Iron, NO, and Lipid Peroxides in Photodynamic Therapy

光动力疗法（PDT）是一种多组分癌症治疗，其中肿瘤暴露于致命的单线态 局部敏化药物诱导的氧（1 O2）介导的光氧化应激。我们已经了解了很多 各种敏化剂的肿瘤细胞光杀伤机制，在一些细胞中发生凋亡（程序性细胞死亡）， 例和坏死（非程序性死亡）。然而，代谢和环境因素在 PDT诱导的细胞凋亡与坏死性细胞死亡仍然没有很好的理解。获现有拨款资助的研究项目包括 重点关注一氧化氮（NO）在这方面的影响。使用代谢致敏的乳腺肿瘤细胞， 原卟啉IX（PpIX），我们发现（i）在照射过程中提供的NO（NO-now）保护了细胞免受坏死， 通过抑制质膜（PM）脂质的自由基（链）过氧化而产生的光杀伤作用;重要的是， 杀伤从坏死转变为凋亡;（ii）NO在照射过程中更早递送，不再存在 （NO-那么）也抑制光杀伤，初步数据表明涉及铁信号机制。中 在脂质体体系中，NO还能保护PpIX不被光降解，从而延长其降解寿命。的 提出的研究将更深入地研究NO的这些新效应，提出以下假设： 邻近微血管细胞产生的NO-now和NO-then可增强肿瘤细胞对PDT杀伤的抵抗力; (b)通过抑制PM脂质链过氧化，NO-now可以通过减少离子泵失活促进细胞凋亡， 膜透化，从而支持促凋亡能量代谢;（c）通过也保护膜透化， 与自由基介导的降解结合的致敏剂，NO-现在可以导致促细胞凋亡的“选择”！O2 目标的用于测试这些假设的拟议体外研究将涉及模型膜、两个人类乳房 肿瘤细胞株（COH-BR 1，MCF-7），PpEX和mercury 540（MC 540）作为敏化剂，化学和细胞 （巨噬细胞，内皮）NO供体，以及诸如荧光显微术，荧光分光光度法， 免疫印迹、电泳迁移率变动分析以及高效液相和薄层色谱， 电化学和磷光成像检测。具体计划是调查（i）致敏物 保护无现在与延长！模型系统和细胞中的O2光生成;（ii）NO-现在的能力， 促进细胞凋亡的光杀伤，同时抑制坏死;（iii）机制，NO-现在的代谢三聚体; (iv)NO-然后诱导的光电阻的特性;和（v）NO-然后诱导的电阻的潜在机制。 虽然肿瘤血管系统中的巨噬细胞和内皮细胞产生大量NO，但目前对NO的研究还很少 它会如何影响光动力疗法的疗效这些研究将提供重要的新见解沿着这些路线， NO-now的情况可能提示PDT中调节细胞凋亡的新方法，该终点是优选的 因为炎症被最小化了。

Photodynamic therapy (PDT) is a multicomponent cancer treatment in which tumors are exposed to lethal singlet oxygen ('O2)-mediated photooxidative stress induced by a localized sensitizing drug. Much has been learned about mechanism of tumor cell photokilling by various sensitizers, apoptosis (programmed cell death) occurring in some cases and necrosis (non-programmed death) in others. However, the role of metabolic and environmental factors in PDT-induced apoptotic vs. necrotic cell death are still not well understood. Studies supported by the existing grant have focused on the effects of nitric oxide (NO) in this regard. Using breast tumor cells metabolically sensitized with protoporphyrin IX (PpIX), we found that (i) NO delivered during irradiation (NO-now) protected against necrotic photokilling by inhibiting free radical (chain) peroxidation of plasma membrane (PM) lipids; importantly, residual killing was switched from necrosis to apoptosis; (ii)NO delivered much earlier and no longer present during irradiation (NO-then) inhibited photokilling as well, preliminary data suggesting involvement of an iron signalingmechanism. In a liposome system, NO also protected PpIX from photodegradation, thus prolonging its 'Degenerating lifetime. The proposed studies will delve more deeply into these novel effects of NO with the following hypotheses proposed: (a) NO-now and NO-then generated by neighboring microvascularcells can enhance tumor cell resistance to PDT killing; (b) By inhibiting PM lipid chain peroxidation, NO-now can foster apoptosis by reducing ion pump inactivation and membrane permeabilization, thereby supporting pro-apoptotic energy metabolism; (c) By also protecting membrane- bound sensitizer from free radical-mediated degradation, NO-now can result in a "selection" for pro-apoptotic !O2 targets. The proposed in vitro studies for testing these hypotheses will involve model membranes, two human breast tumor lines (COH-BR1, MCF-7), PpEX and merocyanine 540 (MC540) as sensitizers, chemical and cellular (macrophage, endothelial) NO donors, and techniques such as fluorescence microscopy, spectrofluorimetry, immunoblotting, electrophoretic mobility shift assays, and high-performanceliquid and thin layer chromatography with electrochemical and phosphorimaging detection, respectively. The specific plan is to investigate (i) sensitizer protection by NO-now with prolonged !O2 photogeneration in model systems and cells; (ii)ability of NO-now to facilitate apoptotic photokilling while inhibiting necrosis; (iii) mechanisms by which NO-now accommodatesapoptosis; (iv) characteristics of NO-then-induced photoresistance; and (v) underlying mechanisms of NO-then-induced resistance. Although significant NO is produced by macrophages and endothelial cells in tumor vascular systems, little isknown about how it might impact PDT efficacy. These studies will provide important new insights along these lines, and in the case of NO-now may suggest novel approaches for accommodating apoptosis in PDT,this end-point beingpreferred over necrosis because inflammation is minimized.