Kolar Satya Sree N, Barhoumi Rola, Lupton Joanne R, Chapkin Robert S
Faculty of Nutrition, Texas A&M University, College Station, Texas 77843-2253, USA.
Cancer Res. 2007 Jun 1;67(11):5561-8. doi: 10.1158/0008-5472.CAN-06-4716.
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 Ca2+ homeostasis was examined. Mouse colonocytes were treated with 50 micromol/L DHA or linoleic acid (LA) for 72 h +/- butyrate (0-10 mmol/L) for the final 24 h. Cytosolic and mitochondrial Ca2+ levels were measured using Fluo-4 and Rhod-2. DHA did not alter basal Ca2+ or the intracellular inositol trisphosphate (IP3) pool after 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHA- and butyrate-treated cultures exhibited a 25% and 38% decrease in cytosolic Ca2+ compared with LA and butyrate. Chelation of extracellular Ca2+ abolished the effect of thapsigargin on the IP3-releasable Ca2+ pool. DHA and butyrate cotreatment compared with untreated cells increased the mitochondrial-to-cytosolic Ca2+ ratio at 6, 12, and 24 h by 73%, 18%, and 37%, respectively. The accumulation of mitochondrial Ca2+ preceded the onset of apoptosis. RU-360, a mitochondrial-uniporter inhibitor, abrogated mitochondrial Ca2+ 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 Ca2+ -mediated intrinsic mitochondrial pathway.
我们之前已经表明，丁酸盐作为一种短链脂肪酸纤维发酵产物，可通过非线粒体、Fas介导的外源性途径诱导结肠细胞凋亡。有趣的是，可发酵纤维与含有二十二碳六烯酸（DHA，22:6n-3）的鱼油联合使用时，诱导凋亡和预防结肠肿瘤发生的能力增强。为了确定其分子作用机制，研究了DHA和丁酸盐联合处理对细胞内Ca2+稳态的影响。将小鼠结肠细胞用50微摩尔/升DHA或亚油酸（LA）处理72小时，在最后24小时加入丁酸盐（0-10毫摩尔/升）。使用Fluo-4和Rhod-2测量细胞质和线粒体Ca2+水平。在丁酸盐联合处理6小时后，DHA不会改变基础Ca2+或细胞内三磷酸肌醇（IP3）池。相反，在12小时和24小时时，与LA和丁酸盐处理的培养物相比，DHA和丁酸盐处理的培养物细胞质Ca2+分别降低了25%和38%。细胞外Ca2+的螯合消除了毒胡萝卜素对IP3可释放Ca池的影响。与未处理的细胞相比，DHA和丁酸盐联合处理在6小时、12小时和24小时时分别使线粒体与细胞质Ca2+比率增加了73%、18%和37%。线粒体Ca2+的积累先于凋亡的发生。线粒体单向转运体抑制剂RU-360消除了线粒体Ca2+的积累，并且也部分阻断了DHA和丁酸盐联合处理细胞中的凋亡。总体而言，这些数据表明，与单独使用丁酸盐相比，DHA和丁酸盐的组合通过额外激活Ca2+介导的内源性线粒体途径进一步增强了凋亡。
