Sulfhydryl switches and free-radical scavenger involveme

原理：细胞内氧化还原平衡被认为是维持所有类型细胞正常功能的关键，包括调节细胞周期进程、增殖和对细胞毒性挑战的反应。氧化还原状态的畸变对许多领域都有影响，包括衰老和癌症进展。氧化还原状态决定应激反应的一种机制是通过细胞内信号传导。半胱氨酸氨基酸残基存在于许多肽链中，含有巯基键，其作用类似于氧化还原“开关”。这些“开关”及其组成的蛋白质根据氧化刺激被激活或抑制，包括葡萄糖剥夺、过氧化氢暴露、化疗药物治疗和电离辐射（IR）暴露。我们研究了氧化还原信号因子调节对肿瘤细胞氧化应激反应的影响。研究简介：糖酵解代谢对维持细胞功能至关重要，并涉及电子传递，我们研究了这一过程的氧化还原成分。用葡萄糖模拟物2-脱氧-d -葡萄糖（2DG）改变HeLa细胞对IR的糖酵解，用自由基清除剂n -乙酰半胱氨酸（N-acetyl-cyteine）预处理可逆转这一效应（Cancer Res 63: 3413-3417）。这一结果重申了电子转移和氧化还原状态在代谢和细胞毒性中的作用。此外，我们最近根据细胞内通路的氧化还原调节的潜力，重新检查了我们的结果，包括热诱导的放射致敏。在这种情况下，热和辐射似乎对两种方式都产生了独特的反应，当在时间上接近时，可能有很大的氧化还原成分（Mattson等人，接受发表）。作为这些观察的必然结果，我们试图调节肿瘤细胞中其他氧化还原反应元件的功能。当硫氧还蛋白还原酶（TR）被氧化还原失衡激活时，它能够减少和激活其客户蛋白硫氧还蛋白（TRX）， TRX反过来激活氧化还原敏感信号机制，包括AP-1和NF-kB （Cancer Res 60: 6688-6695和Oncogene 21: 6317-6327）。此外，我们发现基于影响TR活性的遗传和药理学模型的转录因子激活存在差异。转录因子调节与多种生理效应相关，包括细胞周期进展、细胞凋亡、坏死和氧化应激存活（Ortiz等人，在准备中）。因此，包括TR和TRX在内的氧化还原敏感信号通路可能是介入治疗的可行治疗分子靶点，可以增强肿瘤对氧化损伤的反应。项目角色：我们在这个项目上的工作是在与dr。Douglas Spitz（之前就职于华盛顿大学，最近就职于爱荷华大学）和Rebecca Aft（华盛顿大学）。四份描述我们发现的手稿已经出版或正在印刷中，一份正在准备提交。由于我积极参与了假设的形成、实验的执行、在科学会议上展示初步结果以及为同行评审准备结果，因此我是这些研究的主要作者。

Rationale: Intracellular redox balance is believed critical to maintaining normal functioning of cells of all types, including regulation of cell cycle progression, proliferation, and response to cytotoxic challenges. Aberrations in redox state have implications for a variety of fields, including aging and cancer progression. One mechanism through which redox state determines stress response is through intracellular signaling. Cysteine amino acid residues present in many peptide chains contain sulfhydryl bonds which behave as redox "switches." These "switches," and the proteins they compose, are activated or suppressed depending on oxidative stimuli, including glucose deprivation, hydrogen peroxide exposure, chemotherapeutic agents treatment, and ionizing radiation (IR) exposure. We investigated the effect of redox signaling factor modulation on the tumor cellular response to oxidative stressors.Research Synopsis: As glycolytic metabolism is essential to maintaining cell function and involves electron transport, we investigated the redox component of this process. Altering glycolysis with a glucose mimetic, 2-deoxy-D-glucose (2DG), in HeLa cells instilled sensitivity to IR, an effect that was reversed by pretreatment with a free radical scavenger, N-acetyl-cyteine (Cancer Res 63: 3413-3417). This result reaffirms the role of electron transfer and redox state in metabolism and cytotoxicity. Additionally, we have recently re-inspected our results involving heat-induced radiosensitization in light of a potential for redox modulation of intracellular pathways. In this case also, heat and radiation appear to instill a unique response to both modalities when administered in close temporal proximity that likely has a large redox component (Mattson et al, accepted for publication). As a corollary of these observations, we attempted to modulate the function of additional redox-responsive elements in tumor cells. When thioredoxin reductase (TR) is activated by redox imbalance, it is able to reduce and activate its client protein, thioredoxin (TRX), which in turn activates redox-sensitive signaling machinery, including AP-1 and NF-kB (Cancer Res 60: 6688-6695 and Oncogene 21: 6317-6327). Additionally, we found differences in transcription factor activation based on genetic and pharmacological models of affecting TR activity. Transcription factor modulation correlates with a variety of physiological effects, include cell cycle progression, apoptosis, necrosis, and oxidative stress survival (Ortiz et al, in preparation). Redox-sensitive signaling pathways, including TR and TRX, may therefore represent viable therapeutic molecular targets for interventional therapy that could enhance the tumor response to oxidative damage.Project Roles: Our work on this project has been performed in close collaboration with Drs. Douglas Spitz (previously of Washington University and recently of the University of Iowa) and Rebecca Aft (Washington University). Four manuscripts depicting our findings have been published or are in press and one is in preparation for submission. I am a contributing author on each of these, due to my active roles in formation of the hypotheses, performing the experiments, presenting preliminary results at scientific conferences, and preparing the results for peer-review.