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. 2001 Jul;49(1):97–105. doi: 10.1136/gut.49.1.97

Effects of cellular redox balance on induction of apoptosis by eicosapentaenoic acid in HT29 colorectal adenocarcinoma cells and rat colon in vivo

P Latham 1, E Lund 1, J Brown 1, I Johnson 1
PMCID: PMC1728373  PMID: 11413117

Abstract

BACKGROUND AND AIMS—Epidemiological evidence suggests n-3 polyunsaturated lipids may protect against colorectal neoplasia. Consumption of fish oil modulates crypt cytokinetics in humans, and crypt apoptosis in animal models. To explore these effects, we investigated involvement of caspase enzymes and cellular redox balance in the induction of apoptosis by eicosapentaenoic acid (EPA) in HT29 cells, and in rat colon in vivo.
METHODS—Survival of HT29 cells grown with EPA in the presence of caspase inhibitors, antioxidants, or buthionine sulphoximine, an inhibitor of glutathione neosynthesis, was determined. The effects of EPA enriched fish oil and glutathione depletion on apoptosis in rat colon were assessed using microdissected crypts.
RESULTS—Treatment of HT29 cells with EPA reduced viable cell number and activated caspase 3, prior to cell detachment. Antioxidants and caspase inhibitors blocked HT29 cell death whereas glutathione depletion increased it. Rats fed fish oil had higher crypt cell apoptosis than those fed corn oil, and glutathione depletion enhanced this effect.
CONCLUSIONS—Incorporation of EPA into colonic epithelial cell lipids increases apoptosis. The results of this study, using both an animal and cell line model, support the hypothesis that this effect is mediated via cellular redox tone, and is sensitive to glutathione metabolism. The data suggest a mechanism whereby polyunsaturated fatty acids may influence the susceptibility of colorectal crypt cells to induction or progression of neoplasia.


Keywords: eicosapentaenoic acid; apoptosis; glutathione; caspase; redox; colorectal cancer; rat

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Figure 1  .

Figure 1  

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Adherent cell viability of HT29 cells following treatment with increasing concentrations of eicosapetaenoic acid (EPA 15-45 µM) for 24 or 48 hours. Cell viability was assessed using the neutral red assay, and data are expressed as per cent of the optical density (OD)550nm of untreated control cultures (mean (SEM)). A minimum of 16 OD550nm readings were taken for each treatment. EPA significantly reduced cell viability after 24 and 48 hours at all concentrations tested (p<0.05) compared with untreated cells. Reduction in viable cell number was significantly associated with EPA treatment and time (p<0.001).

Figure 2  .

Figure 2  

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Activity of DEVDase caspases (chiefly caspase 3) in HT29 cells following treatment with increasing concentrations of eicosapetaenoic acid (EPA 15-45 µM) for 24 or 48 hours. Positive control cells were also treated with 25 µM C2 ceramide (three hours) or 12 000 J/cm2 (UV, 30 minutes). Data are expressed as mean (SEM) pNA liberated from a DEVDase consensus cleavage site per hour per 106 cells of four samples per treatment group. Caspase activity was significantly higher in all EPA treated cells than in untreated cells after 24 and 48 hours (p<0.05).

Figure 3  .

Figure 3  

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Adherent HT29 cell viability following treatment with eicosapetaenoic acid (EPA) (15-45 µM), EPA+100 µM buthionine sulphoximine (BSO), or EPA+BSO+40 µM ebselen for 48 hours. Cell viability was assessed using the neutral red assay, and data are expressed as mean (SEM) per cent of the optical density (OD)550nm of untreated control cultures. A minimum of eight OD550nm readings were taken for each treatment. EPA significantly reduced cell viability after 48 hours at all concentrations tested (p<0.05) compared with untreated cells. BSO alone did not significantly affect cell viability. Exposure to EPA (15-30 µM)+BSO significantly reduced cell viability (p<0.05) but addition of ebselen to EPA+BSO cultures significantly increased cell viability (p<0.05).

Figure 4  .

Figure 4  

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Mitotic cells per crypt expressed as mean (SEM) in crypts isolated from the distal colon of rats given corn oil (CO) or fish oil (FO) in the diet with or without 5 mM buthionine sulphoximine (BSO) in drinking water. There was a significant reduction in mitotic cells/crypt associated with FO consumption at all time points (p<0.001) but no significant effect of BSO in the drinking water.

Figure 5  .

Figure 5  

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Apoptotic cells per crypt expressed as mean (SEM) in crypts isolated from the distal colon of rats given corn oil (CO) or fish oil (FO) in the diet with or without 5 mM buthionine sulphoximine (BSO) in the drinking water. The total number of apoptotic cells is represented by total column height; the number in the top 40% of the crypt is represented in black and in the bottom 60% as the shaded region. Both FO and BSO significantly increased the total number of apoptotic cells/crypt at all time points (p<0.001).

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