Molecular Pathways for Optimizing PDT

焦脱镁叶绿酸盐基化合物已被开发为第二代光敏剂(PS)， 在各种癌症的光动力疗法(PDT)中具有低光毒性但高效率，例如 肺和呼吸道。这个项目的目标是阐明细胞和分子 这些PS在上皮性肿瘤细胞中启动信号从而导致应激反应和 关于细胞死亡或存活的决定。在本供资期间，确定了以下诊断标记 以剂量依赖的方式与细胞对光动力疗法的即时反应相关。这些标记包括 信号转导蛋白氧化交联、细胞因子和生长因子受体失活 功能，减少细胞蛋白的磷酸化和应激蛋白激酶的激活。这些变化 在PDT存活细胞中是暂时的，在一段时间的停滞后，细胞恢复了信号传入的能力 对炎性细胞因子的反应和恢复增殖。我们已经使用这些标记来识别新的 设计的PS衍生物具有增强摄取，改变亚细胞分布，增加单线态氧 生产和改进的肿瘤控制。我们假设PS与碳水化合物和 金属改变PS的吸收和亚细胞积累，导致细胞活动增强；这种变化 在细胞膜上，受体和细胞内信号在肿瘤细胞存活过程中起主要作用。 在多细胞水平上识别调节机制有助于设计组合 抑制肿瘤的疗法，在光动力疗法反应中幸存下来。提出了以下具体目标进行测试 这些预测：1)在小鼠和人类上皮细胞中确定通过什么机制 碳水化合物和金属结合的PS通过决定摄取来实现其细胞类型特异性的作用 细胞器优先积累以及ATP结合盒转运体G2对细胞的相对贡献 PS的稳态水平；2)确定光动力疗法前后的上皮性肿瘤细胞内的反应 确定生存、从应激中恢复、重建动态平衡和恢复增殖； 3)构建上皮、间质、内皮细胞三维共培养体系 细胞，这将允许识别导致炎症释放的调节途径 对PDT治疗的肿瘤细胞作出反应的介质。在组织中关于光动力疗法效应的发现 培养将在临床前动物模型的肿瘤和接受PDT治疗的患者中得到验证 皮肤癌和头颈癌是这项计划的一部分。

Pyropheophorbide-based compounds have been developed as second generation photosensitizers (PS) that have low phototoxicity but high efficacy in photodynamic therapy (PDT) of various cancer forms, such as of the lung and aerodigestive tract. The goal of this project is to elucidate the cellular and molecular mechanisms by which these PS initiate signaling in epithelial tumor cells that leads to stress reactions and decision over cell death or survival. In the current funding period, diagnostic markers were identified that correlate in a dose-dependent fashion with the immediate cellular response to PDT. These markers include oxidative crosslinking of signal-transducing proteins, inactivation of cytokine and growth factor receptor functions, reduced cellular protein phosphorylation and activation of stress protein kinases. These changes are transient in PDT-surviving cells and, after a period of arrest, cells recover the capability to signal in response to inflammatory cytokines and resume proliferation. We have used these markers to identify newly designed derivatives of PS with enhanced uptake, altered subcellular distribution, increased singlet oxygen production and improved tumor control. We hypothesize that conjugation of PS with carbohydrates and metals alters PS uptake and subcellular accumulation resulting in enhanced cellular activity; that alterations in plasma membrane receptors and intracellular signaling play a major role in tumor cell survival following PDT; and that identification of regulatory mechanisms at multi-cellular level helps in devising combination therapies suppressing tumor, which survive the PDT reaction. Following specific aims are proposed to test these predictions: 1) To identify in mouse and human epithelial cells the mechanisms by which carbohydrate- and metal-conjugated PS achieve their cell type-specific action by determining uptake, cell organelle-preferred accumulation, and the relative contribution of ATP-binding cassette transporter-G2 to the steady state level of PS; 2) to define the peri- and post-PDT reactions within epithelial tumor cells that determine survival, recovery from stress, reestablishment of homeostasis, and resumption of proliferation; and 3) to develop a reconstituted three-dimensional co-culture system of epithelial, stromal or endothelial cells, which will permit the identification of the regulatory pathways that lead to the release of inflammatory mediators in response to PDT-treated tumor cells. The findings regarding PDT effect made in the tissue culture will be verified in tumors of preclinical animal models and in patients undergoing PDT treatment for skin and head/neck cancers as part of this program.