Enhanced ultrasound-induced apoptosis and cell lysis by a hypotonic medium

Purpose: To test the hypothesis that non-lethal hypotonia will enhance ultrasound-induced cell killing in vitro and that the mechanism is mechanical in nature. Materials and methods: Hypotonic RPMI medium (146 mOsm) was used to induce non-lethal osmotic swelling of human myelomonocytic leukaemia U937 cells. Hypotonia for 10 min was started just before exposure to 1 MHz ultrasound at 0.5 or 1.0 W cm^-2 for 10 min, or 5 min before exposure to 2.0 W cm^-2 for 1 min. Surviving intact cells were then determined by the trypan blue dye exclusion test immediately after treatment. After 6-h incubation of the treated cells, early apoptosis and secondary necrosis were measured using a flow cytometer. Intracellular free calcium ion imaging by Fura-2 fluorescence and cellular ion scanning using a secondary ion mass spectrometer were also performed. Results: Enhancement of ultrasound-induced cell lysis was observed at all intensities, and most prominently at 2.0 W cm^-2, while apoptosis induction was significantly enhanced at intensities of 0.5 and 1.0 W cm^-2, but not at 2.0 W cm^-2. The enhanced cell lysis is attributed to the increased susceptibility of the cells to mechanical damage. This is consistent with previous reports describing the effects of mechanical stresses on cell membranes. Cellular ion scanning images also suggest that hypotonia has an effect on the membrane damage-and-repair mechanism of the cells. Conclusions: The results support the hypothesis that non-lethal hypotonia can enhance ultrasoundinduced cell killing. These findings also suggest the ʹsonomechanicalʹ nature of the effects on the cells.