Docosahexaenoic acid and butyrate synergistically modulate intracellular calcium compartmentalization to induce colonocyte apoptosis

Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a short-chain fatty acid fiber-fermentation product, protect against colon tumorigenesis in part by coordinately inducing apoptosis. We have demonstrated that the combination of these two bioactive compounds demonstrates an enhanced ability to induce colonocyte apoptosis by potentiating mitochondrial lipid oxidation. In order to explore the potential involvement of intracellular Ca2+ in the pro-apoptotic effect of DHA and butyrate, young adult mouse colonocytes (YAMC) and human colonocytes (HCT-116: p53+/+ and p53- /-) were treated with DHA or linoleic acid (LA) for 72 h ± butyrate for the final 6, 12 or 24 h. Cytosolic and mitochondrial Ca2+ levels were measured using Fluo-4 and Rhod-2. In addition, IP3 pool, store-operated channel (SOC)-mediated changes and apoptosis were measured. DHA did not alter basal Ca2+ or apoptosis following 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHA and butyrate treated cultures exhibited a decrease in cytosolic Ca2+ and enhanced apoptosis compared to LA and butyrate. DHA and butyrate also increased the mitochondrial-to-cytosolic Ca2+ ratio at 6, 12 and 24 h. The accumulation of mitochondrial Ca2+ preceded the onset of apoptosis which increased only following 12 h of butyrate co-treatment. RU-360, a mitochondrial uniporter inhibitor, abrogated mitochondrial Ca2+ accumulation and also partially blocked apoptosis in DHA and butyrate co-treated cells. p53+/+ and p53-/- cells demonstrated similar data with respect to all parameters. Additionally, mitochondrial Ca2+ measurements were also made in rat primarycolonocyte- culture. Rats were fed semipurified diets containing either fish oil (a source of DHA) or corn oil (a source of LA), and colonic crypts were incubated in butyrate exvivo and mitochondrial Ca2+ was quantified. Crypts from rats fed fish oil incubated in butyrate exhibited an increase in the mitochondrial-to-cytosolic Ca2+ ratio compared to fish oil only. In summary, our results indicate for the first time that the combination of DHA and butyrate, compared to butyrate alone, further enhances apoptosis by additionally recruiting a p53-independent Ca2+-mediated intrinsic mitochondrial pathway. These data explain in part why fermentable fiber when combined with fish oil exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis