In vitro study of low intensity ultrasound combined with different doses of PDT: Effects on C6 glioma cells

The aim of this study was to study the effects of killing C6 glioma cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy combined with photodynamic therapy (SPDT). In the SPDT group, the cells were treated with sonication at an intensity of 0.5 W/cm(2) and a frequency of 1 MHz, followed by different doses of light irradiation. The growth inhibition rate following treatment was determined by MTT assay. The apoptotic rate was examined by a flow cytometry. Cleavage of caspase 3, 8 and 9 was investigated by immunoblotting. Reactive oxygen species (ROS) were measured by a fluorescence microplate reader. The effect of SPDT on the glioma cells was also studied in the absence or presence of various ROS scavengers. The growth inhibition rate of C6 glioma cells treated with SPDT was significantly higher compared with sonodynamic therapy (SDT) or photodynamic therapy (PDT) alone at light doses <200 J/cm(2). The growth inhibition rate of C6 glioma cells treated with SPDT did not rise significantly when the light dose increased to >120 J/cm(2). The apoptosis rate was the highest in the SPDT group, when the light dose was at 80 J/cm(2). A greater amount of ROS were generated in the SPDT group than in the groups treated with SDT or PDT alone. The addition of NaN3 or mannitol resulted in a decrease in the growth inhibition rate with SPDT. In conclusion, our data indicate that SPDT powerfully kills C6 glioma cells in vitro through the synergistic effects of SDT and PDT. The pathway of PDT inducing C6 glioma cell apoptosis includes both the mitochondrial and death receptor pathways. Furthermore, ROS may play an important role in SPDT.