Light-induced Apoptosis Involves a Defined Sequence of Cytoplasmic and Nuclear Calcium Release in AlPcS4-photosensitized Rat Bladder RR 1022 Epithelial Cells¶
Light-induced Apoptosis Involves a Defined Sequence of Cytoplasmic and Nuclear Calcium Release in AlPcS4-photosensitized Rat Bladder RR 1022 Epithelial Cells¶
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DOI：
10.1562/0031-8655(2000)072
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发表时间：
2000-08
期刊：
影响因子：
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通讯作者：
A. Rück;K. Heckelsmiller;R. Kaufmann;N. Grossman;E. Haseroth;N. Akgün
中科院分区：
文献类型：
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作者：
A. Rück;K. Heckelsmiller;R. Kaufmann;N. Grossman;E. Haseroth;N. Akgün
摘要光敏剂的光激活引起的氧化应激被认为在光动力学治疗（PDT）过程中触发细胞死亡途径中起作用。活性氧被认为是一种信号转导分子，激活导致细胞凋亡的下游反应。主要争论的是其他信号系统，如钙或pH值的合作作用。目前的工作有助于这一讨论，通过研究PDT的影响，在细胞培养的大鼠膀胱上皮细胞的亲水性四磺酸铝酞菁（AlPcS4）。将细胞与光敏剂和钙敏感探针Fluo-3共孵育。用共聚焦激光扫描显微镜分析光诱导的反应。在光激活过程中的动态观察与亚细胞分辨率。在照射过程中检测到瞬时钙升高，特别是在细胞核中，随后是更持续的增加。在与光敏剂孵育1和24 h后，对能量-剂量依赖性光毒性的评价显示，当药物存在24 h时，光毒性增强。令人惊讶的是，当药物孵育24小时时，在非常低的光剂量（0.2 J/cm 2）下观察到细胞增殖的刺激（细胞活力160%）。在1和3 J/cm 2之间的通量照射后可以观察到细胞凋亡的诱导。用异硫氰酸荧光素标记的膜联蛋白V鉴定凋亡细胞，膜联蛋白V在其易位到外质膜后与磷脂酰丝氨酸结合。在存在的抗氧化剂吡咯烷二硫代氨基甲酸酯的瞬时钙升高完全抑制，是随后的PS易位。与此相反，N-乙酰-L-半胱氨酸没有抑制瞬时钙增加。我们的数据可能是一致的钙调节过程中AlPcS4-PDT和氧自由基的参与。
Abstract Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose–dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate–labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-l-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.