Forbes Neil S, Meadows Adam L, Clark Douglas S, Blanch Harvey W
Department of Chemical Engineering, University of California, Berkeley, Berkeley, CA 94720, USA.
Metab Eng. 2006 Nov;8(6):639-52. doi: 10.1016/j.ymben.2006.06.005. Epub 2006 Jun 30.
Selective estrogen receptor (ER) modulators are highly successful breast cancer therapies, but they are not effective in patients with ER negative and selective estrogen receptor modulator (SERM)-resistant tumors. Understanding the mechanisms of estrogen-stimulated proliferation may provide a route to design estrogen-independent therapies that would be effective in these patients. In this study, metabolic flux analysis was used to determine the intracellular fluxes that are significantly affected by estradiol stimulation in MCF-7 breast cancer cells. Intracellular fluxes were calculated from nuclear magnetic resonance (NMR)-generated isotope enrichment data and extracellular metabolite fluxes, using a specific flux analysis algorithm. The metabolic pathway model used by the algorithm includes glycolysis, the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. The pathway model also incorporates mitochondrial compartmentalization and reversible trans-mitochondrial membrane reactions to more accurately describe the role of mitochondria in cancer cell proliferation. Flux results indicate that estradiol significantly increases carbon flow through the pentose phosphate pathway and increases glutamine consumption. In addition, intra-mitochondrial malic enzyme was found to be inactive and the malate-aspartate shuttle (MAS) was only minimally active. The inactivity of these enzymes indicates that glutamine is not oxidized within mitochondria, but is consumed primarily to provide biosynthetic precursors. The excretion of glutamine carbons from the mitochondria has the secondary effect of limiting nicotinamide adenine dinucleotide (NADH) recycle, resulting in NADH buildup in the cytosol and the excretion of lactate. The observed dependence of breast cancer cells on pentose phosphate pathway activity and glutamine consumption for estradiol-stimulated biosynthesis suggests that these pathways may be targets for estrogen-independent breast cancer therapies.
选择性雌激素受体（ER）调节剂是非常成功的乳腺癌治疗药物，但它们对雌激素受体阴性和对选择性雌激素受体调节剂（SERM）耐药的肿瘤患者无效。了解雌激素刺激增殖的机制可能为设计对这些患者有效的非雌激素依赖性疗法提供途径。在本研究中，代谢通量分析用于确定在MCF-7乳腺癌细胞中受雌二醇刺激显著影响的细胞内通量。使用特定的通量分析算法，根据核磁共振（NMR）产生的同位素富集数据和细胞外代谢物通量计算细胞内通量。该算法使用的代谢途径模型包括糖酵解、三羧酸循环（TCA循环）、磷酸戊糖途径、谷氨酰胺分解代谢、丙酮酸羧化酶和苹果酸酶。该途径模型还纳入了线粒体区室化和可逆的跨线粒体内膜反应，以更准确地描述线粒体在癌细胞增殖中的作用。通量结果表明，雌二醇显著增加通过磷酸戊糖途径的碳流并增加谷氨酰胺消耗。此外，发现线粒体内的苹果酸酶无活性，而苹果酸-天冬氨酸穿梭（MAS）仅具有最低活性。这些酶的无活性表明谷氨酰胺在线粒体内未被氧化，而是主要被消耗以提供生物合成前体。谷氨酰胺碳从线粒体的排泄具有限制烟酰胺腺嘌呤二核苷酸（NADH）循环的次要作用，导致NADH在细胞质中积累并导致乳酸排泄。观察到乳腺癌细胞对磷酸戊糖途径活性和谷氨酰胺消耗的依赖性以进行雌二醇刺激的生物合成，这表明这些途径可能是雌激素非依赖性乳腺癌治疗的靶点。
