Effects of ultrasound on apoptosis induced by anti-CD20 antibody in CD20-positive B lymphoma cells

Aim: The present study was conducted to examine the thermal and non-thermal effects of ultrasound on apoptosis induced by anti-CD20 monoclonal antibody (rituximab). Materials and methods: SU-DHL-4 cells, a CD20-positive cell line derived from B cell lymphomas with a BCL2 gene rearrangement, were exposed to continuous 1 MHz ultrasound for therapeutic use under an air- or CO₂-saturated condition to control cavitation. Early apoptosis (EA) and secondary necrosis (SN) were examined by flow cytometry. Cavitation was determined by detecting the hydroxyl radicals derived from pyrolysis of water molecules using electron paramagnetic resonance-spin trapping. To assess thermal effects, cells were treated in a temperature-controlled water bath. Results: There was a significant additive increase in EA and EA + SN observed in cells treated with rituximab combined with heat at 42 °C or non-thermal ultrasound at 0.5 W/cm² under an air-saturated condition, where heat or ultrasound induced some cell death. A significant synergistic increase in EA and EA + SN was observed in cells treated with rituximab and ultrasound at 2.5 W/cm² under CO₂-saturated conditions, where inertial cavitations were completely suppressed. No enhancement was observed at a temperature less than 40 °C or ultrasound at 0.5 W/cm² under CO₂-saturated conditions. Conclusion: These results suggest that the immuno-therapeutic application of ultrasound at relatively high-intensities combined with rituximab thus produces synergistic effects under conditions where the non-thermal and non-cavitational effects are predominant.