ABSTRACT
AIM:To investigate the effect of low-intensity ultrasound combined with microbubble contrast agent on autophagic death of thyroid cancer cells,and to analyze the mechanism of autophagy activation and its effect on cell via-bility. METHODS:Human thyroid cancer cell line TPC1 was treated with low-intensity ultrasound at 20 kHz frequency and 80 mW intensity combined with microbubbles. The cell death and viability were analyzed by Live/Dead assay and CCK-8 assay 60,120 and 240 s after the treatment. The protein levels of microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),autophagy-related protein 5 (ATG5) and SQSTM1/P62 were determined by Western blot. The number of in-tracellular autophagosomes was measured by the methods of monodansylcadaverine(MDC) staining,green fluorescent pro-tein (GFP)-LC3 transfection and transmission electron microscopy. The level of reactive oxygen species(ROS) was mea-sured and the effect of ROS on autophagy activation was evaluated by N-acetyl-L-cysteine (NAC) treatment. The effect of ATG5 siRNA transfection on autophagy was analyzed for determining the role of autophagic death. RESULTS:Low-intensi-ty ultrasound combined with microbubbles significantly promoted TPC1 cell death and inhibited TPC1 cell viability (P<0.05) in a time-dependent manner. Compared with low-intensity ultrasound group and microbubble group,ultrasound com-bined with microbubbles significantly increased the protein levels of LC3-Ⅱ and ATG5, but inhibited the protein level of P62 (P<0.05). The results of MDC staining,GFP-LC3 transfection and transmission electron microscopy showed that ul-trasound combined with microbubbles significantly increased the number of autophagosomes in the TPC1 cells. Compared with low-intensity ultrasound group and microbubble group, ultrasound combined with microbubbles increased the level of ROS,while NAC significantly reduced the protein level of LC3-Ⅱ (P<0.05). Thansfection with ATG5 siRNA inhibited the autophagy,significantly decreased the percentage of cell death and increased cell viability (P<0.05). CONCLU-SION:Low-intensity ultrasound combined with microbubbles promotes the autophagic cell death by increasing the level of ROS in thyroid cancer cells,leading to death of thyroid cancer cells.