Early Changes Induced by Low Intensity Ultrasound in Human Hepatocarcinoma Cells

To determine whether low intensity ultrasound irradiation can induce early changes in human hepatocarcinoma cells and investigate the effects of ultrasound intensity, sonication time and incubation time post-sonication on US induced early changes, HepG2 cells were irradiated by low intensity US, at different intensity with different sonication time and incubation time post-sonication, respectively, in this study. Morphologic changes of cells were examined by light and fluorescent microscopy respectively. The percentage of viable, apoptosis and secondary necrosis cells were detected by flow cytometry with double staining of FITC-Annexin V/PI. Percentages of cells in different states were analyzed by L9 33 orthogonal test to find out most effective parameters for US induced early changes. The morphological characteristics of early apoptosis and secondary necrosis are found in US irradiated cells, however few in control. The fluorescent photos also prove early changes, such as phosphatidylserine externalization, increase significantly after US irradiation. The results from flow cytometry show the percentages of early apoptosis and secondary necrosis cells increase significantly after US irradiation (P<0.05). Orthogonal analysis reveals US induced early changes increase with the increase of ultrasound intensity; Moreover, US induced early changes may be an early time-dependent event with early apoptosis reaches its maximum at about 12 h. In this study, it is suggested that optimal early apoptosis with minimal necrosis is acquired when HepG2 cells were exposed to ultrasound at 5 W/cm2 for 1 min and incubated for 12 h post-sonication. It reveals that ultrasound induced apoptosis is a preferred mode of killing cancer cells and suggests the optimal parameters setup for both in vitro experiments and its clinical application.