Apoptosis induced by microbubbles-assisted ultrasound in leukamia cells via mitochondrial permeability transition pore

• 1、The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Department of Biomedical Engineering, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049

Abstract：Objective: Microbubbles-assited ultrasound cavitation has presented to induce tumor cell apoptosis closely associated with mitochondrial dysfunction. The exact mechanisms behind irreversible mitochondria damage are still not fully understood. Methods: Here, low-intensity pulsed ultrasound (1 MHz, 0.3 Mpa peak negative pressure, 10% duty cycle and 1 kHz pulse repetition frequency) was applied to K562 chronic myelogenous leukemia cells for 1 min with 10% v/v Sonovue microbubbles to enhance the effects of ultrasound cavitation. After ultrasound irradiation, the apoptosis index was determined by flow cytometry with Annexin V-FITC/PI. In addition, the mitochondria membrane potential (△Ψm) was determined by JC-1 assay. The translocation of apoptosis-associated protein cytochrome c (Cyt c) was evaluated by western blotting.Results: In the present study, we found that microbubbles-assisted ultrasound cavitation could increase cellular apoptosis index, mitochondrial depolarization and Cyt c release in K562 cells compared with ultrasound alone treatment. Furthermore, the mitochondrial dysfunctions were significantly inhibited by cyclosporine A (CsA), which is a classical inhibitor of mitochondrial permeability transition pore (mPTP). However, the inhibitor of Bax protein, Bax-inhibiting peptide, could not suppress these effects.Conclusions: This paper suggested that mPTP opening was involved in mitochondrial dysfunctions after exposure to microbubbles-assisted ultrasound cavitaiton. Moreover, the release of Cyt c from mitochondria was dependent on CsA-sensitive mPTP opening, but not the formation of Bax-voltage dependent anion channel (VDAC) complex or Bax oligomeric pore. The data provide more insight to understand the mechanisms of mitochondrial dysfunctions induced by ultrasound cavitation, which can be used as a basis for therapeutic purposes.

Keywords： Ultrasound Cavitation, Microbubble, mPTP, Cyt c,CsA