Mechanisms of Cell Death after Photodynamic Therapy

描述(申请人提供)：光动力疗法(PDT)与酞菁光敏剂PC4导致迅速产生的活性氧(ROS)，线粒体通透性转变(MPT)，去极化和肿胀，释放细胞色素c，并激活坏死和caspase依赖的细胞凋亡。然而，PC-4和最近合成的PC-4衍生物PC-181也定位于内质网(ER)和溶酶体。我们推测，PC4和PC181对这些细胞器的破坏会导致额外的扰动，如钙离子、铁和蛋白酶的释放，最终促进PDT后MPT依赖的细胞杀伤。我们的目标是进一步确定MPT在PDT诱导的癌细胞杀伤中的作用，并确定内质网损伤和溶酶体在促进死亡途径中的相互作用。在目标1中，我们将利用隔室定位荧光指示剂和共聚焦显微镜来表征PDT后胞浆、线粒体和内质网钙离子的变化。通过特定的干预措施，我们将确定内质网钙库的耗竭、线粒体钙摄取的抑制和线粒体内的螯合是否对PDT后的MPT起抑制作用，并防止随后的细胞杀伤。我们还确定了细胞内钙离子的增加是否可能激活钙依赖的钙蛋白酶，导致BID加工和移位到线粒体，从而导致线粒体功能障碍、MPT和细胞死亡。在目标2中，我们将研究溶酶体铁和蛋白酶的释放在MPT和细胞死亡中的作用。我们将使用特定的荧光指示剂测量胞浆和线粒体可螯合铁，并确定隔室负载铁络合剂的效果。类似地，我们将评估蛋白水解酶释放到胞浆中，并研究特定的组织蛋白酶指示剂和敲除细胞所提供的保护。我们期望通过激活BID裂解和移位到线粒体，从溶酶体释放铁和蛋白酶协同促进线粒体的通透性。在目标3中，我们将研究在培养细胞和分离的线粒体中增强PDT毒性的策略。我们将评估水杨酸盐在PDT后降低MPT发病阈值和增强肿瘤杀伤力的能力。建议的研究将加深我们对如何增强PDT诱导的癌细胞杀伤的理解，并进一步提高PDT转化的有效性，从而造福于癌症患者。

DESCRIPTION (provided by applicant): Photodynamic therapy (PDT) with the phthalocyanine photosensitize PC 4 causes rapid generation of reactive oxygen species (ROS), mitochondrial permeability transition (MPT), depolarization and swelling, release of cytochrome c, and activation of both necrosis and caspase-dependent apoptosis in tumor cells. However, PC 4 and a recently synthesized PC 4 derivative PC 181 also localize to endoplasmic reticulum (ER) and lysosomes. We hypothesize that damage to these organelles by PC 4 and PC 181 leads to additional perturbations, such as Ca2+, iron and protease release that ultimately promote MPT-dependent cell killing after PDT. Our goal is to further characterize the role of the MPT in PDT-induced killing of cancer cells and to determine the interactions of damage to ER and lysosomes in promotion of death pathways. In Aim 1, we will characterize changes of cytosolic, mitochondrial and ER Ca2+ after PDT using compartmentally localized fluorescence indicators and Confocal microscopy. Using specific interventions, we will determine whether depletion of ER Ca2+ stores, inhibition of mitochondrial Ca2+ uptake and intramitochondrial chelation act to suppress MPT after PDT and prevent subsequent cell killing. We also determine whether increased cytosolic Ca2+ may activate Ca2+-dependent calpains, to cause Bid processing and translocation to mitochondria with con- sequent mitochondrial dysfunction, MPT, and cell death. In Aim 2, we will investigate the contribution of lysosomal iron and protease release in relation to the MPT and cell death. We will measure cytosolic and mitochondrial chelatable iron using specific fluorescent indicator and determine the effects of compartmentally loaded iron chelators. Similarly, we will assess protease release into the cytosol and investigate the protection conferred by specific cathepsin indicators and knock-out cells. We expect iron and protease release from lysosomes to synergistically promote mitochondrial permeabilization by activating Bid cleavage and translocation to mitochondria. In Aim 3, we will investigate strategies to enhance PDT toxicity in cultured cells and in isolated mitochondria. We will evaluate the ability of salicylate to decrease the threshold of MPT onset and to enhance tumor killing after PDT. The proposed studies will enhance our understanding how to enhance PDT-induced killing of cancer cells and further increase the efficacy of PDT translation to the benefit for cancer patients.