To verify the effect of echo-contrast agent (ECA) on apoptosis induced by ultrasound, leukemia cell lines (Jurkat, Molt-4 and U937) were sonicated at intensities previously shown to induce optimal apoptosis with or without Levovist, an ECA. The results showed that loss of viability and apoptosis can be induced in all three cell lines, apoptosis highest with Molt-4, based on viability and DNA fragmentation assay. Such finding was supported by corresponding increase of cells with low mitochondrial membrane potential, high superoxide production, increased intracellular calcium concentration, and phosphorylation of histone H2AX after sonication. Optimal ultrasound condition was 0.3 W/cm2, 1 MHz, 10% duty factor pulsed at 100 Hz; but in the presence of Levovist, an apparent shift of cell killing induction was observed at 0.2 W/cm2. While these results further confirmed previous findings on ultrasound-induced apoptosis, they also suggest that use of an enhancing factor, such as addition of ECA, may be useful in cancer therapy when a much lower intensity is desired.
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