Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, CONICET-UBA, Junín 956, C1113AAD, Buenos Aires, Argentina.
Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina.
Apoptosis. 2019 Feb;24(1-2):119-134. doi: 10.1007/s10495-018-01512-w.
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.
黑色素瘤是一种侵袭性皮肤癌，对传统疗法具有高度抗性。光动力疗法（PDT）是一种非侵入性治疗程序，可对恶性细胞产生选择性细胞毒性。在这项工作中，我们评估了一种阳离子锌（II）酞菁（Pc13）作为光敏剂对一组黑色素瘤细胞的影响。用 Pc13 孵育并照射会引起浓度和光剂量依赖性光毒性。为了研究与 Pc13 相关的细胞死亡的机制，并比较不同剂量 PDT 的效果，使用最敏感的黑色素瘤 B16F0 细胞。通过共聚焦成像，我们表明 Pc13 靶向溶酶体和线粒体。照射后，观察到细胞内活性氧的明显增加，并且在抗氧化剂 Trolox 的存在下完全阻止了 Pc13 的光毒性。吖啶橙/溴化乙锭染色显示了表明凋亡和坏死的形态变化。在照射后短时间内观察到凋亡的生化特征标志物，包括 Bcl-2、Bcl-xL 和 Bid 的表达水平显著降低以及线粒体膜通透性增加。随后，细胞色素 c 向细胞质释放和 caspase-3 激活导致 PARP-1 切割和 DNA 片段化。同时，处理细胞的细胞外腔中乳酸脱氢酶的剂量依赖性增加揭示了坏死的特征性质膜损伤。总之，这些结果表明，Pc13 PDT 诱导的氧化应激引发了双重凋亡和坏死反应，表明细胞死亡的联合机制可能导致黑色素瘤治疗的有效替代方法。
