In vitro activation of mitochondria-caspase signaling pathway in sonodynamic therapy-induced apoptosis in sarcoma 180 cells.
Sonodynamic therapy (SDT) has been shown to mediate apoptosis in many experimental systems, but the detailed mechanism of this process is unclear. In this study, we aim to investigate the potential participation of the mitochondria-caspase signaling pathway in the SDT-induced apoptosis in isolated sarcoma 180 (S180) cells. The cell suspension was treated with 1.75MHz continuous ultrasound (US) at an acoustic intensity (I(SATA)) of 1.4W for 3min in the absence or presence of 20mug/ml hematoporphyrin (Hp). At different times after the SDT-treatment, the apoptotic cells were identified under a scanning electron microscope, and the apoptosis index (AI) was determined by flow cytometry. In addition, the mitochondrial membrane potential, permeabilization of the inner mitochondrial membrane, and translocation of apoptosis-related proteins were assessed by confocal microscopy. Simultaneously, the activation of some special apoptosis-associated proteins [caspase-9, caspase-3, polypeptide poly (ADP-ribose) polymerase (PARP), and Bax] was evaluated by western blotting. Our results indicate that the ultrasonically activated Hp can cause obvious cell apoptosis (AI, 57.66%) at 3h after treatment, and this effect can be significantly reduced by caspase-9 inhibitor (AI, 20.76%) and the oxygen scavenger NaN(3) (20.11%). However, the apoptosis induced by ultrasound alone was relatively lower (28.33%) and was not reduced by NaN(3). Further, SDT caused an 82.1% reduction in the mitochondrial membrane potential and a 70.7% reduction in the permeabilization of the inner mitochondrial membrane immediately after treatment, and these two effects were obviously prevented by NaN(3). In comparison with the control cells, the SDT-treated cells showed obvious cytochrome-c and Bax translocations, caspase activation, Bax expression, and PARP cleavage at 1h after SDT-treatment. However, in the cells treated with ultrasound alone, these phenomena partially and weakly occurred 3h after exposure. These results primarily showed that the mitochondria-caspase signaling pathway in S180 cells was activated in the US- and SDT-induced apoptosis. Moreover, Hp significantly accelerates the process of apoptosis and enhances the cytotoxic effect of ultrasonic treatment. Singlet oxygen may be responsible for the mitochondrial damage and the activation of the apoptotic signaling pathway.
Tang
Wei
W
College of Life Sciences, Shaanxi Normal University, Shaanxi, Xi'an 710062, China.
Liu
Quanhong
Q
Zhang
Jing
J
Cao
Bing
B
Zhao
Ping
P
Qin
Xiaofei
X
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000502:physiology*; D004789:Enzyme Activation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D051379:Mice; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008928:Mitochondria / Q000502:physiology*; D012510:Sarcoma 180 / Q000473:pathology; D015398:Signal Transduction / Q000502:physiology*; D026082:Singlet Oxygen / Q000494:pharmacology; D014159:Transducers; D014407:Tumor Cells, Cultured; D014464:Ultrasonic Therapy
D026082:Singlet Oxygen; D020169:Caspases