Choi Hongran, Lim Wonbong, Kim Ji-Eun, Kim Inae, Jeong Jinan, Ko Youngjong, Song Jongwoon, You Sunyeol, Kim Doman, Kim Misook, Kim Byung-Kuk, Kim Okjoon
Department of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University, Bug-Gu, Gwangju, Korea.
Photomed Laser Surg. 2009 Jun;27(3):453-60. doi: 10.1089/pho.2008.2334.
The objective of this study is to investigate the effect of intracellular photosensitizer distribution on tumor cell death after photodynamic therapy (PDT).
The photosensitizer accumulates in tumor tissue during PDT, and generates intracellular reactive oxygen species (ROS), resulting in tumor cell death.
This study was carried out to elucidate the effects of PDT in a KB oral cancer cell line using hematoporphyrin with irradiation at 635 nm and 5 mW/cm(2). After irradiation, the MTT reduction method, agarose gel electrophoresis, flow cytometry, and Diff-Quick staining were performed. The intracellular ROS level was measured by DCF-DA. Intracellular hematoporphyrin was monitored with a confocal microscope, and Western blot and caspase activity assays were performed.
In our study, cell survival was reduced by about 50% after 3 h of hematoporphyrin incubation time. In DNA fragmentation, flow cytometry, and Diff-Quick assay, necrosis was identified within 12 h and apoptosis soon thereafter. Confocal microscopy revealed that hematoporphyrin was localized in the cell membrane, cytoplasm, and nucleus as time passed. The quantities of intracellular ROS correlated with the time of hematoporphyrin accumulation. Additionally, Western blot analysis of Bcl-2/Bax, the release of cytochrome C, and activity of caspase-3 and caspase-9 showed that apoptosis followed the mitochondria-dependent pathway.
PDT with hematoporphyrin in the KB cell line showed morphological changes of cell necrosis and apoptosis, which were associated with the time of distribution and localization of hematoporphyrin. Also, the apoptosis evoked followed the mitochondria-dependent pathway.
本研究旨在探讨光动力疗法（PDT）后细胞内光敏剂分布对肿瘤细胞死亡的影响。
在PDT过程中，光敏剂在肿瘤组织中蓄积，并产生细胞内活性氧（ROS），导致肿瘤细胞死亡。
本研究采用血卟啉对KB口腔癌细胞系进行PDT，照射波长为635nm，功率密度为5mW/cm²，以阐明其作用效果。照射后，进行MTT还原法、琼脂糖凝胶电泳、流式细胞术和Diff-Quick染色。采用DCF-DA检测细胞内ROS水平。用共聚焦显微镜监测细胞内血卟啉，并进行蛋白质免疫印迹和半胱天冬酶活性检测。
在我们的研究中，血卟啉孵育3小时后细胞存活率降低约50%。在DNA片段化、流式细胞术和Diff-Quick检测中，12小时内可识别出坏死，随后很快出现凋亡。共聚焦显微镜显示，随着时间推移，血卟啉定位于细胞膜、细胞质和细胞核。细胞内ROS的量与血卟啉积累时间相关。此外，对Bcl-2/Bax的蛋白质免疫印迹分析、细胞色素C的释放以及半胱天冬酶-3和半胱天冬酶-9的活性表明，凋亡遵循线粒体依赖性途径。
KB细胞系中血卟啉介导的PDT表现出细胞坏死和凋亡的形态学变化，这与血卟啉的分布和定位时间有关。而且，所引发的凋亡遵循线粒体依赖性途径。
