(PQA1)Molecular mechanisms by which the diabetic drug metformin kills cancer cell

(PQA1)糖尿病药物二甲双胍杀死癌细胞的分子机制

• 批准号: 9063480
• 负责人: W KEITH MISKIMINS
• 金额: $ 29.51万
• 依托单位: SANFORD RESEARCH/USD
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-02 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9063480
• 关键词: Address; Affect; Anabolism; Antineoplastic Agents; Aspirin; Binding; Cancer Patient; Carbohydrates; Cell Cycle Arrest; Cell Death; Cell Survival; Cells; Clinical Trials; Culture Media; Data; Deacetylase; Dissociation; Drug Targeting; Drug usage; Energy Metabolism; Enzymes; Event; Glucose; Glycolysis; Goals; Health; Hexokinase 2; Human; Incidence; Malignant Neoplasms; Malignant neoplasm of ovary; Measures; Mediating; Metabolic; Metabolic Pathway; Metabolism; Metformin; Mitochondria; Molecular; Molecular Mechanisms of Action; Niacinamide; Normal Cell; Oxygen Consumption; Pathway interactions; Peptides; Pharmaceutical Preparations; Phosphotransferases; Process; Protein Acetylation; Proteins; Publishing; Repression; Research; Role; Signal Pathway; Testing; Work; antitumor effect; base; cancer cell; cancer therapy; cell killing; cytotoxic; cytotoxicity; deprivation; diabetic; drug efficacy; drug mechanism; genetic approach; hexokinase; improved; in vivo; inhibitor/antagonist; insight; ketogenic diet; killings; malignant breast neoplasm; mortality; mouse model; novel; overexpression; prevent; tumor; tumor growth

DESCRIPTION (provided by applicant): The broad goal of this proposal is to understand, and take advantage of, metabolic changes that are involved in cancer cell death induced by the common diabetic drug metformin. Metformin treatment of cancer cells leads to accumulation of dysfunctional mitochondria which is associated with cells death. Our new preliminary data show that metformin causes hexokinase II (HKII) to dissociate from mitochondria and promotes depletion of cellular ATP and NAD+. These events, as well as metformin-mediated cell death, are strongly enhanced by glucose deprivation. This is not observed in non-transformed cells. NAD+ depletion following metformin treatment also appears to be associated with changes in protein acetylation. Finally, addition of exogenous NAD+ or overexpression of NAMPT, the rate limiting enzyme in NAD synthesis, protects cells against metformin cytotoxicity. Based on these findings, we hypothesize that metformin-mediated cancer cell death is associated with depletion of ATP and NAD+ and specific effects on a key glycolytic enzyme, HKII, and on NAD-dependent sirtuin protein deacetylase pathways. This hypothesis will be tested through two specific aims. Aim 1 is to dissect the role of glucose and glycolysis on metformin-mediated cell death of cancer cells, to determine the significance of HKII dissociation from mitochondria, and to establish a mouse model to examine the interaction between glucose levels and metformin in treating cancer. Genetic approaches will be used to alter expression of HKII and then the affect on metformin cytotoxicity will be measured. The importance of mitochondrial association by HKII will be examined by expressing deletion constructs that lack the mitochondrial binding domain or by using peptides and compounds that are known to disrupt HKII binding to mitochondria. A mouse model will be developed using a carbohydrate-restricted ketogenic diet to reduce glucose availability to determine if this enhances metformin's anti-tumor activity in vivo. Also metformin will be combined with drugs that target hexokinase activity or localization to determine if this improves the anti-tumor effects. Aim 2 is to determine how NAD+ and NAMPT protect cells against metformin cytotoxicity. We will examine the effects of NAMPT overexpression, or inhibition, on metformin-mediated changes in energy metabolism and cell killing. We will determine if the inhibition of specific sirtuin deacetylases is involved in metforin-mediated changes in metabolism and cell survival. We will identify acetylated proteins that change in abundance upon metformin treatment of cancer cells. We will use mouse models to examine the effects of NAMPT expression and NAD+ precursors on metformin inhibition of tumor growth. We will determine the potential for inhibitors of NAMPT and sirtuin deacetylases to potentiate the action of metformin against tumor growth. With the completion of this work we will have a more complete understanding of the molecular mechanism of action of metformin on cancer cells. We will have an improved rationale for re-purposing of metformin for cancer therapy and we will have new insights on how to improve the efficacy of the drug.

描述（由申请人提供）：该提案的总体目标是了解并利用常见糖尿病药物二甲双胍诱导的癌细胞死亡所涉及的代谢变化。二甲双胍治疗癌细胞会导致功能失调的线粒体积聚，从而导致细胞死亡。我们的新初步数据表明，二甲双胍会导致己糖激酶 II (HKII) 从线粒体解离，并促进细胞 ATP 和 NAD+ 的消耗。这些事件以及二甲双胍介导的细胞死亡会因葡萄糖剥夺而强烈增强。在非转化细胞中没有观察到这一点。二甲双胍治疗后 NAD+ 的消耗似乎也与蛋白质乙酰化的变化有关。最后，添加外源 NAD+ 或过表达 NAMPT（NAD 合成中的限速酶）可保护细胞免受二甲双胍的细胞毒性。基于这些发现，我们假设二甲双胍介导的癌细胞死亡与 ATP 和 NAD+ 的消耗以及对关键糖酵解酶 HKII 和 NAD 依赖性去乙酰化酶途径的特定影响有关。这一假设将通过两个具体目标进行检验。目标 1 是剖析葡萄糖和糖酵解在二甲双胍介导的癌细胞死亡中的作用，确定 HKII 与线粒体解离的重要性，并建立小鼠模型来检查葡萄糖水平和二甲双胍在治疗癌症中的相互作用。将使用遗传方法改变 HKII 的表达，然后测量对二甲双胍细胞毒性的影响。 HKII 与线粒体关联的重要性将通过表达缺乏线粒体结合结构域的缺失构建体或通过使用已知破坏 HKII 与线粒体结合的肽和化合物来检查。将使用限制碳水化合物的生酮饮食来开发小鼠模型，以减少葡萄糖的利用率，以确定这是否会增强二甲双胍的体内抗肿瘤活性。此外，二甲双胍还将与针对己糖激酶活性或定位的药物联合使用，以确定这是否可以提高抗肿瘤效果。目标 2 是确定 NAD+ 和 NAMPT 如何保护细胞免受二甲双胍的细胞毒性。我们将研究 NAMPT 过度表达或抑制对二甲双胍介导的能量代谢和细胞杀伤变化的影响。我们将确定特定沉默调节蛋白脱乙酰酶的抑制是否与二甲福林介导的代谢和细胞存活变化有关。我们将鉴定在二甲双胍治疗癌细胞时发生大量变化的乙酰化蛋白质。我们将使用小鼠模型来检查 NAMPT 表达和 NAD+ 前体对二甲双胍抑制肿瘤生长的影响。我们将确定 NAMPT 和沉默调节蛋白脱乙酰酶抑制剂增强二甲双胍抗肿瘤生长作用的潜力。随着这项工作的完成，我们将对二甲双胍对癌细胞作用的分子机制有更全面的了解。我们将更好地重新利用二甲双胍用于癌症治疗，并且我们将对如何提高药物疗效有新的见解。