Biological consequences of protein oxidation

摘要：过氧化氢（H2O2）等氧化剂参与介导多种人类疾病，包括动脉粥样硬化、癌症和神经退行性疾病。氧化剂通过破坏生物分子和改变细胞代谢来促进疾病进程。氧化损伤的关键靶点是结构蛋白和酶。为了了解氧化应激如何导致疾病，重要的是要发现哪些蛋白质受到氧化应激的影响，它们被修饰到什么程度，以及修饰的功能后果。蛋白质氧化可能在被癌症化疗药物诱导死亡的肿瘤细胞中发挥重要的生理作用。据推测，细胞内氧化剂（活性氧；ROS）在接受癌症化疗药物治疗的细胞中产生，并且这些是药物诱导的细胞凋亡过程的重要组成部分。如果是这样，那么这些氧化剂可能是通过改变细胞生存所需的细胞内分子来起作用的。我们特别感兴趣的是寻找可能被凋亡相关氧化剂修饰的特定蛋白质，因为它们可能催化增殖或抑制凋亡级联的反应。我们的研究涉及使用人类B淋巴瘤和乳腺癌细胞作为肿瘤模型，我们使用癌症化疗药物，这些药物有时被引用为通过促氧化机制起作用，如阿霉素，VP-16（依托泊苷）和顺铂。蛋白质氧化是通过测定(1)蛋白质羰基通过免疫印迹法，(2)蛋白质蛋氨酸亚砜残基通过氨基酸分析，(3)蛋白质巯基氧化通过免疫印迹法或荧光活化细胞分选（FACS）。我们还测量了f2 -异前列腺素的形成作为脂质过氧化的标志，并使用氧化剂敏感染料DCFDA和DHR 123测量了细胞内ROS的产生。我们的研究结果表明，化疗药物在没有任何可检测的蛋白质或脂质氧化的情况下诱导广泛的细胞凋亡，在细胞的细胞质和线粒体区室中测量。H2O2通过非凋亡途径杀死细胞，导致蛋白质和脂质氧化增加。三种不同的抗氧化剂化合物（n -乙酰半胱氨酸、Tempol和MnTBAP）不能抑制药物诱导的细胞凋亡，而抑制h2o2诱导的细胞死亡。我们得出结论，药物诱导的细胞凋亡发生的机制不涉及氧化剂，也不需要蛋白质氧化。使用小鼠临床前模型治疗乳腺癌的体内研究进一步表明，在治疗方案中加入抗氧化剂不会干扰阿霉素抑制肿瘤生长的能力。这些发现可能对改进化疗方案具有重要的影响，因为它们表明，通过在治疗方案中添加膳食抗氧化剂，可以安全地减少癌症化疗中氧化介导的副作用。

Summary: Oxidants such as hydrogen peroxide (H2O2) are implicated in mediating a wide array of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. Oxidants contribute to disease processes by causing damage to biomolecules and altering cellular metabolism. Key among the targets for oxidative damage are structural proteins and enzymes. In order to understand how oxidative stress can cause disease, it is important to discover which proteins become affected by oxidative stress, to what degree they are modified, and the functional consequences of the modifications. One place where protein oxidation could play a significant physiological role would be in tumor cells induced to die by cancer chemotherapy drugs. It has been hypothesized that intracellular oxidants (reactive oxygen species; ROS) are generated in cells treated with cancer chemotherapy drugs and that these are an essential component of the drug-induced apoptotic process. If so, then these oxidants likely act by modifying intracellular molecules that are required for cell viability. We are particularly interested in finding specific proteins that might be modified by apoptosis-associated oxidants as these are likely to catalyze the reactions that propagate or inhibit the apoptotic cascade. Our studies involve use of human B lymphoma and breast cancer cells as tumor models and we employ cancer chemotherapy drugs which have sometimes been cited as working through a pro-oxidant mechanism such as doxorubicin (adriamycin), VP-16 (etoposide), and cisplatin. Protein oxidation is assessed by measuring (1) protein carbonyl groups by Western blot immunoassay, (2) protein methionine sulfoxide residues by amino acid analysis, (3) protein sulfhydryl oxidation by Western blot immunoassay or fluorescence activated cell sorting (FACS). We also measure formation of F2-isoprostanes as a marker of lipid peroxidation, and intracellular ROS production using the oxidant-sensitive dyes DCFDA and DHR 123. Our results show that chemotherapy drugs induce extensive apoptosis in the absence of any detectable protein or lipid oxidation, measured in both the cytosolic and mitochondrial compartments of the cell. H2O2, which kills the cells by non-apoptotic pathways, causes increases in both protein and lipid oxidation. Three different antioxidant compounds (N-acetyl cysteine, Tempol, and MnTBAP) fail to inhibit drug-induced apoptosis while inhibiting H2O2-induced cell death. We conclude that drug-induced apoptosis occurs using a mechanism that does not involve oxidants and does not require protein oxidation. In vivo studies using a mouse pre-clinical model for treatment of breast cancer showed further that inclusion of an antioxidant in the treatment protocol does not interfere with the ability of doxorubicin to inhibit tumor growth. These finding may have important ramifications for improving chemotherapy protocols because they suggest that oxidant-mediated side-effects from cancer chemotherapy might safely be diminished by addition of dietary antioxidants to the treatment protocols.