Singer S, Neuringer L J, Thilly W G, Chen L B
Department of Surgery, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts 02115.
Cancer Res. 1993 Dec 1;53(23):5808-14.
Rhodamine 123 is a lipophilic cationic compound that is selectively taken up by cancer cell mitochondria. This compound is toxic to epithelial cancer cells in vitro and displays significant anticancer activity in vivo. However, the mechanism of action of rhodamine 123 in intact, actively metabolizing cell preparations is unknown. We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to quantitatively characterize how rhodamine 123 affects the energetics of human colon cancer cells (HCT-116) and spontaneously immortalized normal epithelial cells (CV-1). Rhodamine 123 differentially altered the phosphorus and glucose metabolism of HCT-116 and CV-1 cells. 31P-nuclear magnetic resonance detected mitochondrial poisoning in the HCT-116 human colon cancer cell line in its early stages after selective uptake of rhodamine 123. When we compared administration of rhodamine 123 and [1-C13]glucose to administration of [1-C13]glucose alone in the HCT-116 cells, we noted a marked decrease in intracellular pH to 6.7 +/- 0.06 (mean +/- SD) units, a 2.2-fold increase in lactate production, and a 1.8-fold increase in glucose consumption after 10 h. In addition, we found a 2-fold rise in intracellular free magnesium 12 h after rhodamine 123 administration. These results suggest that when rhodamine 123 inhibits mitochondrial ATP production, it initially stimulates cytoplasmic glycolysis in an attempt to maintain cellular energy demands. The marked fall in intracellular pH and rise in intracellular free magnesium after administration of rhodamine 123 may inhibit activity of several glycolytic enzymes: this effect would inhibit cytoplasmic ATP generation and interfere with multiple cell enzymatic processes, leading to cell death. The CV-1 cells showed no change in intracellular pH, intracellular free magnesium, or magnesium-bound ATP levels over the 24-h period following rhodamine 123 administration. Rhodamine 123 also failed to alter glucose utilization and lactate production levels significantly in the CV-1 cells. These results prove the usefulness of 31P- and 13C-nuclear magnetic resonance spectroscopy for quantifying differing effects of rhodamine 123 on the high energy phosphate metabolism and glucose metabolism of HCT-116 and CV-1 cells.
罗丹明123是一种亲脂性阳离子化合物，可被癌细胞线粒体选择性摄取。该化合物在体外对上皮癌细胞有毒性，并在体内显示出显著的抗癌活性。然而，罗丹明123在完整的、活跃代谢的细胞制剂中的作用机制尚不清楚。我们使用31P和13C核磁共振波谱法定量表征了罗丹明123如何影响人结肠癌细胞（HCT-116）和自发永生化的正常上皮细胞（CV-1）的能量代谢。罗丹明123对HCT-116和CV-1细胞的磷和葡萄糖代谢有不同的影响。在选择性摄取罗丹明123后的早期阶段，31P核磁共振检测到HCT-116人结肠癌细胞系中的线粒体中毒。当我们将罗丹明123和[1-C13]葡萄糖的给药与单独给予[1-C13]葡萄糖在HCT-116细胞中的情况进行比较时，我们注意到10小时后细胞内pH值显著下降至6.7±0.06（平均值±标准差）单位，乳酸产量增加2.2倍，葡萄糖消耗量增加1.8倍。此外，我们发现罗丹明123给药12小时后细胞内游离镁增加了2倍。这些结果表明，当罗丹明123抑制线粒体ATP产生时，它最初会刺激细胞质糖酵解，试图维持细胞能量需求。罗丹明123给药后细胞内pH值的显著下降和细胞内游离镁的增加可能会抑制几种糖酵解酶的活性：这种作用会抑制细胞质ATP的产生并干扰多个细胞酶促过程，导致细胞死亡。在罗丹明123给药后的24小时内，CV-1细胞的细胞内pH值、细胞内游离镁或镁结合ATP水平没有变化。罗丹明123也未能显著改变CV-1细胞中的葡萄糖利用率和乳酸产生水平。这些结果证明了31P和13C核磁共振波谱法在定量罗丹明123对HCT-116和CV-1细胞的高能磷酸盐代谢和葡萄糖代谢的不同影响方面的有用性。
