Study of Cytotoxic Effects of K562/Adm Cells by Low Frequency Ultrasound Enchance Adriamycin.

Objective The aim of this study is to investigate and analysis the effect of ultrasound potentiate the cytotoxicity of adriamycin and reverse drug resistance on leukemia drug resistance K562/Adm cell line in vitro, to find out the mechanism of the reverse effect of ultrasound exposure.

Methods Human leukemia adriamycin resistant strain K562/Adm as target cells, were treated by adriamycin singly in group Adm, by ultrasound exposure singly in group US and by adriamycin prior to ultrasound exposure in group Adm+US. The dosages that didn’t attribute to cell killing immediately were detected. Trypan blue dye exclusion test and MTT assay were used to determine the sensitivity of K562/Adm. Wright’s staining and transmission electron microscope were used to detect the apoptosis and structure change of K562/Adm cells. Flow cytometry was used to analysis intracellular drug concentration and electron microscopic scanning was used to observe the membrane change. Immunocytochemistrial method was used to evaluate the expressions of P-gp.

Results At 0.17W•cm⁻² and lower acoustic intensity, ultrasound didn’t result in K562/Adm acute cells destruction; and 0.5 W•cm⁻² ultrasonic intention could make cell killed rapidly after K562/Adm cells were irritated by ultrasound exposure singly. Significant differences were obtained between ultrasound treated and untreated cells in the presence of various concentrations of adriamycin. If the same concentration of cytotoxic agents were used, more cells were killed if sonication was applied. ultrasound for 30s at 20kHz, 0.17W•cm⁻² intensity almost could not damage K562/Adm cells but dramatically decrease adriamycin concentration which induce cell achieve IC50;There were some morphological alterations in cells irradiated by ultrasound, Nearly all the treated cells by ultrasound exhibited small holes with diameter about 1~2 μm in the K562/Adm cell surface; the intracellular adriamycin accumulation in group Adm+US were prompted compared with Adm group and controlled group. Many apoptotic phenomena were observed in Adm+US group, show many vesicle and the form of apoptotic body, but there were no change in US group compared with controlled group. And the expression of P-gp protein had no significant difference between before and after ultrasound eradiated.

Conclusions

1. Higher ultrasound intensity could make K562/Adm cells killed rapidly, and lower ultrasonic level could potentiate the cytotoxicity of adriamycin to K562/Adm cells and reverse drug resistance on K562/Adm cells.

2. Ultrasonic cavitation and sonoporation are the main mechanisms of the synergism between adriamycin and low-level sonication; the ultrasonically induced increase in intracellular drug accumulation.

3. The expression of P-gp had no change in US group compare with controlled group.