Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.
Department of Biophysics, Jagiellonian University, Krakow, Poland.
Cell Biochem Biophys. 2020 Jun;78(2):149-156. doi: 10.1007/s12013-020-00909-2. Epub 2020 Apr 17.
Iron-catalyzed, free radical-mediated lipid peroxidation may play a major role in tumor cell killing by photodynamic therapy (PDT), particularly when membrane-localizing photosensitizers are employed. Many cancer cells exploit endogenous iNOS-generated NO for pro-survival/expansion purposes and for hyper-resistance to therapeutic modalities, including PDT. In addition to inhibiting the pro-oxidant activity of Fe(II) via nitrosylation, NO may intercept downstream lipid oxyl and peroxyl radicals, thereby acting as a chain-breaking antioxidant. We investigated this for the first time in the context of PDT by using POPC/Ch/PpIX (100:80:0.2 by mol) liposomes (LUVs) as a model system. Cholesterol (Ch or [C]Ch) served as an in-situ peroxidation probe and protoporphyrin IX (PpIX) as photosensitizer. PpIX-sensitized lipid peroxidation was monitored by two analytical methods that we developed: HPLC-EC(Hg) and HPTLC-PI. 5α-hydroperoxy-Ch (5α-OOH) accumulated rapidly and linearly with irradiation time, indicating singlet oxygen (O) intermediacy. When ascorbate (AH) and trace lipophilic iron [Fe(HQ)] were included, 7α/7β-hydroperoxy-Ch (7-OOH) accumulated exponentially, indicating progressively greater membrane-damaging chain lipid peroxidation. With AH/Fe(HQ) present, the NO donor SPNO had no effect on 5α-OOH formation, but dose-dependently inhibited 7-OOH formation due to NO interception of chain-carrying oxyl and peroxyl radicals. Similar results were obtained when cancer cells were PpIX/light-treated, using SPNO or activated macrophages as the NO source. These findings implicate chain lipid peroxidation in PDT-induced cytotoxicity and NO as a potent antagonist thereof by acting as a chain-breaking antioxidant. Thus, unless NO formation in aggressive tumors is suppressed, it can clearly compromise PDT efficacy.
铁催化的自由基介导的脂质过氧化可能在光动力疗法（PDT）杀伤肿瘤细胞中起主要作用，特别是当使用膜定位的光敏剂时。许多癌细胞利用内源性诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）来实现生存/扩张，并对治疗方式（包括 PDT）产生超耐受力。除了通过亚硝基化抑制 Fe(II)的促氧化剂活性外，NO 还可以拦截下游的脂质氧自由基和过氧自由基，从而起到链断裂抗氧化剂的作用。我们首次在 PDT 背景下对此进行了研究，使用 POPC/Ch/PpIX（100:80:0.2 摩尔比）脂质体（LUV）作为模型系统。胆固醇（Ch 或 [C]Ch）作为原位过氧化探针，原卟啉 IX（PpIX）作为光敏剂。我们开发了两种分析方法来监测 PpIX 敏化的脂质过氧化：HPLC-EC（Hg）和 HPTLC-PI。5α-过氧代胆固醇（5α-OOH）随着辐照时间的增加而迅速且呈线性积累，表明存在单线态氧（O）中间体。当存在抗坏血酸（AH）和痕量亲脂性铁[Fe（HQ）]时，7α/7β-过氧代胆固醇（7-OOH）呈指数积累，表明膜破坏性链脂质过氧化作用逐渐增强。在 AH/Fe（HQ）存在下，NO 供体 SPNO 对 5α-OOH 的形成没有影响，但由于 NO 拦截了携带链的氧自由基和过氧自由基，其对 7-OOH 的形成呈剂量依赖性抑制。当用 SPNO 或激活的巨噬细胞作为 NO 来源，用 PpIX/光处理癌细胞时，也得到了类似的结果。这些发现表明，在 PDT 诱导的细胞毒性中，链脂质过氧化起重要作用，而 NO 作为一种有效的链断裂抗氧化剂，可拮抗其作用。因此，除非在侵袭性肿瘤中抑制 NO 的形成，否则它显然会降低 PDT 的疗效。
