Iron, NO, and Lipid Peroxide in Photodynamic Therapy

描述（由申请人提供）：光动力疗法（PDT）是一种独特的抗肿瘤方式，涉及增敏剂、光激光和分子氧，其特点是局部生成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细胞死亡，关于这是如何受到敏化剂定位和有毒氧化剂生成效率等因素的影响，人们已经了解了很多。然而，代谢和环境因素的影响，仍然没有很好地了解。现有赠款支持的研究主要集中在这方面一氧化氮（NO）的作用。已知低剂量一氧化氮合酶（NOS）产生的NO对各种肿瘤具有促生存和促进生长的作用。利用5-氨基乙酰丙酸（ALA）为基础的PDT和化学NO供体的体外模型，我们发现NO可以通过清除脂质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光杀伤，从而导致氧化铁的消耗。我们最近发现，ALA/光应激乳腺肿瘤细胞由于诱导NOS的快速和长时间上调而过量产生NO，这大大增加了细胞对内在凋亡的光杀伤的抵抗力。这一建议在很大程度上源于这一新的观察结果，并基于以下假设：在PDT应激下，许多肿瘤会过度表达NOS和NO作为细胞保护反应，这可能会影响PDT的疗效。我们验证这一假设的总体计划是研究：(i)在ALA/光胁迫下，各种已建立的乳腺癌、前列腺癌和皮肤癌细胞过度表达细胞保护性NOS/NO的相对能力；（ii）光胁迫诱导NOS的机制；（iii）应激诱导NO的细胞保护机制；（iv）该NO对旁观者细胞的影响；(v) PDT诱导小鼠异种移植物模型的NOS/NO及NOS抑制作用。计划的方法包括：培养细胞敏化、辐照和凋亡评估；使用NOS抑制剂、NO清除剂和化学NO供体；RNA干扰;免疫印迹;共焦显微镜;人肿瘤植入免疫抑制小鼠的PDT；除ALA-PDT外，还将使用经典的Photofrin-PDT。这一建议具有重要的创新意义，原因如下：(i)虽然NOS抑制剂在动物肿瘤PDT中的积极作用已经有报道，但没有已知的证据表明PDT导致内源性NOS/ no上调；（ii）考虑到至少有一种待研究药物GW274150（作为抗哮喘药）的人体试验已被报道，最终使用NOS抑制剂改善临床PDT结果的前景是有利的。

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.