The objective of this study was to investigate the role of intracellular calcium overload in the in vitro apoptosis of C6 glioma cells mediated by low level ultrasound and hematoporphyrin monomethyl ether (HMME) therapy. The frequency of ultrasound was optimized by the cell viability assay using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The apoptotic rate, reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP) were determined by flow cytometry. Morphological changes were observed by the transmission electron microscope. Concentrations of intracellular Ca2+, [Ca2+]i were detected by a confocal microscopic laser scanning, and the release of cytochrome-c (cyt-c) was measured by western blotting.
Results: The SDT-mediated apoptotic effect involved an overload of [Ca2+]i derived from the intra- and extracellular sources during the early progression of apoptotosis. The process was associated with an increased ROS production, a decreased MMP, and a release of cyt-c. In conclusion,the combined use of low level ultrasound and HMME improved the apoptotic rate of C6 glioma cells mediated by ultrasound alone. The [Ca2+]i overload involving activation of mitochondrial signaling played a pivotal role in the SDT-induced apoptosis.