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疗法，涉及增敏剂、光激发光和分子氧，其特点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，许多肿瘤会死于细胞凋亡，关于增敏剂的定位和有毒氧化剂生成效率等因素是如何影响这一点的，已经有了很多研究。然而，代谢和环境因素的影响，仍然不是很清楚。现有赠款支持的研究主要集中在一氧化氮(NO)在这方面的影响。一氧化氮合酶(NOS)在低剂量下产生的NO对多种肿瘤具有促进生存和促进生长的作用。利用基于5-氨基酮丙酸(ALA)的光动力疗法和化学NO供体的体外模型，我们已经证明，NO可以通过清除脂质衍生的自由基或通过信号转导血红素加氧酶-1和铁蛋白诱导而保护肿瘤细胞免受坏死性光杀伤，从而导致氧化剂铁的耗竭。我们最近发现，由于诱导型一氧化氮合酶的快速和长期上调，ALA/光应激乳腺肿瘤细胞产生过量的NO，这大大增加了细胞对内在凋亡的光杀伤的抵抗力。这一建议很大程度上源于这一新的观察，并基于以下假设：在PDT应激下，许多肿瘤将过度表达NOS和NO作为一种细胞保护反应，这可能会损害PDT的疗效。我们测试这一假说的总体计划是研究：(I)各种已建立的乳腺、前列腺和皮肤癌细胞在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；以及(V)在小鼠异种移植模型中PDT诱导NOS/NO的作用和NOS抑制剂的作用。计划中的方法包括：培养细胞的敏化、辐射和凋亡评估；使用NOS抑制剂、NO清除剂和化学NO供体；RNA干扰；免疫印迹；共聚焦显微镜；以及植入免疫抑制小鼠的人类肿瘤的PDT；除了ALA-PDT，还将使用经典的Photofrin-PDT。这项建议具有重大意义和创新意义，原因如下：(I)尽管已有报道称一氧化氮合酶抑制剂对动物肿瘤光动力疗法有积极作用，但尚无已知证据表明内源性一氧化氮合酶/一氧化氮因光动力疗法而上调；(Ii)鉴于已有报道称至少有一种待研究的GW274150(作为平喘剂)已进行人体试验，最终使用一氧化氮合酶抑制剂来改善临床光动力疗法的结果是有利的。

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.