Thornburg Joshua Marshall, Nelson Kristin K, Clem Brian F, Lane Andrew N, Arumugam Sengodagounder, Simmons Allan, Eaton John W, Telang Sucheta, Chesney Jason
Department of Medicine, J.G. Brown Cancer Center, University of Louisville, KY 40202, USA.
Breast Cancer Res. 2008;10(5):R84. doi: 10.1186/bcr2154. Epub 2008 Oct 15.
Glycolysis is increased in breast adenocarcinoma cells relative to adjacent normal cells in order to produce the ATP and anabolic precursors required for survival, growth and invasion. Glycolysis also serves as a key source of the reduced form of cytoplasmic nicotinamide adenine dinucleotide (NADH) necessary for the shuttling of electrons into mitochondria for electron transport. Lactate dehydrogenase (LDH) regulates glycolytic flux by converting pyruvate to lactate and has been found to be highly expressed in breast tumours. Aspartate aminotransferase (AAT) functions in tandem with malate dehydrogenase to transfer electrons from NADH across the inner mitochondrial membrane. Oxamate is an inhibitor of both LDH and AAT, and we hypothesised that oxamate may disrupt the metabolism and growth of breast adenocarcinoma cells.
We examined the effects of oxamate and the AAT inhibitor amino oxyacetate (AOA) on 13C-glucose utilisation, oxygen consumption, NADH and ATP in MDA-MB-231 cells. We then determined the effects of oxamate and AOA on normal human mammary epithelial cells and MDA-MB-231 breast adenocarcinoma cell proliferation, and on the growth of MDA-MB-231 cells as tumours in athymic BALB/c female mice. We ectopically expressed AAT in MDA-MB-231 cells and examined the consequences on the cytostatic effects of oxamate. Finally, we examined the effect of AAT-specific siRNA transfection on MDA-MB-231 cell proliferation.
We found that oxamate did not attenuate cellular lactate production as predicted by its LDH inhibitory activity, but did have an anti-metabolic effect that was similar to AAT inhibition with AOA. Specifically, we found that oxamate and AOA decreased the flux of 13C-glucose-derived carbons into glutamate and uridine, both products of the mitochondrial tricarboxylic acid cycle, as well as oxygen consumption, a measure of electron transport chain activity. Oxamate and AOA also selectively suppressed the proliferation of MDA-MB-231 cells relative to normal human mammary epithelial cells and decreased the growth of MDA-MB-231 breast tumours in athymic mice. Importantly, we found that ectopic expression of AAT in MDA-MB-231 cells conferred resistance to the anti-proliferative effects of oxamate and that siRNA silencing of AAT decreased MDA-MB-231 cell proliferation.
We conclude that AAT may be a valid molecular target for the development of anti-neoplastic agents.
与相邻正常细胞相比，乳腺腺癌细胞中的糖酵解作用增强，以产生生存、生长和侵袭所需的三磷酸腺苷（ATP）和合成代谢前体。糖酵解也是细胞质中烟酰胺腺嘌呤二核苷酸（NADH）还原形式的关键来源，NADH对于将电子穿梭到线粒体中进行电子传递是必需的。乳酸脱氢酶（LDH）通过将丙酮酸转化为乳酸来调节糖酵解通量，并且已发现其在乳腺肿瘤中高度表达。天冬氨酸转氨酶（AAT）与苹果酸脱氢酶协同作用，将电子从NADH转移穿过线粒体内膜。草氨酸是LDH和AAT的抑制剂，我们推测草氨酸可能会破坏乳腺腺癌细胞的代谢和生长。
我们研究了草氨酸和AAT抑制剂氨基氧乙酸（AOA）对MDA-MB-231细胞中13C-葡萄糖利用、耗氧量、NADH和ATP的影响。然后我们确定了草氨酸和AOA对正常人乳腺上皮细胞和MDA-MB-231乳腺腺癌细胞增殖的影响，以及对无胸腺BALB/c雌性小鼠体内MDA-MB-231细胞肿瘤生长的影响。我们在MDA-MB-231细胞中异位表达AAT，并研究其对草氨酸抑制细胞生长作用的影响。最后，我们研究了AAT特异性小干扰RNA（siRNA）转染对MDA-MB-231细胞增殖的影响。
我们发现草氨酸并未如预期通过其LDH抑制活性减弱细胞乳酸生成，但确实具有与AOA抑制AAT相似的抗代谢作用。具体而言，我们发现草氨酸和AOA减少了13C-葡萄糖衍生碳进入谷氨酸和尿苷的通量，这两种都是线粒体三羧酸循环的产物，同时也降低了耗氧量，耗氧量是电子传递链活性的一个指标。相对于正常人乳腺上皮细胞，草氨酸和AOA还选择性地抑制了MDA-MB-231细胞的增殖，并减少了无胸腺小鼠体内MDA-MB-231乳腺肿瘤的生长。重要的是，我们发现在MDA-MB-231细胞中异位表达AAT可赋予细胞对草氨酸抗增殖作用的抗性，并且AAT的siRNA沉默降低了MDA-MB-231细胞的增殖。
我们得出结论，AAT可能是开发抗肿瘤药物的一个有效的分子靶点。
