Hiruma Hiromi, Katakura Takashi, Takenami Tamie, Igawa Satoshi, Kanoh Maho, Fujimura Takao, Kawakami Tadashi
Department of Physiology, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara 228-8555, Japan.
J Photochem Photobiol B. 2007 Jan 3;86(1):1-8. doi: 10.1016/j.jphotobiol.2006.08.003. Epub 2006 Sep 18.
Acridine orange (AO), a weakly basic fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured malignant melanoma cells during illumination with blue light. Immediately after the start of the illumination, the successive disruption of vesicles was observed as a flash of fluorescence, and shortly after that, blebs were formed on the plasma membrane. These cells died within 5 min. Vesicle disruption was completely inhibited when cells were treated with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 followed by loading with AO, but not when bafilomycin A1 was treated after AO loading. Thus, the filling of AO in the vesicle, which is driven by vacuolar H(+)-ATPase, is initially required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Acute cell death was inhibited by treatment with bafilomycin A1 before but not after AO loading. Thus, AO- and blue light-induced acute cell death is associated with vesicle disruption rather than bleb formation. Both the vesicle disruption and the formation of plasma membrane blebs were inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not inhibited by the hydroxyl radical scavenger dimethyl thiourea. Acute cell death was also prevented by singlet oxygen scavengers but not by dimethyl thiourea. Thus, these phenomena are likely caused at least in part by the generation of singlet oxygen. The photosensitive features of plasma and vesicle membranes observed in the present study may be based on the use of the photodynamic effect, such as cancer therapy.
吖啶橙（AO）是一种弱碱性荧光染料，可透过质膜和囊泡膜，并优先保留在细胞内酸性区域。利用荧光显微镜，我们观察了蓝光照射下负载AO的培养恶性黑色素瘤细胞的动态变化。光照开始后立即观察到囊泡的连续破裂，表现为荧光闪烁，随后不久，质膜上形成了泡状突起。这些细胞在5分钟内死亡。当细胞先用液泡H(+)-ATP酶抑制剂巴弗洛霉素A1处理，然后负载AO时，囊泡破裂被完全抑制，但在AO负载后再用巴弗洛霉素A1处理则不然。因此，由液泡H(+)-ATP酶驱动的AO在囊泡中的填充是囊泡破裂最初所必需的。相比之下，巴弗洛霉素A1并不能阻止质膜形成泡状突起，这表明泡状突起的形成与囊泡破裂无关。在AO负载前用巴弗洛霉素A1处理可抑制急性细胞死亡，而在AO负载后处理则不能。因此，AO和蓝光诱导的急性细胞死亡与囊泡破裂有关，而不是与泡状突起的形成有关。从细胞环境中去除氧气以及使用单线态氧清除剂叠氮化钠、抗坏血酸和L-组氨酸可抑制囊泡破裂和质膜泡状突起的形成，但羟基自由基清除剂二甲基硫脲则不能。单线态氧清除剂也可防止急性细胞死亡，但二甲基硫脲则不能。因此，这些现象可能至少部分是由单线态氧的产生引起的。本研究中观察到的质膜和囊泡膜的光敏特性可能基于光动力效应的应用，如癌症治疗。
