Evans Andrew, Bates Victoria, Troy Helen, Hewitt Stephen, Holbeck Susan, Chung Yuen-Li, Phillips Roger, Stubbs Marion, Griffiths John, Airley Rachel
Tumour Metabolism and Therapeutics Group, School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool L3 3AF, UK.
Cancer Chemother Pharmacol. 2008 Mar;61(3):377-93. doi: 10.1007/s00280-007-0480-1. Epub 2007 May 23.
The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. "Positive" COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis ((31)P-MRS and (1)H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.
易化型葡萄糖转运体Glut-1在多种实体瘤中过度表达，并预示着不良预后。在人类肿瘤样本中常见的坏死周围表达模式与其在缺氧条件下由缺氧诱导因子HIF-1进行的转录调控或通过氧化磷酸化速率降低有关。缺氧调节基因有望成为新的治疗靶点，作为一种预防源自经化学和放射治疗后存活下来的残留化学和放射抗性缺氧细胞的组织增殖和最终转移扩散的手段。抑制Glut-1的表达或功能可能是特异性靶向肿瘤内缺氧细胞的一种方法，这些细胞依赖于高糖摄取来进行无氧糖酵解。我们使用了NCI-60细胞系面板的一系列福尔马林固定、石蜡包埋样本，对Glut-1进行免疫组织化学检测，并通过与NCI多样性筛选中的药物进行COMPARE分析来选择可能的候选先导化合物，这些药物可能通过抑制Glut-1或Glut-1依赖性过程发挥作用。“阳性”COMPARE命中结果大多是结合表皮生长因子受体（EGFR）的缀合假单胞菌毒素。然而，与标准抗癌药物的相关性几乎都是阴性，这表明Glut-1与化疗耐药性之间存在联系。使用从膀胱EJ138、人纤维肉瘤HT 1080以及肝癌野生型Hepa和HIF-1B缺陷型c4肿瘤细胞系产生的稳定的、Glut-1过表达细胞系进行的MTT增殖试验显示，相对于野生型和空载体对照，Glut-1过表达的EJ138对达卡巴嗪、长春新碱和生物还原剂EO9的化疗耐药性呈细胞系依赖性增加。使用从Glut-1过表达的Hepa和c4细胞系产生的细胞裂解物和异种移植瘤进行的代谢组学分析（（31）P-MRS和（1）H MRS）显示，相对于亲代肿瘤提取物，Glut-1过表达的c4中葡萄糖水平更高，而乳酸水平没有增加，而在Glut-1过表达的Hepa异种移植瘤中乳酸水平又显著更高。这意味着在HIF-1调节的糖酵解酶没有协同增加的情况下，Glut-1过表达会增加葡萄糖摄取，但不会增加糖酵解速率。Glut-1过表达的异种移植瘤还显示出更高水平的磷酸二酯（PDE），这与磷脂的代谢物周转有关，并参与膜脂质降解，表明Glut-1可能增加细胞周转的一种机制。
