Study Of Apoptosis Induced By Nonsecond Steep Pulsed Fileds Through Mitochondrial Pathway In SKOV3 Cells

People's healthy has been seriously threatened by malignant tumor, however, fine treatment was still a big issue in the whole word. Recently, nonsecond pulsed electric filds(nsPEFs) induced apoptosis in tumor cells has a promising clinical applicative perspective. Studies have been revealed that nsPEF may trigger intracellular processes, such as apoptosis, when the duration of PEF was down to nonsecond, and the electric field amplitudes was up to tens of kV/cm, which was called intracellular electo-manpulation(IEM). Apoptosis, also named cell programmed death, Mitochondria played an important role in the process of apoptosis. This study sets out mitochondria pathway to study apoptosis induced by nsPEFs. Two parts included: first part studies effects of nsPEFs-induced apoptosis in SKOV3 cells and finds the best parameters of nanopulses; second part reveals changes of function and apoptotic related factors of mitochondria after SKOV3 cells exposed to nsPEFs.1 Studies the effects of nsPEF-induced apoptosis and the best parameters of nanopulses.1.1 Materials and methods1.1.1 SKOV3 cells were exposed to 10, 30, 60 or 80 pulses with durations of 50 ns, 100 ns or 200 ns, and electric field amplitudes of 50 kV/cm. Effects of nsPEF-induced apoptosis were detected using both annexin V/PI double staining by flow cytometry(FCM) and DNA cleavage by agarose gel electrophoresis.1.1.2 SKOV3 cells exposed to nsPEF (50kV/cm, 100ns, 30 pulses) for 30 min, 2 h, 6 h or 12 h, and then western blot was used to detect the expression of active caspase-3(P﹥0.05).1.2 Results1.2.1 Compared with the control group, SKOV3 cells were mainly stained with Annexine V+/PI-, when cells exposed to duration at 100ns or 50ns. While, SKOV3 cells were mainly stained with Annexine V~+/PI~+, when cells exposure to duration at 200ns. Cells in the groups exposed to 50 ns and 100 ns nsPEFs had degraded DNA as shown by the characteristic"ladder"pattern. In 200 ns groups, however, the DNA degradation was heterogenous and no discrete bands were formed.1.2.2 Compared with the control group, the expression of cleaved caspase-3 was increased significantly(P﹤0.05).1.3 ConclusionNsPEFs has an effect of apoptosis to SKOV3 cells, and the number of apoptotic cells was directly proportional to the pulse duration and number of pulses: the duration less than 100ns, the electric field amplitudes more than 50 kV/cm.2 Changes of mitochondrial function and apoptotic related factors during the process of nsPEF-induced apoptosis2.1 Materials and methods2.1.1 SKOV3 cells exposed to nsPEF (50kV/cm, 100ns, 30 pulses) for 30 min, 2 h, 6 h or 12 h, and then incubated with DCFH. Flow cytometry (FCM) was used to monitor the fluorescence intensity.2.1.2 SKOV3 cells were loaded with JC-1 after cells exposed to nsPEF (50kV/cm, 100ns, 30 pulses) for 30 min, 2 h, 6 h or 12 h. The fluorescence was detected using FCM.2.1.3 After treatment with electric fields (100 ns, 50k V/cm, 30 pulses) for 6h, immunofluorescence was used to determine the subcellular location Cyt c and AIF. Western blot was used to quantify the altered expressions of Cyt c and AIF in the cytoplasm, after cells treated with nsPEFs(100 ns, 50k V/cm, 30 pulses) at 30min, 2h, 6h, 12h.2.1.4 Western blot was used to measure the expressions of Bax and Bcl-2, after cells treated with nsPEFs(100 ns, 50k V/cm, 30 pulses) at 30min, 2h, 6h, 12h.2.2 Results2.2.1 Exposure of SKOV3 cells to 30 pulses of 100 ns, 50 kV/cm nsPEFs led to about a three-fold increase in ROS generation immediately compared to the control group (P﹤0.05). This gradually decreased over time to the basal level at 12 h.2.2.2 The mitochondrial membrane potential collapsed after exposed the SKOV3 cells to 30 pulses of 100 ns, 50 kV/cm. The fluorescence intensity decreased until 12h (P﹤0.05).2.2.3 Compared with the control group, Cyt c and AIF released from mitochondria to cytoplasm in treated group after exposure to nsPEFs 6h. The results of western blot showed that Cyt c and AIF increased markedly in cytoplasm (P﹤0.05).2.2.4 The expression of Bax was increased after 2 h (P﹤0.05), and steadily increased to a peak at 6 h (P﹤0.05). After 6 h, the expression declined slightly but was still markedly higher than that of the control cells (P﹤0.05). The expression of Bcl-2 was subtly attenuated; however, the ratio of Bcl-2/Bax decreased markedly in SKOV3 cells 6 h after treatment with nsPEF (P﹤0.05).2.3 ConclusionMitochondrial function and apoptotic related factors changed after exposure to nsPEFs. We inferred that mitochondrial pathway play an important role in nsPEFs-induced apoptosis in SKOV3 cells.