Docosahexaenoic acid and butyrate synergistically induce colonocyte apoptosis by enhancing mitochondrial calcium accumulation

Docosahexaenoic acid (DHA) from fish oil, and butyrate, a short‐chain fatty acid fiber fermentation product, protect against colon cancer in part by coordinately inducing apoptosis. Since Ca 2+ signals govern vital cell functions, we determined the effect of DHA and butyrate co‐treatment on mitochondrial Ca 2+ homeostasis. Immortalized mouse colonocytes (YAMC) and isogenic p53 wild‐type (p53+/+) and null (p53−/−) human colon tumor (HCT116) cell lines were treated with DHA (50 μM) or linoleic acid (LA) for 72 h; with or without butyrate (0–10 mM for the final 6, 12 or 24 h). Cytosolic and mitochondrial Ca 2+ levels were measured using Ca 2+ sensitive dyes Fluo‐4 and Rhod‐2. Only the combination of DHA and butyrate increased (p<0.01) intra‐mitochondrial Ca 2+ levels at 6, 12 and 24 h in YAMC, p53+/+ and p53−/− cells. Butyrate co‐treatment (12 h) in DHA vs LA incubated cells also increased apoptosis (p<0.05) by 64, 44 and 35% in YAMC, p53+/+ and p53−/− cells, respectively. RU360, a mitochondrial Ca 2+ uniporter inhibitor, and MitoQ, a mitochondrially‐targeted antioxidant attenuated (p<0.05) DHA and butyrate‐induced mitochondrial Ca 2+ accumulation and apoptosis by ~40% in all cell lines. Based on these data, we propose a p53‐independent, oxidation sensitive, mitochondrial Ca 2+ ‐dependent apoptotic pathway in colonic epithelium that is selectively primed by DHA and triggered by butyrate. (NIH CA59034 and P30ES09106) .