Study on Effect of Low Frequency and Low Intensity Ultrasound on Leukemic Cell Lines K562 and Human Hematopoietic Cells.

Objective: To investigate the effect of low frequency and low intensity ultrasound on leukemia cell lines K562 and to clarify the role of Caspase-3 in the effect and to study the effect of low frequency and low intensity ultrasound on human hematopoietic cells.

Methods: K562 cells in log phase were divided into 2 groups: control group and experimental group. The tensity of low frequency(20kHz) and low intensity ultrasound were 0.03W/m² 0.1W/m² and 0.25W/m² respectively. The exposure time were 30s and 60s respectively. K562 cells were incubated in 24-well culture plates for different time periods (6h,24h,48h) after sonication and the number of vital cells was tested by MTT assay. The morphology of apoptosis were analyzed by Wright’s stain and transmission electron microscope. The pencentage of apoptosis were studied by flow cytometry (FCM). The activation of Caspase-3 were tested by Caspase-3 kit with spectrophotometric method. All the hematopoietic cells were classified to two groups: control and experimental groups. The intensity of low frequency(20kHz) and low intensity ultrasound were 0.03W/m² 0.1W/m² and 0.25W/m² respectively. The exposure time were 30s and 60s respectively. The cells were incubated in 24-well culture plates for different time periods (6h, 24h, 48h) after sonication and the number of vital cells were tested by typhan blue exclusion. The ratio of apoptosis and morphology of apoptosis were analyzed by FCM, Wright’s stain and transmission electron microscope.

Results: The absorbance value of MTT decreased significantly after exposure. The highest apoptosis was found in 0.25W group after 30-s sonication and 6-h incubation. Morphological alterations observed in cells after exposure to ultrasound included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation. The result of FCM included: control 7.6%; 0.03W,30s,6h 11.57%; 0.1W,30s,6h 35.95%;0.25W,30s,6h 38.87%. There were no significant difference of activation of Caspase-3 between control and experimental group(0.25W group after 30-s sonication and 6-h incubation). The result of typhan blue exclusion was: the difference between each group was not significant. FCM, Wright’s stain and transmission electron microscope did not find apoptosis in each group.

Conclusions: Low frequency and low intensity ultrasound can induce apoptosis in K562 cell lines after exposure. The effect went significantly when the intensity of ultrasound increase, without relation to the exposure time. The low frequency and low intensity ultrasound has no significant effect on human hematopoietic cells.