Abstract：
Melanoma is an aggressive form of skin carcinoma, highly resistant to traditional therapies. Photodynamic therapy (PDT) is a non-invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. In this work we evaluated the effect of a cationic zinc(II) phthalocyanine (Pc13) as photosensitizer on a panel of melanoma cells. Incubation with Pc13 and irradiation induced a concentration and light dose-dependent phototoxicity. In order to study the mechanism underlying Pc13-related cell death and to compare the effect of different doses of PDT, the most sensitive melanoma B16F0 cells were employed. By confocal imaging we showed that Pc13 targeted lysosomes and mitochondria. After irradiation, a marked increase in intracellular reactive oxygen species was observed and a complete protection from Pc13 phototoxicity was reached in the presence of the antioxidant trolox. Acridine orange/ethidium bromide staining showed morphological changes indicative of both apoptosis and necrosis. Biochemical hallmarks of apoptosis, including a significant decrease in the expression levels of Bcl-2, Bcl-xL and Bid and mitochondrial membrane permeabilization, were observed at short times post irradiation. The consequent release of cytochrome c to cytosol and caspase-3 activation led to PARP-1 cleavage and DNA fragmentation. Simultaneously, a dose dependent increase of lactate dehydrogenase in the extracellular compartment of treated cells revealed plasma membrane damage characteristic of necrosis. Taken together, these results indicate that a dual apoptotic and necrotic response is triggered by Pc13 PDT-induced oxidative stress, suggesting that combined mechanisms of cell death could result in a potent alternative for melanoma treatment.
摘要：
黑色素瘤是一种侵袭性的皮肤癌,对传统疗法有很强的抵抗力。光动力疗法（PDT）是一种非侵入性治疗方法，可以对恶性细胞发挥选择性的细胞毒活性。在这项工作中，我们评估了阳离子锌（II）酞菁（Pc13）作为光敏剂对一组黑色素瘤细胞的作用。与Pc13一起孵育和辐照会引起浓度和光剂量依赖性的光毒性。为了研究Pc13相关细胞死亡的潜在机制，并比较不同剂量的PDT的效果，使用最敏感的黑色素瘤B16F0细胞。通过共聚焦成像，我们显示Pc13靶向溶酶体和线粒体。辐照后，观察到细胞内活性氧的显着增加，并且在抗氧化剂trolox的存在下实现了对Pc13光毒性的完全保护。吖啶橙/溴化乙锭染色显示细胞凋亡和坏死的形态学变化。细胞凋亡的生化标志，包括Bcl-2，Bcl-xL和Bid的表达水平和线粒体膜通透性的显着降低，在辐照后的短时间内观察到。随后细胞色素c释放到细胞质和caspase-3激活导致PARP-1裂解和DNA片段化。同时,治疗细胞胞外区乳酸脱氢酶的剂量依赖性增加揭示了坏死的质膜损伤特征。一起来看,这些结果表明，细胞凋亡和坏死的双重反应是由Pc13PDT诱导的氧化应激触发的，这表明细胞死亡的联合机制可能导致黑素瘤治疗的有效替代方案。
