Iron, NO, and Lipid Peroxide in Photodynamic Therapy

描述（申请人提供）：光动力疗法（PDT）是一种独特的抗肿瘤治疗方法，涉及增敏剂、光激发光和分子氧，其特征是局部产生单线态氧和其他细胞毒性氧化剂。当受到PDT诱导的氧化应激时，许多肿瘤屈服于凋亡性细胞死亡，并且已经了解到这是如何受到诸如敏化剂定位和毒性氧化剂生成效率等因素的影响的。然而，代谢和环境因素的影响，仍然没有得到很好的理解。由现有拨款资助的研究主要集中在一氧化氮（NO）在这方面的影响。已知低剂量的一氧化氮合酶（NOS）产生的NO对各种肿瘤具有促存活和生长促进作用。使用基于5-氨基乙酰丙酸（ALA）的PDT和化学NO供体的体外模型，我们已经表明，NO可以通过清除脂质衍生的自由基或通过血红素加氧酶-1和铁蛋白诱导的信号传导来保护肿瘤细胞免受坏死性光杀伤，从而导致促氧化剂铁的耗尽。我们最近发现，由于诱导型NOS的快速和长期上调，ALA/光应激乳腺肿瘤细胞过度产生NO，这大大增加了细胞对内在凋亡光杀伤的抵抗力。这一提议主要是从这一新的观察结果发展而来，并基于以下假设：在PDT应激下，许多肿瘤将过度表达NOS和NO作为细胞保护反应，这可能会损害PDT的疗效。我们检验这一假设的总体计划是研究：（i）各种已建立的乳腺癌、前列腺癌和皮肤癌细胞在ALA/光应激下过表达细胞保护性NOS/NO的相对能力;（ii）光应激诱导NOS的机制;（iii）应激诱导的NO的细胞保护机制;（iv）这种NO对旁观者细胞的影响;（iv）光应激诱导的NOS/NO的表达。和（v）小鼠异种移植模型中NOS/NO的PDT诱导和NOS抑制剂作用。计划的方法包括：培养的细胞致敏、辐照和凋亡评价; NOS抑制剂、NO清除剂和化学NO供体的使用; RNA干扰;免疫印迹;共聚焦显微镜;以及植入免疫抑制小鼠中的人肿瘤的PDT;除了ALA-PDT之外，还将使用经典的Photofrin-PDT。（i）虽然已经报道了NOS抑制剂在动物肿瘤PDT中的积极作用，但是没有已知的证据表明PDT导致内源性NOS/NO上调;（ii）最终使用NOS抑制剂来改善临床PDT结果的前景是有利的，因为至少一种待研究的NOS抑制剂的人体测试，GW 274150（作为抗哮喘药），已有报道。 公共卫生相关性：光动力疗法（PDT）是一种治疗癌症的独特方法，其中敏化剂、可见光和分子氧相互作用以破坏肿瘤细胞并防止肿瘤进一步生长。一氧化氮（NO）是由一氧化氮合酶（NOS）天然产生的小分子气体，具有多种生物学功能，包括促进肿瘤细胞生长和再分布的能力。我们在这个实验室最近发现，培养的人乳腺肿瘤细胞在给予PDT样刺激时迅速过度产生诱导型NOS（iNOS）和NO，这使得细胞对光杀伤更具抵抗力。本研究的主要目的是阐明光胁迫下诱导型一氧化氮合酶的诱导机制以及产生的一氧化氮如何发挥细胞保护作用。研究iNOS抑制剂在体外和动物模型中促进肿瘤细胞杀伤的能力是该项目的重要组成部分，因为此类抑制剂的未来临床使用可以显着提高PDT在癌症治疗中的有效性。

DESCRIPTION (provided by applicant): Photodynamic therapy (PDT), a unique antitumor modality involving a sensitizing agent, photoexciting light and molecular oxygen, is characterized by local generation of singlet oxygen and other cytotoxic oxidants. When subjected to PDT-induced oxidative stress, many tumors succumb to apoptotic cell death, and much has been learned about how this is affected by factors such as sensitizer localization and efficiency of toxic oxidant generation. However, the influence of metabolic and environmental factors, is still not well understood. Studies supported by the existing grant have focused largely on the effects of nitric oxide (NO) in this regard. Nitric oxide synthase (NOS)-generated NO in low doses is known to have pro-survival and growth-promoting effects on various tumors. Using in vitro models of 5- aminolevulinic acid (ALA)-based PDT and chemical NO donors, we have shown that NO can protect tumor cells against necrotic photokilling by either scavenging lipid-derived radicals or by signaling for heme oxygenase-1 and ferritin induction, leading to depletion of prooxidant iron. We recently discovered that NO is overproduced by ALA/light-stressed breast tumor cells due to rapid and prolonged upregulation of inducible NOS and that this substantially increases cell resistance to intrinsic apoptotic photokilling. This proposal developed largely from this novel observation and is based on the following hypothesis: Under PDT stress, many tumors will overexpress NOS and NO as a cytoprotective response, and this can compromise PDT efficacy. Our overall plan for testing this hypothesis is to study: (i) relative abilities of various established breast, prostate, and skin carcinoma cells to overexpress cytoprotective NOS/NO under ALA/light stress; (ii) mechanisms of NOS induction by photostress; (iii) cytoprotective mechanisms of stress-induced NO; (iv) effects of this NO on bystander cells; and (v) PDT induction of NOS/NO in a mouse xenograft model and NOS inhibitor effects. Planned methods include: cultured cell sensitization, irradiation, and apoptosis evaluation; use of NOS inhibitors, NO scavengers, and chemical NO donors; RNA interference; immunoblotting; confocal microscopy; and PDT of human tumors implanted in immunosuppressed mice; in addition to ALA-PDT, classical Photofrin-PDT will be used. This proposal is significant and innovative for the following reasons: (i) Although positive effects of NOS inhibitors in animal tumor PDT have been reported, there is no known evidence for endogenous NOS/NO upregulation due to PDT; (ii) The prospect of eventually using NOS inhibitors to improve clinical PDT outcomes is favorable, given that human testing of at least one of those to be studied, GW274150 (as an anti-asthmatic), has been reported. PUBLIC HEALTH RELEVANCE: Relevance to Public Health Photodynamic therapy (PDT) is a unique approach for treating cancer in which a sensitizing agent, visible light, and molecular oxygen interact to destroy tumor cells and prevent further tumor growth. Nitric oxide (NO), a small gaseous molecule produced naturally by nitric oxide synthase (NOS) enzymes, has a variety of biological functions, including the ability to promote growth and redistribution of tumor cells. We in this laboratory recently discovered that cultured human breast tumor cells rapidly overproduce inducible NOS (iNOS) and NO when given a PDT-like challenge, and that this makes the cells more resistant to photokilling. The major goal of this project is to elucidate the mechanism of iNOS induction under photostress and how the resulting NO acts cytoprotectively. Studying the ability of iNOS inhibitors to promote tumor cell killing in vitro and in animal models is a crucial part of the project because future clinical use of such inhibitors could dramatically improve PDT effectiveness in cancer treatment.