Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
PLoS One. 2012;7(11):e50082. doi: 10.1371/journal.pone.0050082. Epub 2012 Nov 26.
Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.
原卟啉 IX（PpIX）在恶性细胞中的积累是 5-氨基酮戊酸（ALA）介导的光动力疗法的基础。我们研究了可能影响 ALA 介导的 PpIX 积累的蛋白质的表达，即寡肽转运蛋白-1 和 -2、亚铁螯合酶和 ABC 转运体 G2（ABCG2），在几种肿瘤细胞系中。在这些蛋白质中，只有 ABCG2 与 ALA 介导的 PpIX 积累呈负相关。亚细胞分级分离研究和共聚焦激光显微镜分析表明，ABCG2 不仅分布在质膜上，还分布在内质网等细胞器，包括线粒体。此外，线粒体 ABCG2 调节 ALA 介导的 PpIX 在线粒体中的含量，ABCG2 的特异性抑制剂 Ko143 增强了线粒体 PpIX 的积累。为了阐明线粒体 ABCG2 的可能作用，我们对过表达 ABCG2 的稳定转染-HEK（ST-HEK）细胞进行了特征描述。在这些 ST-HEK 细胞中，检测到功能性 ABCG2 在线粒体中，并用 Ko143 处理可增加 ALA 介导的线粒体 PpIX 积累。此外，从 ST-HEK 细胞中分离的线粒体可能通过 ABCG2 输出阿霉素，因为 Ko143 抑制了阿霉素的输出。对位于线粒体中的 ABCG2 对蛋白酶 K、内切糖苷酶 H 和肽-N-糖苷酶 F 的敏感性表明，线粒体部分中的 ABCG2 被 N-聚糖修饰，并通过内质网和高尔基体运输，最终定位于线粒体中。因此，发现位于线粒体中的 ABCG2 是一种功能性转运蛋白，并且线粒体 ABCG2 通过将 PpIX 从线粒体输出到细胞质来调节 ALA 介导的 PpIX 水平。
