5-氨基酮戊酸介导的光动力疗法抑制 A375 和 A431 细胞的存活活性并促进其凋亡。
5-aminolevulinic acid mediated photodynamic therapy inhibits survival activity and promotes apoptosis of A375 and A431 cells.
机构信息
Central Laboratory, The Union Hospital of Fujian Medical University, Fuzhou 350001, China; Department of Clinical Laboratory, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, 362001, China.
Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Provincial Key Laboratory for Photonics Technology, Fujian Normal University, Fuzhou 350007, China.
出版信息
Photodiagnosis Photodyn Ther. 2018 Mar;21:257-262. doi: 10.1016/j.pdpdt.2018.01.004. Epub 2018 Jan 5.
OBJECTIVES
The purpose of this study was to investigate the effects of 5-aminolaevulinic acid mediated photodynamic therapy (ALA-PDT) on the survival activity and apoptosis of human melanoma cell line A375 and non-melanoma skin carcinoma cell line A431 cells. The mechanism for cellular apoptosis was explored.
METHODS
The cell survival activity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the proportion of apoptotic cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression levels of Bcl-2, Bax, caspase-3, caspase-8 and caspase-9 protein were assessed by western blot. The subcellular localization of cytochrome c was comparatively investigated by immunohistochemistry between pre-ALA-PDT and post- ALA-PDT.
RESULTS
ALA-PDT significantly inhibited the survival activity of A375 cells and A431 cells in a dose- and time-dependent manner. The optimum inhibition efficiencies for A375 cells and A431 cells were obtained at 0.6 mM ALA at 4 h and 8 h after ALA-PDT, respectively. The phenomena of apoptosis were observed in ALA-PDT treated cells by TUNEL assay. The apoptotic rates of A375 cells and A431 cells were 90.0% and 61.5% at 6 h after ALA-PDT, respectively. Apoptosis induced by ALA-PDT involved in down-regulation of Bcl-2 protein, up-regulation of Bax protein and cleaved-PARP protein. It was observed that the expression of cleaved- caspase-3, caspase-8 and caspase-9 proteins in A375 cells and A431 cells gradually increased in 2 h and 4 h but decreased at 4-6 h and 6-8 h after ALA-PDT, respectively. In apoptosis cells immunohistochemical localization show that cytochrome C diffused from the mitochondria into the cytosol.
CONCLUSION
ALA-PDT could significantly inhibit the survival activity of A375 and A431 cells. The apoptosis induced by ALA-PDT in A375 and A431 cells was related to the caspase-dependent death-receptor pathway and Cytochrome c-dependent mitochondrial pathway.
目的
本研究旨在探讨 5-氨基酮戊酸介导的光动力疗法（ALA-PDT）对人黑色素瘤细胞系 A375 和非黑素瘤皮肤癌细胞系 A431 细胞存活活性和凋亡的影响。探索细胞凋亡的机制。
方法
通过 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴盐（MTT）测定法测定细胞存活活性，通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）染色检测凋亡细胞的比例。通过蛋白质印迹法评估 Bcl-2、Bax、caspase-3、caspase-8 和 caspase-9 蛋白的表达水平。通过免疫组织化学比较预 ALA-PDT 和后 ALA-PDT 之间细胞色素 c 的亚细胞定位。
结果
ALA-PDT 以剂量和时间依赖性方式显著抑制 A375 细胞和 A431 细胞的存活活性。A375 细胞和 A431 细胞的最佳抑制效率分别在 0.6 mM ALA 后 4 小时和 8 小时获得。ALA-PDT 处理后的细胞通过 TUNEL 测定法观察到凋亡现象。ALA-PDT 后 6 小时，A375 细胞和 A431 细胞的凋亡率分别为 90.0%和 61.5%。ALA-PDT 诱导的凋亡涉及 Bcl-2 蛋白下调、Bax 蛋白上调和裂解型 PARP 蛋白。观察到 A375 细胞和 A431 细胞中 cleaved-caspase-3、caspase-8 和 caspase-9 蛋白的表达在 ALA-PDT 后 2 小时和 4 小时逐渐增加，但分别在 ALA-PDT 后 4-6 小时和 6-8 小时减少。在凋亡细胞中免疫组织化学定位显示细胞色素 C 从线粒体扩散到细胞质。
结论
ALA-PDT 可显著抑制 A375 和 A431 细胞的存活活性。ALA-PDT 在 A375 和 A431 细胞中诱导的凋亡与 caspase 依赖性死亡受体途径和细胞色素 c 依赖性线粒体途径有关。
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