Induction Of Apoptosis By Low-Power Low-Frequency Ultrasound In C₆ Glioma Cell

Objective:Discussion on low-power low-frequency ultrasound-induced apopto- sis of rat C6 glioma cells (Abbreviations: C6 cells, the same below), the ultrasonic mechanism on the induction of apoptosis in C6 glioma cells.Methods:those methods (including MTT,AO/EB double staining,TUNEL,FCM,technology of EM) were adopted to study different power low- frequency ultrasound-inducted apoptosis in C6 cells.⑴MTT assay: detection of the values of OD on 0h, 6h, 12h, 24h and 48h time points after different power low-frequency ultrasonic irradiation in C6 cells, and calculation of inhibitory rates in C6 cells; and preliminary determination of the required ultrasound parameters.⑵AO/EB double staining: identification of the control group and the irradiation groups apoptosis/necrosis/living C6 cells, and calculation th- ese rates of apoptosis and necrosis,and determination on final ultrasound parameters of next experiments.⑶TUNEL: observation of the control group and the radiation group on the immunological changes in C6 cells, and calculation of these two apoptotic indexes.⑷Flow cytometry: analyses of apoptosis rates(FITC-Annexin V/PI double staining),cell cycle and APR(PI staining)in the control group and the irradiation group.⑸Transmission and scanning electron microscopy: observation of ultrastrural changes in the control group and the irradiation group.Results:⑴MTT assay: analyses show C6 glioma Cells'proliferation were inhibited at different time points after different power low-frequency ultrasonic irradiation in a dose-dependent manner. The values of IR on 0h, 6h, 12h, 24h, 48h time points after ultrasonic irradiation can be found: C6 cells'proliferation were inhibited obviously on 6 hour time point in 1.75W/cm2 group, 2.0W/cm2 group and 2.5W/cm2 group. The following experiments used 6 hour time point as the observation point.⑵AO/EB double staining: analyses showed only a very small number of viable cells in the control group, and apoptotic cells after ultrasonic irradiation increased significantly. apoptotic rates in 1.75W/cm2 group, 2.0W/cm2 group and 2.5W/cm2 group were 17.9±0.65%. 13.4±0.94% and 1.25±0.67%, necrotic rates in 1.75W/cm2 group, 2.0W/cm2 group and 2.5W/cm2 group were 1.3±0.61%, 8±0.54% and 20±0.63% .⑶TUNEL: analyses showed only a very small amount of apoptotic cells (stained brown) in the control group and significantly increased apoptotic cells (stained brown) in the irradiation group. Compared to AI in the control group, AI in the irradiation group very significantly increased (P <0.01).⑷Flow cytometry: analyses showed comparison of apoptotic rate in the control group, apoptotic rate in the radiation group increased very sig- nificantly (P<0.01); No hypodiploid apoptotic peak (Ap peak, apoptotic peak) in the control group emerged before the normal diploid DNA peak, but a significant apoptotic peak in the radiation group emerged before the normal diploid DNA peak. Comparison to APR in the control group, APR in the radiation group significantly increased (P <0.01).⑸Transmission and scanning electron microscopy: Observations by TEM showed no apoptotic ultrastructural changes in the control group, and various stages of apoptotic changes and typical apoptotic bodies in the irradiation group; Observations by SEM showed normal structure and cell borders clearly in the control group, normal structure and cellular membrane perforation phenomenons in the irradiation group.Conclusion:low-power low-frequency ultrasound can inhibit the proliferation of C6 cells in a dose-dependent manner.C6 cells after 1.75W/cm2 irradiation continued to build 6 hours had the highest apoptotic rate and the lowest necrotic rate. It was the best parameter of ultrasound-induced apoptosis in C6 cells. When ultrasonic power of irradiation continue to increase, cell apoptotic rate has not increased, while cell necrotic rate has increased significantly;C6 cells blocked by low-power low-frequency ultrasound in G0-G1 phase, C6 cells reduced in S phase so that the inhibition of cell proliferati- on;Low-power low-frequency ultrasound may cause the membrane per- foration through sonoporation to induce apoptosis in C6 cells.In summary,low-power low-frequency ultrasound in Oncotherapy has good potential.