Lin C W, Shulok J R, Kirley S D, Cincotta L, Foley J W
Urology Research Laboratory, Massachusetts General Hospital, Boston.
Cancer Res. 1991 May 15;51(10):2710-9.
Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells.
尼罗蓝衍生物已被证明是恶性肿瘤光动力治疗中潜在有效的光敏剂。先前一项研究的结果表明，这些染料在细胞中的高积累可能是染料聚集、在膜脂中分配和/或在亚细胞器中隔离的结果。在本报告中，展示了对这些染料在体外细胞中的亚细胞定位和积累机制进行研究的结果。使用了视频增强荧光显微镜，观察到点状荧光模式，其中大部分位于核周区域，细胞外染料浓度在1至100 nM之间。这些颗粒类似于用标准溶酶体染料鉴定的特征颗粒。在较高染料浓度（1 microM或更高）下，核周区域的荧光过于强烈，无法分辨为离散的细胞结构，而包括线粒体和细胞膜在内的其他细胞结构中的荧光可见。在更高的染料浓度（10 - 100 microM）下，用光学显微镜观察到尼罗蓝衍生物为蓝色颗粒，其大小和位置类似于上述点状荧光。进一步表明溶酶体是染料定位主要部位的结果包括：(a) 用溶酶体标记酶酸性磷酸酶对负载染料的细胞进行组织化学染色，显示酶染色和含染料颗粒的定位相似；(b) 对负载染料的细胞进行光处理消除了大部分酸性磷酸酶染色颗粒；(c) 用影响膜pH梯度的试剂处理减少了染料的摄取并增强了染料的外流，而改变细胞膜电位的试剂对染料积累没有影响。染料的摄取被氧化磷酸化抑制剂部分抑制，表明该过程至少部分是能量依赖的。这些发现，连同先前的结果表明这些染料的细胞摄取具有高度浓缩性且在很宽范围内与细胞外染料浓度成正比，与染料主要通过离子捕获机制定位在溶酶体中的假设一致。本研究结果还表明，溶酶体可能是尼罗蓝光敏剂介导的肿瘤细胞光动力杀伤的细胞内靶点，溶酶体亲和性光致敏可能是有效和选择性破坏肿瘤细胞的一种策略。
