Proapoptotic effect of nonthermal pulsed ultrasound on prostate cancer cells in a nude mouse model

Abstract

Background Ultrasound (US) can induce cell injury, and we have previously reported that adjusting the pulse repetition frequency (PRF) of ultrasound output can induce prostate cancer cell destruction without causing a rise in the temperature of the irradiated area. In this study, we examined the mechanism of nonthermal ultrasound cell destruction, which was not fully clarified in our previous reports. Methods In vitro, we evaluated postirradiation cells immediately after treatment and examined membrane disruption by proliferation assay, LDH assay, and apoptosis assay. In vivo, we injected mice with human LNCaP and PC‐3 prostate cancer cells and evaluated the therapeutic effects of US irradiation by H‐E staining and immunostaining. Results Proliferation assays showed inhibition at 3 h postirradiation independently of PRF and cell line (p < 0.05). Quantitative assessment of apoptosis/necrosis by flow cytometry showed widely varying results depending on cell type. LNCaP showed an increase in late apoptosis at 0 h independent of PRF (p < 0.05), while PC‐3 showed no significant difference at 0 h. The LDH assay showed an increase in LDH independent of PRF in LNCaP (p < 0.05 respectively), but no significant difference in PC‐3. In vivo, tumor volume was compared and a significant reduction was observed at 10 Hz for LNCaP (p < 0.05) and 100 Hz for PC‐3 (p < 0.001) at 3 weeks after the start of irradiation. The excised tumors were evaluated with Ki‐67, Caspase‐3, and CD‐31 and showed a significant treatment effect independent of cell type and PRF (p < 0.001 respectively). Conclusion Examining the mechanism behind the therapeutic effect of US irradiation revealed that the main effect was achieved by apoptosis induction rather than necrosis.