Combination chemotherapy modulates mitochondrial calcium compartmentalization to induce colonocyte apoptosis.

B67

We have previously demonstrated that the combination of dietary fish oil enriched in docosahexaenoic acid (DHA, 22:6n-3) and butyrate (4:0), a fiber fermentation product in the colon, is protective against experimental colon carcinogenesis via induction of mitochondrial lipid peroxidation and dissipation of mitochondrial membrane potential. Given that alterations in mitochondrial Ca²⁺ homeostasis and cytochrome-C release from the mitochondria have been strongly implicated in the induction of apoptosis, we hypothesized that DHA and butyrate act synergistically to modulate intracellular Ca²⁺ homeostasis, enhancing mitochondrial Ca²⁺ levels. In order to determine the combined effects of DHA and butyrate on Ca²⁺ homeostasis, young adult mouse colonocytes (YAMC) and human colorectal adenoma (HCT-116) cells were treated with a combination of DHA (50 μM, for 72 h) or control polyunsaturated fatty acid, linoleic acid (LA) with or without butyrate (0-10 mM for the final 6, 12 or 24 h). Cytosolic and mitochondrial Ca²⁺ levels were measured by live-cell fluorescence microscopy using Ca²⁺ sensitive dyes Fluo-4 and Rhod-2, respectively. Only the combination of DHA and butyrate decreased (p<0.05) cytosolic Ca²⁺ levels at 12 and 24 h by 25 and 38%, respectively in YAMC cells. Concomitantly, DHA and butyrate synergistically increased (p<0.05) intra-mitochondrial Ca²⁺ levels at 6, 12 and 24 h, by approximately 50% in both YAMC and HCT-116 cells. The inhibition of endoplasmic reticulum/cytosolic-to-mitochondrial Ca²⁺ transport using a specific inhibitor of mitochondrial Ca²⁺ uptake, RU-360, attenuated mitochondrial Ca²⁺ accumulation and nucleosomal DNA fragmentation in both mouse and human colonocyte cell lines. In comparison, RU-360 had no effect on either mitochondrial Ca²⁺ levels or the induction of apoptosis in LA and butyrate-treated cultures. These data suggest that chemopreventive agents are capable of inducing an intracellular reorganization of Ca²⁺, which directly mediates mitochondria-dependent apoptosis. In conclusion, we have shown that DHA and butyrate sensitize mitochondria to rapidly sequester Ca²⁺, thereby triggering an intrinsic form of apoptosis in colonocytes. This may explain why a diet containing highly fermentable fiber is only protective with respect to colon cancer when fish oil is the lipid source. (Supported by grants from NIH CA59034 and P30ES09106).