Massive apoptotic cell death of human glioma cells via a mitochondrial pathway following 5-aminolevulinic acid-mediated photodynamic therapy
Massive apoptotic cell death of human glioma cells via a mitochondrial pathway following 5-aminolevulinic acid-mediated photodynamic therapy
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DOI：
10.1007/s11060-006-9325-8
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发表时间：
2007-07-01
影响因子：
3.9
通讯作者：
Kuroiwa, Toshihiko
中科院分区：
文献类型：
--
作者：
Inoue, Hiroto;Kajimoto, Yoshinaga;Kuroiwa, Toshihiko
5-氨基乙酰丙酸（5-ALA）介导的光动力疗法（ALA-PDT）诱导胶质瘤细胞死亡的基本机制尚未完全阐明。在这项研究中，ALA-PDT诱导的细胞死亡机制的细节进行了研究在三个人胶质瘤细胞系（U251 MG，U87 MG，和U118 MG）在体外。为了评估原卟啉IX（PpIX）的积累方式，在与5-ALA孵育后通过流式细胞术测量细胞内PpIX含量。采用末端脱氧核苷酸转移酶介导的dUTP-FITC缺口末端标记法（TUNEL）检测U251 MG细胞的凋亡情况，并检测ALA-PDT后caspase活性的变化。同时测定线粒体膜电位（MMP）和细胞色素c释放。PpIX荧光在暴露于5-ALA后4小时达到平台。死亡细胞的比例随着光剂量的增加而增加。通过TUNEL染色证实这些细胞是凋亡的。细胞经PDT处理后，胞浆中caspase-3和caspase-9的活性增加，MMP的活性降低，细胞色素c的活性显著增加。这些结果表明，MMP功能障碍之后是线粒体细胞色素c释放，其通过线粒体途径触发细胞凋亡。ALA-PDT在人脑胶质瘤细胞中由于线粒体途径的直接激活而诱导大量凋亡，该线粒体途径对许多抗凋亡过程具有抗性。这一发现意味着ALA-PDT是一种很有前途的治疗恶性胶质瘤等难治性肿瘤的方法。
The basic mechanism of cell death induced by 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) (ALA-PDT) in glioma cells has not been fully elucidated. In this study, the details of the cell death mechanism induced by ALA-PDT were investigated in three human glioma cell lines (U251MG, U87MG, and U118MG) in vitro. To evaluate the manner of accumulation of protoporphyrin IX (PpIX), intracellular PpIX contents were measured by flow cytometry after incubation with 5-ALA. To analyze the mechanism of cell death, U251MG cells were assayed by the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling (TUNEL) method, and the caspase activity was measured after ALA-PDT. Furthermore, the mitochondrial membrane potential (MMP) and the release of mitochondrial cytochrome c were determined. PpIX fluorescence reached a plateau 4 h after exposure to 5-ALA. The proportion of dead cells increased with increases in the dosage of light. These cells were confirmed by TUNEL staining to be apoptotic. Increases in the activity of both caspase-3 and -9, a decrease in MMP, and a marked increase in cytochrome c in the cytosolic fraction were found after cells were subjected to PDT. These results indicate that a dysfunction of MMP is followed by mitochondrial cytochrome c release, which triggers apoptosis through a mitochondrial pathway. ALA-PDT induces massive apoptosis due to the direct activation of a mitochondrial pathway, which is resistant to many anti-apoptotic processes, in human glioma cells. This finding implies that ALA-PDT is a promising therapy for the treatment of apoptosis-reluctant tumors such as malignant gliomas.