Division of Medical Science, National Cancer Centre Singapore, 169610 Singapore.
Int J Oncol. 2011 Oct;39(4):821-31. doi: 10.3892/ijo.2011.1109. Epub 2011 Jul 1.
The localization of photosensitizers in the subcellular compartments during photodynamic therapy (PDT) plays a major role in the cell destruction; therefore, the aim of this study was to investigate the intracellular localization of Chlorin e6-PVP (Photolon™) in malignant and normal cells. Our study involves the characterization of the structural determinants of subcellular localization of Photolon, and how subcellular localization affects the selective toxicity of Photolon towards tumor cells. Using confocal laser scanning microscopy (CLSM) and fluorescent organelle probes; we examined the subcellular localization of Photolon™ in the murine colon carcinoma CT-26 and normal fibroblast (NHLC) cells. Our results demonstrated that after 30 min of incubation, the distribution of Photolon was localized mainly in the cytoplasmic organelles including the mitochondria, lysosomes, Golgi apparatus, around the nuclear envelope and also in the nucleus but not in the endo-plasmic reticulum whereas in NHLC cells, Photolon was found to be localized minimally only in the nucleus not in other organelles studied. The relationship between subcellular localization of Photolon and PDT-induced apoptosis was investigated. Apoptotic cell death was judged by the formation of known apoptotic hallmarks including, the phosphatidylserine externalization (PS), PARP cleavage, a substrate for caspase-3 and the formation of apoptotic nuclei. At the irradiation dose of 1 J/cm2, the percentage of apoptotic cells was 80%, respectively. This study provided substantial evidence that Photolon preferentially localized in the subcellular organelles in the following order: nucleus, mitochondria, lysosomes and the Golgi apparatus and subsequent photodamage of the mitochondria and lyso-somes played an important role in PDT-mediated apoptosis CT-26 cells. Our results based on the cytoplasmic organelles and the intranuclear localization extensively enhance the efficacy of PDT with appropriate photosensitizer and light dose and support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.
光动力疗法 (PDT) 过程中，光敏剂在亚细胞区室中的定位在细胞破坏中起着重要作用；因此，本研究旨在研究 Chlorin e6-PVP（Photolon™）在恶性和正常细胞中的细胞内定位。我们的研究涉及 Photolon 亚细胞定位的结构决定因素的表征，以及亚细胞定位如何影响 Photolon 对肿瘤细胞的选择性毒性。我们使用共聚焦激光扫描显微镜 (CLSM) 和荧光细胞器探针；研究 Photolon 在鼠结肠癌细胞 CT-26 和正常成纤维细胞 (NHLC) 中的亚细胞定位。我们的结果表明，孵育 30 分钟后， Photolon 的分布主要定位于细胞质细胞器，包括线粒体、溶酶体、高尔基体、核膜周围，也定位于核内，但不在内质网内；而在 NHLC 细胞中，Photolon 仅被发现定位于核内，而不在研究的其他细胞器中。研究 Photolon 的亚细胞定位与 PDT 诱导的细胞凋亡之间的关系。通过形成已知的凋亡特征来判断细胞凋亡死亡，包括磷脂酰丝氨酸外翻（PS）、PARP 切割、 caspase-3 的底物和凋亡核的形成。在 1 J/cm2 的照射剂量下，凋亡细胞的百分比分别为 80%。本研究提供了充分的证据表明， Photolon 优先定位于以下亚细胞细胞器：核、线粒体、溶酶体和高尔基体，随后线粒体和溶酶体的光损伤在 PDT 介导的 CT-26 细胞凋亡中起着重要作用。我们基于细胞质细胞器和核内定位的结果，极大地增强了适当的光敏剂和光剂量的 PDT 疗效，并支持 PDT 通过诱导凋亡有助于消除恶性细胞的观点，这具有生理意义。
