Furre Ingegerd Eggen, Møller Michael T N, Shahzidi Susan, Nesland Jahn M, Peng Qian
Pathology Clinic, Rikshospitalet-Radiumhospitalet HF Medical Center, Faculty Division Radiumhospitalet, University of Oslo, Norway.
Apoptosis. 2006 Nov;11(11):2031-42. doi: 10.1007/s10495-006-0190-x.
Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.
光动力疗法（PDT）是一种基于光敏剂、光和氧气相互作用的癌症治疗方法。由5-氨基乙酰丙酸（ALA）或其衍生物诱导内源性光敏剂原卟啉IX（PpIX）的PDT是这种治疗方式的一种改进，已成功应用于皮肤科。然而，ALA-PDT导致细胞破坏的机制尚未阐明。在本研究中，使用六氨基乙酰丙酸（HAL，ALA的己酯）对人T细胞淋巴瘤Jurkat细胞系进行PDT处理。处理后4小时，近80%的细胞呈现出典型的凋亡特征。通过蛋白质免疫印迹法在亚细胞分级分离样品中评估线粒体促凋亡蛋白。PDT导致细胞色素c的胞质转位和凋亡诱导因子（AIF）的核重新分布，但未观察到线粒体Smac/DIABLO、Omi/HtrA2和EndoG的释放。细胞色素c释放后，半胱天冬酶-9和半胱天冬酶-3及其下游底物被切割，同时出现寡核小体DNA片段化。泛半胱天冬酶抑制剂z-VAD.fmk可防止寡核小体DNA片段化，但未能抑制PDT介导的凋亡。在z-VAD.fmk存在的情况下，HAL-PDT后还发现了由AIF介导的不依赖半胱天冬酶的途径诱导的凋亡，并伴有大规模DNA片段化。这些结果表明，细胞色素c介导的依赖半胱天冬酶的途径和AIF诱导的不依赖半胱天冬酶的途径同时参与了PDT诱导的凋亡。当细胞色素c诱导的依赖半胱天冬酶的途径被阻断时，细胞通过AIF介导的途径进入凋亡，这清楚地表明这种凋亡诱导不需要细胞色素c介导的依赖半胱天冬酶的途径。这一发现可能会对提高PDT的有效性产生影响。
