Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound

Recently, we have reported that ultrasound (US)-induced apoptosis is due to inertial cavitation and that extracellular reactive oxygen species (ROS) generated by inertial cavitation are not directly correlated with the apoptosis (Honda et al. 2002). The molecular mechanism of apoptosis induced by US is not yet sufficiently clear. Here, we examine the role of intracellular calcium ions and the intracellular ROS on apoptosis induced by US. Human myelomonocytic lymphoma U937 cells were exposed to continuous 1-MHz US at an intensity of 4.9 W/cm(2) (I-SPTA) in the presence of air, and changes of intracellular calcium ion concentration ([Ca2+]i) in individual cells by digital imaging, various How cytometric analyses of endpoints of apoptosis (early apoptosis, secondary necrosis, loss of mitochondria membrane potential, superoxide formation, caspase-3 activation) and DNA fragmentation were explored. Furthermore, the effects of an intracellular calcium ion chelator (BAPTA-AM), an antioxidant (N-acetyl-L-cysteine, NAC), a calcium channel blocker (verapamil), Ca2+-free buffer and Levovist(R) were also investigated. These results indicate that: 1. the mitochondria-caspase pathway and the Ca2+-dependent pathway play cardinal roles in apoptosis induced by US because BAPTA-AM partially inhibited DNA fragmentation, loss of mitochondria membrane potential and caspase-3 activation; 2. intracellular ROS generated from mitochondria, rather than extracellular ROS (which were directly produced by inertial cavitation in the medium), are involved in the regulation of apoptosis induced by US because addition of NAC after sonication showed effective suppression of the apoptosis; and 3. increase of [Ca2+]i appears to be due to nonspecific influx from outside the cells because verapamil is not effective and no increase of [Ca2+](i) due to sonication could be observed in the Ca2+-free buffer. (E-mail: kondot@ms.toyama-mpu.ac.jp) (C) 2004 World Federation for Ultrasound in Medicine Biology.