Effect of different concentrations of EPA, DHA and AA on viability of non-cancerous transformed cells.

<p>(A) MCF10A, (B) HEK293 and (C) HaCaT cells were exposed to different concentrations (0–320μM) of either EPA, DHA, AA and MTT assay was performed. Data has been presented as mean±SEM of three independent experiments, performed in 96 well plates. Statistical significance was assayed by one-way ANOVA, followed by a Dunnett's test. ***p<0.01.</p

Concentrations of n6 (AA) and n3 (EPA, DHA) fatty acids used in different ratios at 280 μM total fatty acid concentration.

<p>Concentrations of n6 (AA) and n3 (EPA, DHA) fatty acids used in different ratios at 280 μM total fatty acid concentration.</p

Regulation of MAR binding tumor regulatory proteins by n6/n3 ratios in breast cancer and non-cancerous cells.

<p>Expression of SMAR1 and Cux/CDP proteins was analyzed in MDA-MB-231 (A, B); MCF7 (C,D) and MCF10A (E,F). The effect of low (A, C, E) and high (B, D, F) n6/n3 FA ratios has been shown in MDAMB231, MCF7 and MCF10A cell lines, respectively. The bands were quantified by densitometry using ImageJ 1.44p (National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>) and have been presented as mean±SEM of three different experiments. Ώp<0.05, @p<0.01 and *p<0.001 compared to UC; αp<0.05, ¥p<0.01 and ¤p<0.001 compared to 1:1; ₭p<0.01and Θp<0.001 compared to 1:2.5; #p<0.05 and Øp<0.001 compared to 1:4; $p<0.05 compared to 1:5;ρp<0.05, ∞p<0.01 and Δp<0.001 compared to 2.5:1; ‡p<0.01 and Σp<0.001 compared to 4:1, μp<0.01 and πp<0.001 compared to 5:1.</p

Differential Ratios of Omega Fatty Acids (AA/EPA+DHA) Modulate Growth, Lipid Peroxidation and Expression of Tumor Regulatory MARBPs in Breast Cancer Cell Lines MCF7 and MDA-MB-231

<div><p>Omega 3 (n3) and Omega 6 (n6) polyunsaturated fatty acids (PUFAs) have been reported to exhibit opposing roles in cancer progression. Our objective was to determine whether different ratios of n6/n3 (AA/EPA+DHA) FAs could modulate the cell viability, lipid peroxidation, total cellular fatty acid composition and expression of tumor regulatory Matrix Attachment Region binding proteins (MARBPs) in breast cancer cell lines and in non-cancerous, MCF10A cells. Low ratios of n6/n3 (1:2.5, 1:4, 1:5, 1:10) FA decreased the viability and growth of MDA-MB-231 and MCF7 significantly compared to the non-cancerous cells (MCF10A). Contrarily, higher n6/n3 FA (2.5:1, 4:1, 5:1, 10:1) decreased the survival of both the cancerous and non-cancerous cell types. Lower ratios of n6/n3 selectively induced LPO in the breast cancer cells whereas the higher ratios induced in both cancerous and non-cancerous cell types. Interestingly, compared to higher n6/n3 FA ratios, lower ratios increased the expression of tumor suppressor MARBP, SMAR1 and decreased the expression of tumor activator Cux/CDP in both breast cancer and non-cancerous, MCF10A cells. Low n6/n3 FAs significantly increased SMAR1 expression which resulted into activation of p21^WAF1/CIP1 in MDA-MB-231 and MCF7, the increase being ratio dependent in MDA-MB-231. These results suggest that increased intake of n3 fatty acids in our diet could help both in the prevention as well as management of breast cancer.</p></div

Various ratios of n6 and n3 regulate the cell proliferation of breast cancer and non-cancerous cell lines.

<p>MDA-MB-231(A), MCF7 (B) as well as MCF10A (C) cell lines were treated with low and high n6/n3 ratios for 24h. Next day, the number of viable cells was counted using the trypan blue dye exclusion assay. Data has been presented as mean±SEM of three independent experiments, each conducted in triplicates. ƍp<0.01 and *p<0.001 compared to UC; Ϫp<0.05, ¥p<0.01 and ¤p<0.001 compared to 1:1; Φp<0.01 and Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4; $p<0.05, ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ×p<0.001 and ßp<0.001 compared to 1:10; ρp<0.05 compared to 2.5:1; ‡p<0.01 compared to 4:1.</p

Differential ratios of n6 and n3 fatty acids regulate the viability of breast cancer and non-cancerous cells.

<p>Breast cancer cell lines, (A) MDA-MB-231 and (B) MCF7 as well as immortalized non-tumorigenic human breast epithelial, MCF10A cells (C) were treated with low and high n6/n3 ratios and analyzed for cell viability by MTT assay. Data has been presented as mean±SEM of five independent experiments, each conducted in triplicates. ƍp<0.01 and *p<0.001 compared to UC; ¥p<0.01 and ¤p<0.001 compared to 1:1; Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4;ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ×p<0.01 and ßp<0.001 compared to 1:10; ∞p<0.01 and Δp<0.001 compared to 2.5:1.</p

Relative percentage of total n6/n3 and EPA+DHA/AA ratio in breast cancer cells.

<p>The cells were treated with different ratios of n6 and n3 FA for 24h. The levels of total n6/n3 and EPA+DHA/AA has been shown in MDA-MB-231 (A) and MCF7 (B) cells treated with low and high n6/n3 ratios, respectively. Total n6 fatty acids include Linoleic acid (LA) (18:2n6), Gamma-linolenic acid (GLA) (18:3n6), Dihomo-gamma-linolenic acid (DGLA) (20:3n6), Arachidonic acid (AA) (20:4n6); Total n3 fatty acids include ALA (18:3n3), EPA (20:5n3), DHA (22:6n3), DPA(22:5n3). Each value represents mean±SEM of three independent experiments. %p<0.05 and *p<0.001 compared to UC; Ϫp<0.05, ¥p<0.01 and ¤p<0.001 compared to 1:1; ₭p<0.01, Φp<0.01 and Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4; $p<0.05, ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ×p<0.001 and ßp<0.001 compared to 1:10.</p

Effect of different concentrations of EPA, DHA and AA on viability of breast cancer cells.

<p>(A) MDA-MB-231 and (B) MCF7 cells were treated with different concentration of EPA, DHA and AA for 24 h, followed by cell viability analysis by MTT assay. Data has been presented as mean±SEM of three independent experiments, each performed in 96 well plates. Statistical significance was assayed using one-way ANOVA, followed by a Dunnett's test.*p<0.05, ***p<0.001.</p

Different ratios of n6 and n3 regulate the lipid peroxidation in breast cancer and non-cancerous cells.

<p>(A) MDA-MB-231, (B) MCF7 and (C) MCF10A cells were treated with low and high n6/n3 ratios for 24h. Next day, lipid peroxidation was analyzed by using cis-parinaric acid and the values have been plotted in terms of percentage fluorescent intensity. Decrease of cis-parinaric acid fluorescence is proportional to increase in lipid peroxidation. Data has been presented as mean±SEM of three independent experiments, each conducted in triplicates. ƍp<0.01 and *p<0.001 compared to UC; ¥p<0.01 and ¤p<0.001 compared to 1:1; ₭p<0.01, Φp<0.01 and Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4; $p<0.05, ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ßp<0.001 as compared to 1:10; ∞p<0.01 and Δp<0.001 compared to 2.5:1; ‡p<0.01 as compared to 4:1; Ŧp<0.05 compared to 5:1; μp<0.01 compared to 5:1; €p<0.01 compared to 10:1.</p

Relative percentage of EPA, DHA and AA in breast cancer cell lines.

<p>The cells were treated with different ratios of n6 and n3 FA for 24h. The levels of EPA, DHA and AA has been shown in MDA-MB-231 (A) and MCF7 (B) cells treated with low and high n6/n3 ratios. Each value represents mean±SEM of three independent experiments. %p<0.05, ƍp<0.01 and *p<0.001 compared to UC; Ϫp<0.05, ¥p<0.01 and ¤p<0.001 compared to 1:1; Φp<0.01 and Θp<0.001 compared to 1:2.5; #p<0.05, Λp<0.01 and Øp<0.001 compared to 1:4; $p<0.05, ϒp<0.01 and ϙp<0.001 compared to 1:5; βp<0.05, ×p<0.001 and ßp<0.001 compared to 1:10; Δp<0.001 compared to 2.5:1; Σp<0.001 compared to 4:1; μp<0.01 compared to 5:1.</p