Mechanisms of Cell Death after Photodynamic Therapy

描述（由申请人提供）：使用酞菁光敏剂PC 4的光动力疗法（PDT）引起肿瘤细胞中活性氧（ROS）的快速产生、线粒体通透性转换（MPT）、去极化和肿胀、细胞色素c的释放以及坏死和半胱天冬酶依赖性凋亡的激活。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。我们假设PC 4和PC 181对这些细胞器的损伤导致额外的扰动，如Ca2+、铁和蛋白酶释放，最终促进PDT后的MPT依赖性细胞杀伤。我们的目标是进一步表征MPT在PDT诱导的癌细胞杀伤中的作用，并确定ER和溶酶体损伤在促进死亡途径中的相互作用。在目标1中，我们将使用区室定位荧光指示剂和共聚焦显微镜来表征PDT后细胞质、线粒体和ER Ca 2+的变化。使用特定的干预措施，我们将确定是否耗尽ER Ca2+商店，抑制线粒体Ca2+摄取和线粒体内螯合作用，以抑制PDT后的MPT，并防止随后的细胞杀伤。我们还确定了胞浆Ca 2+增加是否可能激活Ca 2+依赖性钙蛋白酶，导致Bid加工和易位到线粒体，伴随线粒体功能障碍、MPT和细胞死亡。在目标2中，我们将研究溶酶体铁和蛋白酶释放与MPT和细胞死亡的关系。我们将使用特异性荧光指示剂测量细胞溶质和线粒体可螯合铁，并确定隔室负载铁螯合剂的作用。同样，我们将评估蛋白酶释放到胞质溶胶中，并研究特定组织蛋白酶指示剂和敲除细胞所赋予的保护作用。我们预期铁和蛋白酶从溶酶体释放协同促进线粒体透化通过激活Bid裂解和易位到线粒体。在目标3中，我们将研究增强培养细胞和分离线粒体中PDT毒性的策略。我们将评估水杨酸盐降低MPT发作阈值和增强PDT后肿瘤杀伤的能力。拟议的研究将增强我们对如何增强PDT诱导的癌细胞杀伤的理解，并进一步提高PDT翻译的疗效，从而使癌症患者受益。

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.