Akt1 protein, and FS × TRAS interactions were seen for HER2 mRNA and phosphorylated-Akt1 protein. FS, with and without TRAS, increased tumor n-3 PUFA levels and serum lignans indicating potential roles in the observed effect. In conclusion, TRAS reduces tumor growth by influencing HER2 signaling. Dietary FS has minimal tumor-reducing effect, does not interfere with TRAS action, but improves overall survival in athymic mice.

α-Linolenic Acid Reduces Growth of Both Triple Negative and Luminal Breast Cancer Cells in High and Low Estrogen Environments

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.

Growth and gene expression differ over time in alpha-linolenic acid treated breast cancer cells.

SCOPE Heterogeneity of breast cancer (BC) subtypes makes BC treatment difficult. α-linolenic acid (ALA), rich in flaxseed oil, has been shown to reduce growth and increase apoptosis in several BC cell lines, but the mechanism of action needs further understanding. METHODS AND RESULTS Four BC cell lines (MCF-7, BT-474, MDA-MB-231 and MDA-MB-468) were incubated with 75 μM ALA+1 nM 17-β estradiol (E2) or 1 nM E2 only (control) for 24 h. MDA-MB-231 cells were additionally incubated at 6 and 12 h. Viable cell number was measured, and expression of genes related to BC (signaling pathways, cell cycle, apoptosis) was quantified by real-time PCR array. There was a reduction in growth of all ALA treated cell lines after 24 h, and in MDA-MB-231 cells this was time-dependent. Many genes were altered after 24 h, and these differed between cell lines. In MDA-MB-231 cells, several gene expression changes were time-dependent. CONCLUSIONS ALA reduces growth of BC cell lines, by modifying signaling pathways, which differ between BC molecular subtypes. The ALA effect on gene expression is dynamic and changes over time, indicating the significance of incubation period in detecting gene changes.

Beneficial Influence of Diets Enriched with Flaxseed and Flaxseed Oil on Cancer

Dietary flaxseed and flaxseed oil are commonly consumed for their suggested anticancer effects. Flaxseed oil has an exceptionally high level of the omega-3 fatty acid α-linolenic acid and flaxseed is also the richest dietary source of phytoestrogens called lignans. This chapter provides information on flaxseed and flaxseed oil, including their composition and effects on the prevention and treatment of cancer. The major focus is on the effects in breast, colorectal and prostate cancer as observed in preclinical studies in cell culture and animal models, epidemiological and clinical studies. Limited studies on the effects in other forms of cancer are also discussed. Recent evidence supporting a potential anticancer role of flaxseed and flaxseed oil is for breast cancer. Extensive studies in rodent models suggest that flaxseed and its oil can reduce the various stages of carcinogenesis and there is increasing support from epidemiological and clinical studies. Studies in rodent models also suggest that flaxseed and its oil do not interfere with and may rather enhance the action of breast cancer drugs, including tamoxifen and trastuzumab. Regarding colorectal and prostate cancer, there are fewer studies with less consistent results. However, a protective effect is shown in general studies. The research in other forms of cancer is limited, inconsistent and warrants further investigation. Potential mechanisms of the action of flaxseed oil including effects on the properties of the cell membrane, the regulation of transcription, lipid peroxidation and others are discussed. Safety of diets enriched in flaxseed and flaxseed oil and flaxseed’s regulatory status are outlined. Current limitations in the research and future directions are provided.

Abstract 3486: Interaction of flaxseed oil with trastuzumab in HER2 overexpressing human breast tumors (BT-474)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: Trastuzumab (TRAS) is a first line therapy for breast tumors that overexpress the human epidermal growth factor receptor 2 (HER2) but tumors develop resistance within one year of treatment. Flaxseed oil (FO) rich in n-3 fatty acid alpha linolenic acid (ALA) has been shown to reduce HER2 expression and signalling in estrogen receptor positive human breast tumors. Our objective was to determine whether combining dietary FO with TRAS treatment will enhance TRAS effectiveness in reducing tumor growth. METHODS: Ovariectomized mice with established HER2 overxpressing tumors (BT-474) and estrogen pellet implant were fed the basal diet (BD) (control) or treated with TRAS (1 or 2.5mg/kg body weight) and fed either the BD or BD supplemented with 4% FO for 4 weeks. Palpable tumor area was measured weekly. After animal sacrifice, excised tumors were analyzed using immunohistochemistry for cell proliferation (Ki-67) and apoptosis and using Western blot analysis for HER2, MAPK and Akt. RESULTS: Tumors in the control, TRAS1, TRAS2.5, and TRAS1+FO, had palpable tumor areas that significantly increased (p<0.05) by 164%, 104%, 60% and 107%, respectively at the end of the study compared to week 0 while tumors in the FO+TRAS2.5 group did not significantly increase in size. FO+TRAS2.5 treated tumors also had significantly lower (p<0.05) palpable tumor area compared to the TRAS2.5 and other treated tumors at week 4. There was no difference in palpable tumor area between TRAS1 and FO+TRAS1 treated tumors. FO+TRAS2.5 caused a 39% greater increase in apoptosis (p<0.05) and a 61% greater reduction in cell proliferation (p<0.05) compared to TRAS2.5 treatment alone. Western blots support these results and indicate enhanced reductions in HER2, MAPK and Akt expression when TRAS2.5 treatment was combined with dietary FO. CONCLUSIONS: ALA-rich FO enhanced the effectiveness of TRAS at 2.5mg/kg in reducing tumor growth and the mechanisms may involve reduced cell proliferation and increased apoptosis through modulation of HER2 signalling pathways. This is significant as it could lead to a simple, inexpensive complementary treatment for HER2+ patients being treated with TRAS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3486.