Response dynamics of entorhinal cortex in awake, anesthetized, and bulbotomized rats

The steady-state rate of glucose oxidation through the mitochondrial TCA cycle (V(TCA)) was measured in acid extracts of 10- and 30-day-old cerebral cortex of rats receiving [1-13C]glucose intravenously and in neocortical slices superfused in vitro with the same isotope. TCA cycle flux was determined for each age group based on metabolic modeling analysis of the isotopic turnover of cortical glutamate and lactate. The sensitivity of the calculated rates to assumed parameters in the model were also assessed. Between 10 and 30 postnatal days, V(TCA) increased by 4.3-fold (from 0.46 to 2.0 micromol g(-1) min(-1)) in the cortex in vivo, whereas only a 2-fold (from 0.17 to 0.34 micromol g(-1) min(-1)) increase was observed in neocortical slices. The much greater increase in glucose oxidative metabolism of the cortex measured in vivo over that measured in vitro as the cortex matures suggests that function-related energy demands increase during development, a process that is deficient in the slice as a result of deafferentiation and other mechanisms.     

Functional compartmentalization of energy production in neural tissue

Previous work in our laboratory has shown that neural trauma results in a disparity between oxidative and glycolytic rates. In non-neural tissue, glycolysis and oxidative phosphorylation have been shown to work independently of one another, a phenomenon known as "energy compartmentalization". We believe that functional compartmentalization of energy production may also occur in the brain with glycolysis providing energy for membrane bound ionic pumps. Spreading depression, induced in rodent brain by topical KCl application, results in K+ shifts. The restoration of K+ gradients is accomplished by energy dependent Na(+)-K+ pumps. If these pumps depend upon glycolysis, blocking glycolysis should prevent reconstitution of normal [K+]e levels. The present series of experiments were designed to suggest that energy compartmentalization may also exist in brain, and that glycolytic energy production is preferentially used by Na(+)-K+ pumps to maintain normal ionic homeostasis by observing the dynamics of spreading depression induced K+ shifts before and after glycolytic blockade. Spreading depression was associated with increased K+ (48.6 +/- 16.6 mM over control) that normalized within 2.9 +/- 0.3 minutes. Following superfusion with a glycolytic blocking agent, spreading depression produced similar increases in [K+]e (40.6 +/- 12.0 mM over control) but time for reconstitution of the normal [K+]e was 400% longer than controls (2.9 +/- 0.3 to 14.9 +/- 2.1 minutes, P less than 0.001). Time required for recovery of EEG was identical pre- and post-blockade. We believe these data suggest that energy compartmentalization may exist in neural tissue and that glycolytic pathways of energy production are functionally tied to membrane Na(+)-K+ pumps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia

Purpose A high-rate glycolysis is a fundamental property of solid tumors and is associated with an over-expression of glucose transporters and glycolytic enzymes. We hypothesize that over-expression of glucose transporters in tumors prevents apoptosis, promotes cancer cell survival, and confers drug resistance. Inhibition of glucose transporter will preferentially sensitize the anticancer effects of chemotherapeutic drugs to overcome drug resistance in hypoxia. Methods Glucose transporter expressions were detected in cancer tissues and NCI 60 cancer cells with immunostaining and DNA microarray. Glucose uptake was measured with ³H-2-deoxy-glucose. Cytotoxicity of daunorubicin (DNR) in combination of glucose inhibitor was detected by MTS assay under hypoxic condition. Early stage apoptosis was monitored with Annexin V-FITC staining. Results Immunostaining showed that GLUT1 was significantly increased in hypoxic regions of the human colon and breast tumors. The expression profiles of all glucose transporters in NCI 60 cancer cells exhibited distinct expression patterns. Phloretin exhibited more than 60% glucose uptake inhibition. Hypoxia conferred two to fivefold higher drug resistance in SW620 and K562 to DNR. Inhibition of glucose uptake by phloretin sensitized cancer cells to DNR for its anticancer activity and apoptosis to overcome drug resistance only under hypoxia. Conclusion Cancer cells heavily rely on glucose transporters for glucose uptake to facilitate a high-rate glycolysis under hypoxia for their survival and drug resistance. Combination of glucose transporter inhibitors and chemotherapeutic drugs may provide a preferential novel therapeutic strategy to overcome drug resistance in hypoxia.     

Control of cellular proliferation by modulation of oxidative phosphorylation in human and rodent fast-growing tumor cells

The relationship between cell proliferation and the rates of glycolysis and oxidative phosphorylation in HeLa (human) and AS-30D (rodent) tumor cells was evaluated. In glutamine plus glucose medium, both tumor lines grew optimally. Mitochondria were the predominant source of ATP in both cell types (66-75%), despite an active glycolysis. In glucose-free medium with glutamine, proliferation of both lines diminished by 30% but oxidative phosphorylation and the cytosolic ATP level increased by 50%. In glutamine-free medium with glucose, proliferation, oxidative phosphorylation and ATP concentration diminished drastically, although the cells were viable. Oligomycin, in medium with glutamine plus glucose, abolished growth of both tumor lines, indicating an essential role of mitochondrial ATP for tumor progression. The presumed mitochondrial inhibitors rhodamines 123 and 6G, and casiopeina II-gly, inhibited tumor cell proliferation and oxidative phosphorylation, but also glycolysis. In contrast, gossypol, iodoacetate and arsenite strongly blocked glycolysis; however, they did not affect tumor proliferation or mitochondrial metabolism. Growth of both tumor lines was highly sensitive to rhodamines and casiopeina II-gly, with IC(50) values for HeLa cells lower than 0.5 microM, whereas viability and proliferation of human lymphocytes were not affected by these drugs (IC(50) > 30 microM). Moreover, rhodamine 6G and casiopeina II-gly, at micromolar doses, prolonged the survival of animals bearing i.p. implanted AS-30D hepatoma. It is concluded that fast-growing tumor cells have a predominantly oxidative type of metabolism, which might be a potential therapeutic target.     

Inhibition of phosphatidylinositol 3-kinase-mediated glucose metabolism coincides with resveratrol-induced cell cycle arrest in human diffuse large B-cell lymphomas

An abnormally high rate of aerobic glycolysis is characteristic of many transformed cells. Here we report the polyphenolic compound, resveratrol, inhibited phosphatidylinositol 3-kinase (PI-3K) signaling and glucose metabolism, coinciding with cell-cycle arrest, in germinal center (GC)-like LY1 and LY18 human diffuse large B-cell lymphomas (DLBCLs). Specifically, resveratrol inhibited the phosphorylation of Akt, p70 S6K, and S6 ribosomal protein on activation residues. Biochemical analyses and nuclear magnetic resonance spectroscopy identified glycolysis as the primary glucose catabolic pathway in LY18 cells. Treatment with the glycolytic inhibitor 2-deoxy-D-glucose, resulted in accumulation of LY18 cells in G0/G1 -phase, underscoring the biological significance of glycolysis in growth. Glycolytic flux was inhibited by the PI-3K inhibitor LY294002, suggesting a requirement for PI-3K activity in glucose catabolism. Importantly, resveratrol treatment resulted in inhibition of glycolysis. Decreased glycolytic flux corresponded to a parallel reduction in the expression of several mRNAs encoding rate-limiting glycolytic enzymes. These results are the first to identify as a mechanism underlying resveratrol-induced growth arrest, the inhibition of glucose catabolism by the glycolytic pathway. Taken together, these results raise the possibility that inhibition of signaling and metabolic pathways that control glycolysis might be effective in therapy of DLBCLs.     

异常代谢物与肿瘤免疫微环境的改造

代谢重编程是肿瘤常见的特征之一,受到细胞内在因素和肿瘤免疫微环境（TIME）中代谢物的调节。微环境中的肿瘤细胞与免疫细胞对营养物质的利用存在竞争关系,肿瘤细胞代谢活性增强会导致免疫细胞所需关键营养物质被过度消耗,并减少抑制肿瘤免疫的代谢副产物的产生和蓄积,进而导致免疫细胞功能障碍。TIME中的肿瘤细胞、免疫细胞和基质细胞均可通过代谢中间物或产物的消耗和分泌来改变TIME,而改造后的TIME亦可反过来影响这些细胞的功能。本文就TIME中的异常代谢物对其的改造进行简要综述,深入了解并揭示异常代谢物与TIME的关系,以期为肿瘤免疫治疗提供理论基础及新的思路。     

缺氧对食管癌细胞缺氧诱导因子-1α及己糖激酶表达的影响

目的探讨不同缺氧程度对食管癌细胞株TE1中缺氧诱导因子-1α（Hypoxia-inducible factor-1α,HIF-1α）及糖酵解关键酶表达的影响。方法 TE1细胞分别在正常氧分压和缺氧条件下培养,缺氧时间设定为6、12、24及48h,使用Western blot方法检测缺氧培养不同时间后细胞中HIF-1α及糖酵解关键酶己糖激酶II（Hexokinase-II,HK-II）的蛋白水平表达变化。结果常氧及缺氧条件下TE1细胞中HIF-1α及HK-II均有表达,缺氧后表达均较常氧培养时明显增强,且随缺氧时间不同而呈先增多后减少的动态变化。结论低氧能够增加食管癌细胞中HIF-1α及HK-II表达而促进糖酵解进程,联合抑制HIF-1α和糖酵解酶可能成为治疗食管癌的潜在的靶点。     

小牛脾提取物改善环磷酰胺诱导的小鼠白细胞减少症和抑制HL60细胞的糖酵解作用(英文)

小牛脾提取物为临床治疗肿瘤的辅助药物,可以改善患者的生理状态,但是其作用机制尚不明确。我们评价了小牛脾提取物对小鼠白细胞减少症和HL60细胞生长的影响。10 mL/kg(2.5 mg生物活性多肽和190μg核糖/mL)小牛脾提取物可显著增加白细胞减少症小鼠的白细胞数量,尤其是中性粒细胞的数量;小牛脾提取物在体外明显刺激骨髓细胞增殖和下调血清中的GM-CSF水平。此外,小牛脾提取物可以明显抑制HL60细胞增殖并降低乳酸和ATP的生成;同时可以明显诱导HL60细胞凋亡和细胞周期S期阻滞。研究结果提示:小牛脾提取物可以明显升高白细胞数量,其机制可能与直接刺激骨髓细胞增殖相关;小牛脾提取物可以抑制HL60细胞生长,其作用机制可能与诱导细胞凋亡,细胞周期阻滞和抑制细胞糖酵解作用有关。该研究结果揭示了小牛提取物新的作用和机制,对于扩展小牛脾提取物的临床应用有重要价值。     

己糖激酶对肿瘤作用的研究进展

肿瘤细胞的特征性代谢性方式是有氧性糖酵解,即Warburg效应。己糖激酶作为糖酵解的关键酶,在肿瘤细胞中广泛高表达,被认为与肿瘤代谢、凋亡和自噬紧密相关。本文通过对己糖激酶及其上下游的研究,可以找到潜在的能适用于多种肿瘤的基因靶向治疗方法。     

Histone Deacetylase 6 Inhibition Exploits Selective Metabolic Vulnerabilities in LKB1 Mutant,KRAS Driven NSCLC

IntroductionIn KRAS-mutant NSCLC, co-occurring alterations in LKB1 confer a negative prognosis compared with other mutations such as TP53. LKB1 is a tumor suppressor that coordinates several signaling pathways in response to energetic stress. Our recent work on pharmacologic and genetic inhibition of histone deacetylase 6 (HDAC6) revealed the impaired activity of numerous enzymes involved in glycolysis. On the basis of these previous findings, we explored the therapeutic window for HDAC6 inhibition in metabolically-active KRAS-mutant lung tumors.MethodsUsing cell lines derived from mouse autochthonous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo.ResultsSurprisingly, KL-mutant cells revealed reduced levels of redox-sensitive cofactors at baseline. This is associated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and antitumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo.ConclusionsExploring the differential metabolism of KL and KRAS/TP53-mutant NSCLC, we identified decreased metabolic reserve in KL-mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC on the basis of a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for the treatment of KRAS-mutant NSCLC with co-occurring LKB1 mutations.