Effects And Potential Mechanisms Of Nanosecond Pulsed Electric Fields On Apoptosis Induction Of Cisplatin-Sensitive And Cisplatin-Resistant Human Ovarian Cancer Cells

Ovarian cancer is the most lethal malignancy of the female reproductive system and drug resistance is the main factor affecting chemotherapy effect and survival rate, thus overcoming drug resistance is an emergent issue in ovarian cancer treatment.Nanosecond pulsed electric field (nsPEF) emerges as a new kind of pulsed electric field technology characterized by high power and low energy, researchers in the fields of biological engineering and medical science are focusing on its intracellular electromanipulation (IEM) and non-thermal effect in treating cancer, and numerous studies have shown that nanosecond pulsed electric is a potential anticancer strategy.Studies have shown that under certain parameters, mitochondria mediated apoptosis induced by nanosecond pulsed electric field in some cancer cells. According to our preliminary experiment data,we infer nanosecond pulsed electric field may induce mitochondria-mediated apoptosis in COC1 and COC1/DDP cells.This study we chose human ovarian cancer cells COC1 (cisplatin-sensitive cell line) and COC1/DDP (cisplatin-resistant cell line) in vitro as our experimental cells to explore the effects of nanosecond pulsed electric field on subcellular units such as mitochondria and nucleus,we attempt to provide some preliminary proof in applying nanosecond pulsed electric field to treatment of drug resistant cancer.Objective This study explored growth inhibition, apoptosis induction and potential mechanisms of nanosecond pulsed electric fields in human ovarian cancer cells COC1 (cisplatin-sensitive cell line) and C0C1/DDP (cisplatin-resistant cell line) in vitro.Methods COC1 and COC1/DDP cells were divided into two groups, the treated group was exposed to nanosecond pulsed electric field with pulse duration of 32 ns, field strength of 10 kv/cm for 10 min, the control group was sham-exposed. Cell viability was determined with WST-8 assay at indicated time points(0.5h、6h、12h、24h) after treating; In rinding effects and potential mechanisms of nanosecond pulsed electric fields on apoptosis induction at 12h after nsPEF exposure, cellular apoptosis rate was determined by flow cytometry with Annexin V-FITC/PI double staining, at the same time Hochest33342 staining was detected by fluorescence microscopy; Mitochondrial membrane potential was detected with JC-1 assay; Caspase-3 activity was detected by luminescent assay; Cell cycle was determinded by flow cytometry with PI staining.Results 1.Compared to control group, at indicated time points 0.5h、 6h、12h、24h after treating, cell viability in the COC1 treated group remarkably decreased (P=0.001; P=0.001; P=0.001; P=0.013), COC1/DDP treated group also decreased in cell viability (P<0.001; P=0.001; P<0.001; P<0.001), the lower cell viability in both of the two groups occurred at 12h after treating. Compared with COC1 cell at each indicated time points, COC1/DDP cell led a higher cell viability (P=0.001; P<0.001;P<0.001; P=0.026).2.At 12 h after nsPEF treated, compared with control group, early and late apoptosis rate in the COC1 treated group was higher (P=0.001; P=0.001),the same was in COC1/DDP treated group (P=0.005; P=0.016);Compared with COC1 treated group, early and late apoptosis rate in COC1/DDP treated group was lower(P=0.002;P=0.001).Apoptotic bodies and dense particles were markedly in both COC1 and COC1/DDP treated groups observed by fluorescence microscopy with Hochest33342 staining.3.At 12 h after nsPEF treated, orange and diffuse green fluorescence were observed in most COC1/DDP treated cells and diffuse green fluorescence were observed in most COC1 treated cells by JC-1 staining; Compared with the control groups, JC-1 red/green fluorescence ratio in both COC1 and COC1/DDP treated group were lower (P=0.001; P=0.002).4.At 12 h after nsPEF treated, caspase-3 activity in COCl treated group was 1.26±0.043 times of control group, caspase-3 activity in COC1/DDP treated group was 1.16±0.020 times of control group, caspase-3 activity in both COC1 and COC1/DDP treated group were higher. (P=0.000; P=0.000);5.At 12 h after nsPEF treated, compared with control group, cell percentage of G0/G1 phase in COC1 treated group decreased (P=0.002), cell percentage of S phase in COC1 treated group increased (P=0.007), while there were no difference of cell percentage in G0/G1 phase, S phase or G2/M phase between control and treated COC1/DDP groups (P>0.05).Conclusions Nanosecond pulsed electric field could inhibit proliferation and induce apoptosis of COC1 and COC1/DDP cells, induce cell cycle arrest in S phase in COC1 cell. In the process of apoptosis, mitochondrial membrane potential (Δψm) decreased, and caspase-3 activity increased. In together, mitochondria may play an important role in nanosecond pulsed electric field induced apoptosis.