In vitro stimulation of calcium overload and apoptosis by sonodynamic therapy combined with hematoporphyrin monomethyl ether in C6 glioma cells

The present study investigated enhancement of apoptosis induction and the mechanisms underlying calcium overload on C6 glioma cells in vitro, stimulated by low-level ultrasound in combination with hematoporphyrin monomethyl ether (HMME). The optimum frequency of ultrasound was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The apoptotic rate, reactive oxygen species concentration and decreased mitochondrial membrane potential (MMP) were analyzed by flow cytometry. Morphological changes were detected by a transmission electron microscope, and the concentration of intracellular Ca²⁺, [Ca²⁺]_i, was detected by a confocal laser scanning microscope. In addition, the release of cytochrome c (cyt-c) was measured by western blot analysis. The results revealed that an increased apoptotic effect was induced by sonodynamic therapy (SDT), and this was found to correlate with the overloaded [Ca²⁺]_i, derived from the intra- and extracellular sources in the early apoptotic process. The results also revealed an increased level of ROS production, a decreased MMP and an increased release of cyt-c. The present study indicated that low-level ultrasound in combination with HMME improved the apoptotic effect in C6 glioma cells. The overloaded [Ca²⁺]_i was involved in the mechanism by which apoptosis was stimulated and enhanced by SDT.