Sulfhydryl switches and free-radical scavenger involveme

理论基础：细胞内氧化还原平衡被认为对维持所有类型细胞的正常功能至关重要，包括调节细胞周期进程、增殖和对细胞毒性挑战的反应。氧化还原状态下的异常与许多领域有关，包括衰老和癌症进展。氧化还原状态决定应激反应的一个机制是通过细胞内信号传递。存在于许多肽链中的半胱氨酸氨基酸残基含有作为氧化还原“开关”的硫氢键。这些“开关”及其组成的蛋白质根据氧化刺激而被激活或抑制，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对肿瘤细胞氧化应激反应的影响。研究概要：由于糖酵解代谢是维持细胞功能所必需的，并且涉及电子传递，我们研究了这一过程中的氧化还原成分。在HeLa细胞中，用葡萄糖类似物2-脱氧-D-葡萄糖(2DG)改变糖酵解，可向其灌输对IR的敏感性，这种作用可被自由基清除剂N-乙酰半胱氨酸预先逆转(癌症资源63：3413-3417)。这一结果重申了电子转移和氧化还原状态在新陈代谢和细胞毒性中的作用。此外，我们最近根据细胞内途径的氧化还原调节的可能性，重新检查了涉及热诱导放射增敏的结果。在这种情况下，热和辐射似乎对这两种模式都有独特的反应，当在时间上非常接近地给予时，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。当氧化还原失衡激活硫氧还蛋白还原酶(Trr)时，它能够还原并激活其客户蛋白硫氧还蛋白(Trx)，进而激活氧化还原敏感的信号机制，包括AP-1和NF-kB(癌症资源60：6688-6695和癌基因21：6317-6327)。此外，我们还发现了基于遗传和药理学模型影响转录因子活性的差异。转录因子调节与多种生理效应相关，包括细胞周期进程、细胞凋亡、坏死和氧化应激存活(Ortiz等人，正在准备中)。因此，包括TRR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与道格拉斯·斯皮茨博士(之前在华盛顿大学，最近在爱荷华大学)和丽贝卡·阿夫特(华盛顿大学)密切合作完成的。描述我们调查结果的四份手稿已经出版或正在印刷中，其中一份正在准备提交。我是其中每一个的撰稿人，因为我在形成假设、执行实验、在科学会议上展示初步结果以及准备结果供同行审查方面发挥了积极作用。

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.