Iron, NO, and Lipid Peroxides in Photodynamic Therapy

光动力疗法(Pdt)是一种多组分的癌症治疗方法，在这种疗法中，肿瘤暴露在致命的单重态下。 氧(‘O2)介导的由局部致敏药物诱导的光氧化应激。 不同增敏剂对肿瘤细胞光杀伤机制的研究 其他病例和坏死(非程序性死亡)。然而，代谢和环境因素在 光动力疗法诱导的细胞凋亡性死亡与坏死性细胞死亡之间的关系尚不清楚。由现有拨款资助的研究包括 重点研究了一氧化氮(NO)在这方面的影响。使用代谢敏化的乳腺肿瘤细胞 原卟啉IX(PpIX)，我们发现(I)在照射过程中传递的NO(NO-NOW)可防止坏死 通过抑制质膜(PM)脂类的自由基(链)过氧化来进行光杀灭；重要的是，残留 杀戮从坏死转变为凋亡；(2)没有更早的交付，也不再存在于辐射期间 (NO-THEN)也抑制了光杀死，初步数据表明参与了铁信号机制。在一个 脂质体系统，NO还保护PPIX免光降解，从而延长其降解寿命。 拟议的研究将更深入地探讨一氧化氮的这些新影响，并提出以下假设：(A) 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脂链过氧化，NO-NOW可通过减少离子泵失活和 膜通透性，从而支持促凋亡的能量代谢；(C)还通过保护膜- 自由基介导的降解结合的敏化剂，NO-Now可以导致对促凋亡！O2的“选择” 目标。为验证这些假说而提出的体外研究将涉及模型膜、两个人体乳房 作为化学和细胞增敏剂的肿瘤细胞系(COH-BR1、MCF-7)、PPEX和花青素540(MC540) (巨噬细胞，内皮细胞)NO供体，以及荧光显微镜，荧光分光光度， 免疫印迹、电泳迁移率改变分析以及高效液体和薄层色谱 电化学检测和磷成像检测。具体计划是调查(一)敏化剂 通过模型系统和细胞中长时间的！O2光生来保护NO-NOW；(Ii)NO-NOW对 在抑制坏死的同时促进凋亡的光杀伤；(Iii)NO-现在调节细胞凋亡的机制； (Iv)NO诱导的光抗性的特征；(V)NO诱导的抗性的潜在机制。 尽管肿瘤血管系统中的巨噬细胞和内皮细胞产生了大量的一氧化氮，但人们对此知之甚少。 它可能会如何影响光动力疗法的疗效。这些研究将沿着这些思路提供重要的新见解，并在 NO-NOW的情况可能提示在PDT中调节细胞凋亡的新方法，这一终点是首选的 过度坏死是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.