Docosahexaenoic Acid and Butyrate Synergistically Induce Colonocyte Apoptosis by Enhancing Mitochondrial Ca Accumulation

We have previously shown that butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. Interestingly, fermentable fiber when combined with fish oil containing docosahexaenoic acid (DHA, 22:6n-3) exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis. To determine the molecular mechanism of action, the effect of DHA and butyrate cotreatment on intracellular Ca homeostasis was examined. Mouse colonocytes were treated with 50 Mmol/L DHA or linoleic acid (LA) for 72 h F butyrate (0–10 mmol/L) for the final 24 h. Cytosolic and mitochondrial Ca levels were measured using Fluo-4 and Rhod-2. DHA did not alter basal Ca or the intracellular inositol trisphosphate (IP3) pool after 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHAand butyrate-treated cultures exhibited a 25% and 38% decrease in cytosolic Ca compared with LA and butyrate. Chelation of extracellular Ca abolished the effect of thapsigargin on the IP3-releasable Ca pool. DHA and butyrate cotreatment compared with untreated cells increased the mitochondrial-to-cytosolic Ca ratio at 6, 12, and 24 h by 73%, 18%, and 37%, respectively. The accumulation of mitochondrial Ca preceded the onset of apoptosis. RU-360, a mitochondrial-uniporter inhibitor, abrogated mitochondrial Ca accumulation and also partially blocked apoptosis in DHA and butyrate cotreated cells. Collectively, these data show that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca-mediated intrinsic mitochondrial pathway. [Cancer Res 2007;67(11):5561–8]