Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29.
Fish oil has been shown to reduce the induction of colorectal cancer in animal models by a mechanism which may involve suppression of mitosis, increased apoptosis, or both. We used the human colonic adenocarcinoma cell line HT29 to explore the effects of the long-chain n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on cell proliferation and death in vitro. Cells were cultured in media containing EPA at 5, 10, and 15 microg/mL. Cell number and thymidine incorporation were used to quantify proliferation, and cell cycle effects were studied using flow cytometry. Gel electrophoresis, annexin-V binding, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells were treated as two distinct populations. In the presence of EPA at 10 and 15 microg/mL there was a marked reduction in the growth rate of adherent HT29 colonies, owing to an increased detachment of adherent cells. After treatment with 10 or 15 microg/mL EPA the proportion of adherent cells in S-phase increased, indicating either a block in late S-phase or early G2. Floating cells showed evidence of extensive DNA cleavage, but the proportion of floating cells with sub GO DNA content declined on treatment with 10 or 15 microg/mL EPA even though the number of floating cells increased. We conclude that EPA does not inhibit mitosis of adherent cells, but increases the rate at which they become detached from the substrate, probably at an early stage in the initiation of apoptosis. This mechanism may be analogous to "anoikis," or induction of apoptosis in response to loss of cell contact, and may contribute to the anticarcinogenic effects of fish oil in vivo.
Clarke
R G
RG
Institute of Food Research, Norwich Research Park, Colney, United Kingdom.
Lund
E K
EK
Latham
P
P
Pinder
A C
AC
Johnson
I T
IT
D000165:Acridine Orange; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation; D015118:Eicosapentaenoic Acid / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D019073:HT29 Cells; D006801:Humans; D013936:Thymidine / Q000378:metabolism
D017304:Annexin A5; D015118:Eicosapentaenoic Acid; D000165:Acridine Orange; D013936:Thymidine