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

https://doi.org/10.1016/j.ultrasmedbio.2004.02.008Get rights and content

Abstract

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/cm2 (ISPTA) in the presence of air, and changes of intracellular calcium ion concentration ([Ca2+]i) in individual cells by digital imaging, various flow 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® 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: [email protected])

Introduction

Apoptosis was originally described by Kerr et al. (1972), based on the examination of tissue specimens from the adrenal grand. Since then, apoptosis has been extensively studied from many medical and biological points of view. Apoptosis is generally characterized by cell shrinkage, chromatin condensation, inner nucleosomal DNA fragmentation and caspase activation. Apoptosis plays an important role, not only in essential physiologic phenomena, but, also, in the tumor response to treatment with various anticancer modalities (Thompson 1995). However, the mechanism of apoptosis induced by a mechanical stress, ultrasound (US), is not clearly understood.

The first observation of in vitro apoptosis induced by US has been shown in human leukemia cell lines, HL-60, K562, U937 and M1/2 (Ashush et al. 2000). They indicated that 750-kHz high-intensity focused pulsed ultrasound at intensities more than 22.4 W/cm2 caused apoptosis, revealed by morphology and flow cytometry. Furthermore, apoptosis induced by US in vivo has been also reported (Vykhodtseva et al. 2001). However, no report has yet been published on whether or not continuous-wave US for cancer therapy, such as used to induce hyperthermia with relatively low intensities, can induce apoptosis.

Recently, we have shown apoptosis in U937 cells induced by continuous US from a physiotherapy unit (Honda et al. 2002). After sonication, many apoptotic changes, including nuclear chromatin condensation and nuclear fragmentation, are found in sonicated cells, and various apoptotic events such as phosphatidylserine (PS) externalization, decrease of mitochondria membrane potential, superoxide formation, and activation of caspase-3 were confirmed by flow cytometry. We have discovered that apoptosis induced by US was dependent on the dissolved gases and was linked to inertial cavitation itself, but not to the quantities of free radicals generated extracellularly.

From a series of experiments on apoptosis induced by physicochemical stress in U937 cells, we found that U937 cells have Ca2+/Mg2+-dependent endonuclease (Kimura et al. 1998), and that increased intracellular calcium ion concentration ([Ca2+]i) plays a major role in apoptosis induced by hyperthermia (Kameda et al. 2001), its combination with local anesthetics (Arai et al. 2002), a temperature-dependent free radical generator (Li et al. 2001) and also in cadmium-induced apoptosis (Li et al. 2000).

In the present study, the roles of intracellular calcium ions and of intracellular reactive oxygen species (ROS) in sonicated U937 cells were investigated for a better understanding of the molecular mechanism of apoptosis induced by US. In addition, to confirm the role of [Ca2+]i, we examined the effects of the intracellular Ca2+ chelator, BAPTA-AM (Table 1). The effects of an antioxidant and antiapoptotic agent, N-acetyl-l-cysteine (NAC), were also utilized to examine the roles of ROS in the apoptosis.

Section snippets

Chemicals (Table 1)

BAPTA-AM and Fura-2/AM were purchased from Dojindo Laboratory (Kumamoto, Tokyo, Japan). NAC and DiOC6(3) were obtained from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Dihydroethidium (DHET) was from Molecular Probes Inc. (Eugene, OR, USA). Verapamil was purchased from Sigma Chemical Corp. (St. Louis, MO, USA). PhiPhiLux-G1D2 kits and Annexin V-FITC kits were obtained from Oncoimmunin, Inc. (Gaithersburg, MD, USA) and Immunotech (Marseille, France), respectively. Levovist was obtained

Fraction of intact cells and apoptosis

When U 937 cells were exposed to 1-MHz continuous waves at an intensity of 4.9 W/cm2 (ISPTA) (0.6 MPa) for 1 min in air-saturated medium, the fraction of intact cells measured immediately after sonication was 35.5 ± 6.5% (mean ± SD, n = 3). After incubation for 6 h, the fraction of early apoptosis (eA) and secondary necrosis (sN) estimated by flow cytometry were 6.0 ± 2.1% (eA) and 30.6 ± 7.5% (sN) (mean ± SD, n = 5), respectively. The fractions of eA and sN in nonexposed cells were 2.1 ± 0.3%

Discussion

In view of the biological effects and advanced therapeutic application of US, it is important to examine how US can induce cell killing because US has been widely used for medical diagnosis and therapy. Generally, US has been known to induce cell lysis and there has been a question as to whether or not US can induce apoptosis, a gene-regulated cell death, in remaining nonlysed intact cells.

Ashush et al. (2000) first reported that apoptosis could be induced by high-intensity pulsed US in human

Acknowledgements

This study was supported in part by a grant-in-aid for scientific research on priority areas (C)(12217049) from the Ministry of Education, Sports, Culture, Science and Technology, Japan.

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