VDAC Regulation of Warburg Metabolism in Hepatocarcinoma

VDAC 对肝癌 Warburg 代谢的调节

• 批准号: 9186515
• 负责人: Eduardo Nestor Maldonado
• 金额: $ 34.2万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9186515
• 关键词: Adenine Nucleotides; Agonist; Antioxidants; Apoptosis; Binding; Binding Sites; Bioenergetics; CASP3 gene; Cancer cell line; Cell Death; Cell Proliferation; Cells; Cessation of life; Characteristics; Computer Simulation; Confocal Microscopy; Cyclic AMP-Dependent Protein Kinases; Cyclosporine; Data; Dimerization; Electrophysiology (science); Failure; Future; Generations; Glutathione Disulfide; Glycolysis; HepG2; Human; In Situ Nick-End Labeling; Knowledge; Lead; Lipid Bilayers; MAPK8 gene; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Potentials; Metabolic; Metabolism; Mitochondria; Mus; NADH; Necrosis; New Agents; Nude Mice; Outer Mitochondrial Membrane; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Pathway interactions; Permeability; Pharmacology; Phenotype; Phosphorylation; Phosphorylation Site; Positron-Emission Tomography; Primary carcinoma of the liver cells; Protein Dephosphorylation; Protein Isoforms; Protein Kinase A Inhibitor; Proteomics; Reactive Oxygen Species; Regulation; Respiration; Role; Site; Source; Staining method; Stains; Tubulin; Tubulin Interaction; Voltage-Dependent Anion Channel; Warburg Effect; Xenograft Model; Xenograft procedure; adenylate kinase; annexin A5; base; cancer cell; cell injury; cell killing; chemotherapeutic agent; cytotoxic; cytotoxicity; dimer; drug development; erastin; expectation; experimental study; high throughput screening; hydrophilicity; in vivo; inhibitor/antagonist; knock-down; metabolic phenotype; microscopic imaging; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; overexpression; pharmacophore; prevent; public health relevance; reconstitution; response; small molecule; tumor growth

 DESCRIPTION (provided by applicant): Enhanced glycolysis and suppression of mitochondrial metabolism are features of the Warburg phenomenon in hepatocarcinoma and other cancers. Metabolites enter and exit mitochondria through one channel in the outer membrane: the voltage dependent anion channel (VDAC). The central hypothesis of this proposal is that high free tubulin in cancer cells blocks VDAC and suppresses oxidative phosphorylation in Warburg metabolism and that reversal of tubulin inhibition of VDAC has an anti-Warburg effect that enhances oxidative phosphorylation and decreases glycolysis. Based on exciting new preliminary data, we further hypothesize that VDAC-tubulin antagonists, including erastin, reverse tubulin- dependent VDAC inhibition with consequent mitochondrial hyperpolarization, increased ROS generation and cell death. Accordingly in Specific Aim 1, we will characterize the effects of erastin and other VDAC-tubulin antagonists on cellular bioenergetics (ATP, ADP, AMP, Pi, NADH redox state, phospho-AMP kinase, and rates of respiration and glycolysis) in human hepatocarcinoma cells (HepG2, Huh7 and FOCUS). We will also assess in a Huh7 mouse xenograft model the effect of erastin/VDAC-tubulin antagonists on mitochondrial membrane potential (ΔΨ) and the glycolytic phenotype. Erastin-like compounds that emerged from high throughput screening will be confirmed by electrophysiology as VDAC-tubulin antagonists, further evaluated for effects on cellular bioenergetics and used to create a pharmacophore. In Specific Aim 2, we will determine if protein kinase A (PKA) agonists and PKA inhibitors alter the bioenergetics of Huh7, FOCUS and HepG2 cells in the presence and absence of erastin/VDAC- tubulin antagonists and in VDAC isoform double knockdown cells. We will also assess the effects of PKA overexpression/silencing on cellular bioenergetics. Additionally, proteomic analysis will determine specific sites of VDAC isoform phosphorylation. In Specific Aim 3 we will determine mechanisms of erastin-induced ROS formation that leads to cell death. We expect that erastin and other VDAC-tubulin antagonists will increase mitochondrial metabolism, ΔΨ and ROS formation, leading to the mitochondrial permeability transition and mitochondrial bioenergetic failure, culminating in cell death. Ultimately, we expect that antagonizing VDAC- tubulin interaction will suppress tumor growth in vivo. Overall, the project will generate fundamental new knowledge on mechanisms underlying suppression of mitochondrial metabolism in hepatocarcinoma cells and identify new agents that block VDAC-tubulin interaction to revert the pro-proliferative Warburg metabolic phenotype and selectively promote cytotoxic oxidative stress.

 描述（由申请人提供）：糖酵解增强和线粒体代谢抑制是肝癌和其他癌症中瓦尔堡现象的特征。代谢产物通过外膜中的一个通道进入和离开线粒体：电压依赖性阴离子通道（VDAC）。该提议的中心假设是癌细胞中的高游离微管蛋白阻断VDAC并抑制瓦尔堡代谢中的氧化磷酸化，并且逆转微管蛋白对VDAC的抑制具有抗瓦尔堡效应，可增强氧化磷酸化并减少糖酵解。基于令人兴奋的新的初步数据，我们进一步假设VDAC-微管蛋白拮抗剂，包括erastin，逆转微管蛋白依赖性VDAC抑制，随之发生线粒体超极化，增加ROS生成和细胞死亡。因此，在具体目标1中，我们将表征erastin和其他VDAC-微管蛋白拮抗剂对人肝癌细胞（HepG 2、Huh 7和FOCUS）中细胞生物能量学（ATP、ADP、AMP、Pi、NADH氧化还原状态、磷酸-AMP激酶以及呼吸和糖酵解速率）的影响。我们还将在Huh 7小鼠异种移植模型中评估erastin/VDAC-微管蛋白拮抗剂对线粒体膜电位（Δ E）和糖酵解表型的影响。从高通量筛选中出现的erastin样化合物将通过电生理学确认为VDAC-微管蛋白拮抗剂，进一步评价对细胞生物能量学的影响并用于产生药效团。在具体目标2中，我们将确定蛋白激酶A（PKA）激动剂和PKA抑制剂是否在存在和不存在erastin/VDAC-微管蛋白拮抗剂的情况下以及在VDAC同种型双敲低细胞中改变Huh 7、FOCUS和HepG 2细胞的生物能量学。我们还将评估PKA过表达/沉默对细胞生物能量学的影响。此外，蛋白质组学分析将确定VDAC亚型磷酸化的特定位点。在特定目标3中，我们将确定erastin诱导的ROS形成导致细胞死亡的机制。我们预计erastin和其他VDAC-微管蛋白拮抗剂将增加线粒体代谢、ΔΨ和ROS形成，导致线粒体渗透性转变和线粒体生物能衰竭，最终导致细胞死亡。最终，我们预期拮抗VDAC-微管蛋白相互作用将抑制体内肿瘤生长。总体而言，该项目将产生关于肝癌细胞中线粒体代谢抑制机制的基本新知识，并确定阻断VDAC-微管蛋白相互作用的新药物，以逆转促增殖瓦尔堡代谢表型并选择性促进细胞毒性氧化应激。