Vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine orange-loaded malignant melanoma cells.
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.
Hiruma
Hiromi
H
Department of Physiology, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara 228-8555, Japan. hiruma@med.kitasato-u.ac.jp
Katakura
Takashi
T
Takenami
Tamie
T
Igawa
Satoshi
S
Kanoh
Maho
M
Fujimura
Takao
T
Kawakami
Tadashi
T
D000165:Acridine Orange; D016923:Cell Death / Q000528:radiation effects; D002462:Cell Membrane / Q000528:radiation effects*; D002463:Cell Membrane Permeability / Q000528:radiation effects; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000528:radiation effects*; D006801:Humans; D008027:Light; D008545:Melanoma / Q000473:pathology / Q000532:radiotherapy*; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000502:physiology; D010789:Phototherapy / Q000379:methods*; D025262:Vacuolar Proton-Translocating ATPases / Q000502:physiology
D025262:Vacuolar Proton-Translocating ATPases; D000165:Acridine Orange; D010100:Oxygen