Free, ethyl‐ and methyl‐ ester eicosapentaenoic acid inhibit the proliferation of human and murine colon cancer cells in vitro
Authors: Pfister Eric , Lane Michelle A. 
Published: November 2023

In the United States colorectal cancer is the second and third leading cause of cancer related deaths in men and women, respectively. Approximately 50,000 deaths from colon cancer are expected in 2017 alone. Omega‐3 polyunsaturated fatty acids (n‐3 PUFAs) have been extensively researched and shown to have anti‐inflammatory actions. Cancer development and progression is closely related to inflammatory processes, therefore, n‐3 PUFAs are of particular interest in their role in cancer prevention and treatment. Our objectives were to determine if eicosapentaenoic acid (EPA), a common n‐3 PUFA found in fish oil, inhibited the proliferation of colon cancer cells in vitro. One murine colon cancer cell line, MC‐38, and two human colon cancer cell lines, HCT‐116 and SW620, were seeded at a density of 5 × 104 (MC38 and HCT‐116) and 1.25 × 104 (SW620) cells per well on 24 well plates. After 24 hours each plate was treated with 0, 6.25, 12.5, 25, 50, and 100 microM EPA dissolved in ethanol. All cells received the same volume of ethanol. The experimental and control groups were plated in triplicate and cell number was determined 24, 48, and 72 hours after treatment. All data are reported as mean ± SEM for n=3 except for the HCT‐116 which had a n=2. One hundred microM EPA inhibited the proliferation of MC‐38 cells after 24 and 48 hours to 66.6 ± 6.0% and 72.2 ± 15.9% of control, respectively. However, at 72 hours the 100 microM EPA treatment had no effect on the proliferation of MC‐38 cells with 98.9 ± 7.4% of cells remaining when compared to vehicle control. Similarly the HCT‐116 cell line exhibited a decrease in cellular proliferation after 24 and 48 hours of treatment with 100 microM EPA to 64.0 ± 27.3 and 56.3 ± 13.6% of control, respectively. As with the MC‐38 cells, EPA no longer inhibited the proliferation of HCT‐116 cells after 72 hours of treatment, with 115.4 ± 5.2% of cells remaining when compared to vehicle control. These data suggested the EPA was either degraded or metabolized by the cancer cells within 48 hours and therefore had no effect at 72 hours. In order to combat this issue, two more stable forms of EPA, ethyl‐ and methyl‐ester EPA were used as treatments for the SW620 cell line. The experiment was conducted in the same method as the free EPA trials. After 72 hour of treatment with 100 microM free EPA, SW620 cells exhibited a decrease in proliferation to 79.6 ± 3.5 % of control. SW620 cells treated with 100 microM ethyl‐ and methyl‐ester EPA for 72 hours exhibited a larger decrease in cell number to 30.0 ± 13.2 and 32.6 ± 5.8% of control, respectively. In conclusion, the free‐EPA inhibited cellular proliferation up to 48 hours after treatment, after which its effects were diminished. The use of ethyl‐ and methyl‐ester EPA allowed for prolonged inhibition of cellular proliferation indicating that these esterified forms of EPA may be more efficacious in vivo.Support or Funding InformationThis research was supported by donations from the Heather Custer Memorial Fund and Kiel Colon Cancer.