ABSTRACT
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 NaN(3) 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.
ABSTRACT
PURPOSE: Our aim was to study calcium overload-induced apoptosis and its relation to reactive oxygen species (ROS) in rat C6 glioma cells after sonodynamic treatment (SDT). MATERIALS AND METHODS: Hematoporphyrin monomethyl ether (HMME) was used as the sonosensitizer. The concentration of intracellular Ca(2+) ([Ca(2+)](i)) was measured by fluorometry. Apoptosis and necrosis rates were evaluated by a flow cytometry. Moreover, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA(2)), cytochrome c (cyto-c) and cleaved caspase-3 were investigated by immunoblotting. RESULTS: Our study indicated that [Ca(2 +)](i) and ROS increased in cells of SDT group, the apoptosis rate, quantity of cyto-c and cleaved caspase-3 markedly increased after SDT. Furthermore, N-Acetyl-L-cysteine (NAC) or 1,2-bisethane-N,N,N',N'-tetraacetic acid tetrakis ester (BAPTA-AM) could decrease the apoptosis rate, the release of cyto-c and cleaved caspase-3 in SDT group, SERCA(2) degradation was found in SDT group and could also be prevented by the addition of NAC. CONCLUSIONS: Our results show that HMME-SDT can induce C6 cell death through both necrosis and apoptosis. ROS in C6 cells play a decisive role in HMME-SDT-induced cell death. The endoplasmic reticulum (ER) may be a major target of HMME-SDT, ROS can induce SERCA(2) degradation, causing the elevation of [Ca(2+)](i).
