In vitro activation of mitochondria-caspase signaling pathway in sonodynamic therapy-induced apoptosis in sarcoma 180 cells
In vitro activation of mitochondria-caspase signaling pathway in sonodynamic therapy-induced apoptosis in sarcoma 180 cells
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DOI：
10.1016/j.ultras.2009.12.001
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发表时间：
2010-05-01
期刊：
影响因子：
4.2
通讯作者：
Qin, Xiaofei
中科院分区：
文献类型：
--
作者：
Tang, Wei;Liu, Quanhong;Qin, Xiaofei
声动力疗法（SDT）已被证明在许多实验系统中介导细胞凋亡，但这一过程的详细机制尚不清楚。在这项研究中，我们的目的是探讨潜在的参与，在SDT诱导的肉瘤180（S180）细胞凋亡的caspase信号通路。在不存在或存在20 μ g/ml血卟啉（Hp）的情况下，用1.75MHz连续超声（US）以1.4W的声强度（I（SATA））处理细胞悬浮液3分钟。在SDT处理后的不同时间，在扫描电镜下鉴定凋亡细胞，并通过流式细胞术测定凋亡指数（AI）。此外，通过共聚焦显微镜评估线粒体膜电位、线粒体内膜的透化和凋亡相关蛋白的易位。同时，一些特殊的凋亡相关蛋白[caspase-9，caspase-3，多肽聚（ADP-核糖）聚合酶（PARP），和Bax]的活化通过蛋白质印迹法进行了评估。结果表明，超声激活的Hp在作用3 h后可引起明显的细胞凋亡（AI，57.66%），caspase-9抑制剂（AI，20.76%）和氧清除剂NaN（3）（20.11%）可明显减弱这种作用。然而，单独超声诱导的细胞凋亡相对较低（28.33%），NaN（3）不能减少。此外，SDT在处理后立即引起线粒体膜电位降低82.1%和线粒体内膜透性降低70.7%，NaN明显阻止了这两种作用（3）。与对照组相比，SDT处理后1 h细胞出现明显的细胞色素c和Bax易位、caspase活化、Bax表达和PARP裂解。然而，在单独用超声波处理的细胞中，这些现象在暴露后3小时部分且微弱地发生。这些结果初步表明，在超声和SDT诱导的S180细胞凋亡中，caspase信号通路被激活。此外，Hp还能显著加速细胞凋亡的进程，增强超声波的细胞毒作用。单线态氧可能是线粒体损伤和凋亡信号通路激活的原因。(C)2009年爱思唯尔B。V.保留所有权利。
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 1.75 MHz continuous ultrasound (US) at an acoustic intensity (I(SATA)) of 1.4W for 3 min in the absence or presence of 20 mu g/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 3 h 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 1 h after SDT-treatment. However, in the cells treated with ultrasound alone, these phenomena partially and weakly occurred 3 h 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. (C) 2009 Elsevier B. V. All rights reserved.