Calcium overload and in vitro apoptosis of the C6 glioma cells mediated by sonodynamic therapy (hematoporphyrin monomethyl ether and ultrasound)

Abstract

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.

Fig. 1

The viability of C6 glioma cells after treatment with ultrasound of under 1.0 W/cm² for 60 s at frequencies varying from 0 to 1.0 MHz. Compared to the controls, the significant decrease (P < 0.05) in viability caused by 0.05 MHz and 0.8 MHz or 1.0 MHz are represented by * and ** respectively. Data are expressed as mean ± SEM from six independent experiments

Fig. 2

Effect of nimodipine on the change in intracellular free calcium induced by various treatments. a Fluorescent images of C6 glioma cells, b Quantification of [Ca2+]i in different treatment groups. The cells incubated in PBS or HBSS with and without nimodipine were treated with HMME, ultrasound or SDT and imaged by confocal laser scanning microscopy in 1800 s. P < 0.05 was considered as significant. In PBS group:Increase was significant comapared to the controls*, compared to the response of ultrasound alone▼, HBSS group: ** significant compared to the HBSS control, ▼▼significant compared to the ultrasound treatment response

Fig. 3

Apoptotic rate (AR) of the C6 glioma cells mediated by HMME, ultrasound and SDT, and ultrasound treatment in calcium-free PBS or HBSS measured by FCM. Compared to control, the increase was considered significant when *P < 0.05

Fig. 4

TEM images (magnification × 10,000) of ultrastructural changes in C6 glioma cells treated with PBS (control) (CP), HMME-PBS (HP), ultrasound-PBS (UP), SDT-PBS (SP), HBSS (control) (CH), HMME-HBSS (HH), ultrasound-HBSS (UH) and SDT-HBSS (SH)

Fig. 5

ROS production by the C6 glioma cells treated with HMME, ultrasound, and SDT in calcium-free PBS or HBSS, measured by FCM. Compared to the controls, the SDT-treated cells showed significantly increased (P < 0.05) production of ROS group. Asterisk represents increase compared with the control groups.▼represents increase compared to ultrasound treated group

Fig. 6

Effect of calcium in the extracellular medium on MMP reduction in the C6 glioma cells treated with HMME, ultrasound and SDT. The MMP was measured by FCM. The effect was considered significant when *P < 0.05

Fig. 7

Western blot analysis showing the release of cyt-c from HMME, ultrasound and SDT treated C6 glioma cells incubated either in calcium-free PBS or HBSS. A. Western blot, B. Quantification of the released cyt-c showing fold increase by various treatments. Compared to the control, the effect was significan when *P < 0.05