Effects of omega-3 fatty acids on progestin stimulation of invasive properties in breast cancer

Abstract

Clinical studies have shown that progestins increase breast cancer risk in hormone replacement therapy, while we and others have previously reported that progestins stimulate invasive properties in progesterone receptor (PR)-rich human breast cancer cell lines. Based on others' reports that omega-3 fatty acids inhibit metastatic properties of breast cancer, we have reviewed the literature for possible connections between omega-3 fatty-acid-driven pathways and progestin-stimulated pathways in an attempt to suggest theoretical mechanisms for possible omega-3 fatty acid inhibition of progestin stimulation of breast cancer invasion. We also present some data suggesting that fatty acids regulate progestin stimulation of invasive properties in PR-rich T47D human breast cancer cells, and that an appropriate concentration of the omega-3 fatty acid eicosapentaenoic acid inhibits progestin stimulation of invasive properties. It is hoped that focus on the inter-relationship between pathways by which omega-3 fatty acids inhibit and progestins stimulate breast cancer invasive properties will lead to further in vitro, in vivo, and clinical studies testing the hypothesis that omega-3 fatty acids can inhibit progestin stimulation of invasive properties in breast cancer, and ameliorate harmful effects of progestins which occur in combined progestin-estrogen hormone replacement therapy.

Fig. 1

a Effect of 200 μM fatty acid on T47D invasion. T47D cells, obtained from the American Type Culture Collection, and found to exhibit the exquisite progestin-responsiveness characteristic of these cells, were grown in plastic flasks in 5 % CO₂ in air in minimal essential medium, powdered (autoclavable) plus non-essential amino acids, 2 mM l-glutamine, 10 % fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 6 ng/ml insulin (Sigma-Aldrich) until about 90 % confluence. The medium was then changed to fresh and made 200 μM in EPA, AA, or 0.1 % in ethanol (vehicle). Cells were incubated for 72 h, and the medium was made 10 μM in ara-C (arabinofuranosyl cytidine) for the last hour, to stop DNA replication. EPA, AA, and ara-C were purchased from Sigma-Aldrich. The cells of each flask were then harvested and single cell suspensions at 10⁶ cells/ml (300 μl) in the same medium as above (containing ara-C), except without serum and phenol red and with or without 10 nM R5020 (dissolved in ethanol) were placed in the upper insert of a modified Boyden chamber (Cell Biolabs, catalog # CBA-110) to measure invasion through a layer of extracellular matrix and a membrane with 8-μm pores, to a lower chamber containing 500 μl of twice charcoal-stripped serum-containing medium without phenol red. Final ethanol concentration was 0.2 % in all samples. As stated above, the cells were grown to around 90 % confluency in complete growth medium (containing 10 % fetal bovine serum and phenol red), and then incubated with the tested fatty acids in fresh complete growth medium for 72 h. They were then made into single cell suspensions and incubated in the presence of the fatty acids with or without progestin for a further 48 h in the same medium as above, except serum-free and without phenol red, while invading through a layer of extracellular matrix and a porous membrane into 10 % charcoal-stripped serum-containing medium without phenol red. After 48 h, invading cells were stained and counted, according to the manufacturer’s instructions. Results are the average plus SEM of six independent experiments, and were analyzed by ANOVA followed by the Fisher’s LSD multiple comparison procedure. CC no fatty acid or R5020, RC R5020 alone, CE EPA alone, RE R5020 plus EPA, CA arachidonic acid alone, RA R5020 plus arachidonic acid. All samples are different from control (CC) at p < 0.05 except for CA. All other samples are statistically the same except RC, CE, and CA (p < 0.05). *Different from control. **Different from RC and control. ***Different from RC. b Effect of 75 μM fatty acid on T47D invasion. Protocol was the same as in (a), except that fatty acid concentration was 75 μM. Results are the average plus SEM of nine independent experiments for CC and RC, and three experiments for the remaining samples. Results were analyzed by ANOVA followed by the Fisher’s LSD multiple comparison procedure. Abbreviations are the same as in (a). All samples are different from control at p < 0.05. *Different from all others. No other differences are significant. c Effect of 40 μM fatty acid on T47D invasion. Protocol was the same as in (a), except that fatty acid concentration was 40 μM. Results are the average plus SEM of nine experiments for CC and RC, and three experiments for the remaining samples. Abbreviations are the same as in (a). Results were analyzed by ANOVA followed by Fisher’s LSD multiple comparison procedure. *Different from all others at p < 0.05

Fig. 2

Working model for the hypothesis that omega-3 fatty acids can inhibit progestin stimulation of breast cancer invasion. The model is separated into two parts for clarity. a Depicts progestin stimulation of invasion, while b diagrams omega-3 fatty acid pathways which may act to inhibit progestin stimulation of invasive properties. Necessarily, the model is incomplete, yet testable. Parts of the model are colored red to emphasize pathways for which there is the most evidence suggesting interaction of omega-3 fatty acid pathways to inhibit progestin-driven pathways, in particular progestin stimulation of MUC1. Small upward-pointing arrows to the right of gene products indicate up-regulation of these products, whereas downward-pointing arrows indicate down-regulation. Abbreviations: HSP heat shock proteins, PR progesterone receptor, P phosphate group, Src the tyrosine–protein kinase c-Src, Ras the G-protein Ras, EGFR epidermal growth factor receptor 1, EGF epidermal growth factor, MUC-1 mucin-1, TF tissue factor, MAPKs mitogen-activated protein kinases, MnSOD manganese superoxide dismutase, CXCR4 a transmembrane G-protein-coupled chemokine receptor for CXCL-12/stromal cell-derived factor 1, PPARγ peroxisome proliferator-activated receptor gamma, RXR retinoid X receptor, PPRE peroxisome proliferator response element, EZH2 enhancer of zeste homolog 2 (a histone-methylating polycomb group protein), ErbB-2/Her2 human epidermal growth factor receptor 2

Fig. 2

Working model for the hypothesis that omega-3 fatty acids can inhibit progestin stimulation of breast cancer invasion. The model is separated into two parts for clarity. a Depicts progestin stimulation of invasion, while b diagrams omega-3 fatty acid pathways which may act to inhibit progestin stimulation of invasive properties. Necessarily, the model is incomplete, yet testable. Parts of the model are colored red to emphasize pathways for which there is the most evidence suggesting interaction of omega-3 fatty acid pathways to inhibit progestin-driven pathways, in particular progestin stimulation of MUC1. Small upward-pointing arrows to the right of gene products indicate up-regulation of these products, whereas downward-pointing arrows indicate down-regulation. Abbreviations: HSP heat shock proteins, PR progesterone receptor, P phosphate group, Src the tyrosine–protein kinase c-Src, Ras the G-protein Ras, EGFR epidermal growth factor receptor 1, EGF epidermal growth factor, MUC-1 mucin-1, TF tissue factor, MAPKs mitogen-activated protein kinases, MnSOD manganese superoxide dismutase, CXCR4 a transmembrane G-protein-coupled chemokine receptor for CXCL-12/stromal cell-derived factor 1, PPARγ peroxisome proliferator-activated receptor gamma, RXR retinoid X receptor, PPRE peroxisome proliferator response element, EZH2 enhancer of zeste homolog 2 (a histone-methylating polycomb group protein), ErbB-2/Her2 human epidermal growth factor receptor 2