PHOTOSENSITIZED REDUCTION AND DNA ALKYLATION OF ALKYLATING QUINONES AND NITROAR

烷基化醌和硝基芳基的光敏还原和 DNA 烷基化

• 批准号: 8167848
• 负责人: ANTONIO E ALEGRIA
• 金额: $ 21.81万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167848
• 关键词: Adenosine; Air; Alkylating Agents; Alkylation; Anoxia; Apoptosis; Binding; Cations; Cell Death; Cells; Charge; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Adducts; DNA Alkylation; DNA Binding; Detection; Development; Dyes; Electrons; Endoscopy; Environment; Event; Funding; Grant; Guanosine; Hematoporphyrins; Hydrophobicity; Hydroxylamine; Hypoxia; Institution; Knowledge; Lasers; Light; Lipid Binding; Lipids; Malignant Neoplasms; Malignant neoplasm of esophagus; Measures; Membrane Lipids; Methods; Metronidazole; Molecular; Necrosis; Nitroimidazoles; Nucleosides; Oligonucleotides; Organ; Oxidation-Reduction; Oxygen; Pathway interactions; Photochemotherapy; Photosensitizing Agents; Porphyrins; Process; Production; Property; Quinones; Reaction; Relative (related person); Research; Research Personnel; Resources; Rest; Role; Singlet Oxygen; Site; Solid Neoplasm; Source; Sulfhydryl Compounds; Superoxides; Testing; Therapeutic Effect; Tissues; Triplet Multiple Birth; United States National Institutes of Health; Uroporphyrins; Vesicle; Work; Zn(II)-phthalocyanine; absorption; adduct; aqueous; cancer therapy; chlorin; comparative; crosslink; cytotoxicity; design; flash photolysis; flexibility; inorganic phosphate; killings; macromolecule; neoplastic cell; optical fiber; phthalocyanine; physical property; quantum; triplet state; tumor; wasting

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Photodynamic therapy (PDT) is a cancer treatment that uses a combination of red laser light, a photosensitizing agent and molecular oxygen to bring about a therapeutic effect. PDT is particularly promising for treating hollow-organ cancers, for example oesophageal cancers. This is because laser light can now be delivered with great accuracy, via thin flexible optical fibers and endoscopy, to almost any site in the body and with minimal damage to overlying healthy tissue. Porphyrins (POR), phthalocyanines (PC), chlorins (CHL) and others are currently being used in photodynamic treatment (PDT) of tumors due to their large absorption coefficients in the 500-800 nm range. In the presence of air these will photosensitize the production of singlet oxygen and superoxide. Singlet oxygen production, the so-called Type II pathway, is claimed as the most important process which kills tumor cells. However, Type I pathways, those involving photoreduction or photooxidation of substrates, have.also been proposed as photocytotoxic events in PDT, especially in hypoxic environments. In addition, PDT produces in many instances, in cells, apoptosis and necrosis. Solid tumors are often hypoxic. Thus, if photosensitizers are localized inside these tumors, singlet oxygen would not be the reactive species which should eventually kill these tumor cells. Since these dyes are able to photoreduce oxygen, then, these should also photoreduce molecules with nearly equal or more positive redox potentials than oxygen in anoxic/hypoxic cells. If this substrate is a DNA alkylating quinone or nitroarene, which is activated by reduction, it could act as an alkylating species and then DNA alkylation should be expected, with the consequent cell death. Such activation should occur near the DNA site to avoid wasting quinones or nitroarenes by alkylation of other less critical macromolecules. Nitroarenes are reduced to nitrosoarenes (2 electrons), which are highly reactive towards thiols, or further to hydroxylamines (4 electrons), which are reactive species towards Lguanine. In contrast, the aziridinyl-quinones require either 1 or 2 electrons to be activated as alkylating agents. In this regard, since fewer electrons are needed by quinones for alkylating activity aziridinyl-quinones could be more easily photoactivated to a DNA-alkylating species. Photosensitizers are photooxidized, or their triplet states quenched, by nitroimidazoles under anaerobic and hypoxic conditions. This has been demonstrated using flash photolysis methods, even for a nitroimidazole with a much more negative redox potential than oxygen. For example, this was observed using hematoporphyrin and uroporphyrin as photosenzitizers in the presence of metronidazole, with E = -485 mV, while the redox potential of oxygen is -330 mV. However, to our best knowledge, direct detection and characterization of a dye-photoreduced nitroarene or quinone has not occurred. To our best knowledge, previous work on reactions involving photosensitizers and nitroarenes (a) have not dealt with cells under hypoxic conditions, (b) have not considered the importance of the nitroarene redox potential in the yield or quantum yields of photoreduction or cytotoxicity, (c) have not used alkylating quinones instead of nitroaranes in their studies, (d) have not considered the yields or quantum yields of reduced quinone/nitroarene in heterogeneous media vs. aqueous media and (e) have not explored the combination of a DNA-bound (or free) sensitizer with an alkylating quinonelnitroarene in producing DNA adducts. In this work, pyridinium zinc phthalocyanine (PPC) and 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), which are cations and should bind DNA phosphates, will always be included in the development of the following specific aims. Other photosensitizers (hydrophilic or lipophylic and negatively charged) will be included for comparative purposes. Whenever possible, the role of pH will be determined. Special emphasis will also be made on hypoxic or anoxic conditions, although normoxic conditions will be used for comparison. Thus, the following specific aims are designed to fill some of the gaps stated above: 1. To measure binding or distribution constants of POR, PC, CHL and quinones and nitroarenes to DNA or oligonucleotides and multilamellar vesicles (MLVs) of dimiristoylphosphatidylcholine (DMPC) in order to determine the relative hydrophobicity and amount of the photosensitizer and quinone/nitroarene bound to DNA or lipid membrane. 2. To detect intermediates in the photoreactions of POR, PC and CHL in the presence or absence of quinone/nitroarenes and in the presence or absence of nucleosides (guanosine, adenosine). 3. To detect intermediates in the photoreactions of DNA-bound (or oligonucletide-bound), lipid SUVs (small unilamellar vesicles)-bound, and unbound POR, PC and CHL with quinone/nitroarenes. 4. To measure photophysical properties of these intermediates and the interdependence of these properties on the physical properties of the photosensitizer and substrate (redox potentials of substrates, triplet energy of the sensitizer, DNA binding, lipid partition). 5. To identify and quantify photoproducts derived from the quinone/nitroarenes in the photoreactions stated above, in the presence and absence of SUVs or DNA, not including DNA covalent adducts. 6. To identify nucleoside and DNA covalent adducts, including cross-linking, formed in the photoreactions described above. 7. To determine the role of the combination of these photosensitizers with alkylating quinones/nitroarenes on inducing cytotoxicity in tumor cells under hypoxia/anoxia vs.normoxia. Specific aim # 7 will test pairs of sensitizers and quinone/nitroarenes which are successful in the production of intermediates or photoproducts under anoxia or hypoxia measured in Specific Aims 2 to 6. A few of the unsuccessful pairs will also be included as negative controls. Specific Aims 1 and 2 will be worked on during the first years. The rest of the years will essentially be devoted to specific Aims 3 to 7.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 光动力疗法（PDT）是一种结合使用红色激光、光敏剂和分子氧来产生治疗效果的癌症治疗方法。 PDT 对于治疗食道癌等中空器官癌症特别有前景。这是因为激光现在可以通过细的柔性光纤和内窥镜以极高的精度传送到身体的几乎任何部位，并且对覆盖的健康组织的损害最小。卟啉（POR）、酞菁（PC）、二氢卟酚（CHL）等由于在500-800 nm范围内具有较大的吸收系数，目前被用于肿瘤的光动力治疗（PDT）。在空气存在的情况下，这些将光敏化单线态氧和超氧化物的产生。单线态氧的产生，即所谓的 II 型途径，被认为是杀死肿瘤细胞的最重要的过程。然而，涉及底物光还原或光氧化的 I 型途径也被认为是 PDT 中的光细胞毒性事件，尤其是在缺氧环境中。此外，PDT 在许多情况下会导致细胞凋亡和坏死。 实体瘤通常是缺氧的。因此，如果光敏剂位于这些肿瘤内部，单线态氧将不会成为最终杀死这些肿瘤细胞的活性物质。由于这些染料能够光还原氧，因此，它们也应该光还原分子，其氧化还原电位几乎等于或比缺氧/缺氧细胞中的氧更正。如果该底物是 DNA 烷基化醌或硝基芳烃，可通过还原激活，则它可以充当烷基化物质，然后预期会发生 DNA 烷基化，从而导致细胞死亡。这种激活应该发生在 DNA 位点附近，以避免因其他不太重要的大分子的烷基化而浪费醌或硝基芳烃。 硝基芳烃被还原为亚硝基芳烃（2 个电子），其对硫醇具有高度反应性，或进一步还原为羟胺（4 个电子），其是对鸟嘌呤的反应性物质。相反，氮丙啶基醌作为烷化剂需要 1 或 2 个电子才能被激活。在这方面，由于醌的烷基化活性所需的电子较少，氮丙啶基醌可以更容易地光活化为DNA烷基化物质。在厌氧和缺氧条件下，光敏剂被硝基咪唑光氧化，或其三线态猝灭。这已经通过闪光光解方法得到了证明，甚至对于氧化还原电位比氧负得多的硝基咪唑也是如此。例如，在甲硝唑存在下使用血卟啉和尿卟啉作为光敏剂观察到这一点，E = -485 mV，而氧的氧化还原电位为-330 mV。然而，据我们所知，染料光还原硝基芳烃或醌的直接检测和表征尚未发生。 据我们所知，以前涉及光敏剂和硝基芳烃的反应的工作（a）没有处理缺氧条件下的细胞，（b）没有考虑硝基芳烃氧化还原电位在光还原或细胞毒性的产率或量子产率中的重要性，（ c) 在他们的研究中没有使用烷基化醌代替硝基芳烃，(d) 没有考虑异质介质中还原醌/硝基芳烃的产率或量子产率与水介质相比，并且（e）尚未探索DNA结合（或游离）敏化剂与烷基化醌硝基芳烃的组合来产生DNA加合物。在这项工作中，吡啶锌酞菁 (PPC) 和 5,10,15,20-四(1-甲基-4-吡啶基)卟啉四(对甲苯磺酸) (TMPyP) 是阳离子，应结合 DNA 磷酸盐，将始终将其纳入以下具体目标的制定中。其他光敏剂（亲水性或亲脂性和带负电）将包括在内以进行比较 目的。只要有可能，就会确定 pH 值的作用。尽管将使用常氧条件进行比较，但还将特别强调缺氧或缺氧条件。 因此，设计了以下具体目标来填补上述一些空白： 1. 测量 POR、PC、CHL 以及醌和硝基芳烃与 DNA 或寡核苷酸和多层囊泡 (MLV) 的结合或分布常数 二甲酰磷脂酰胆碱 (DMPC) 以确定光敏剂和醌/硝基芳烃与 DNA 或脂质膜结合的相对疏水性和量。 2. 在存在或不存在醌/硝基芳烃以及存在或不存在核苷（鸟苷、腺苷）的情况下检测POR、PC和CHL光反应中的中间体。 3. 检测 DNA 结合（或寡核苷酸结合）、脂质 SUV（小单层囊泡）结合和未结合的 POR、PC 和 CHL 与醌/硝基芳烃的光反应中的中间体。 4.测量这些中间体的光物理性质以及这些性质与光敏剂和底物的物理性质（底物的氧化还原电位、敏化剂的三重态能量、DNA结合、脂质分配）的相互依赖性。 5. 在存在和不存在 SUV 或 DNA（不包括 DNA 共价加合物）的情况下，鉴定和定量上述光反应中衍生自醌/硝基芳烃的光产物。 6. 鉴定在上述光反应中形成的核苷和 DNA 共价加合物，包括交联。 7.确定这些光敏剂与烷基化醌/硝基芳烃的组合在缺氧/缺氧与常氧下诱导肿瘤细胞细胞毒性的作用。 具体目标#7将测试在具体目标2至6中测量的缺氧或缺氧条件下成功生产中间体或光产物的敏化剂和醌/硝基芳烃对。一些不成功的对也将被包括作为阴性对照。具体目标 1 和 2 将在第一年实现。剩下的几年将主要致力于具体目标 3 至 7。