| Objective:Ovarian cancer is very common malignant tumor in female genital carcinomas. It is hard for early discovery and diagnosis. About 60%-70% patients are terminal stages when they come to see a doctor. Though the morbidity of ovarian cancer is lower than cervical cancer and endometrial cancer, the mortality of which is higher than sum of cervical cancer and endometrial cancer. It affects women’s health seriously. Despite a variety of manners are applied to ovarian cancer clinical treatment, the 5-year survival rate is low. Cisplatin is a kind of cell cycle cytotoxic drugs, which distributes in tumor cells indiscriminately. Its main target is DNA. Radiation treatment and chemical therapy aim to damage DNA in tumor cells. It always treatment failure when drug resistance and radiation tolerance gradually appear. Nanosecond electric pulses (nsEP) is a potential treatment. It can affect cellular structures across cell membrane and induce apoptosis, inhibit the angiogenesis and destroy the tumor microenvironment, which can lead to necrosis of tumor cells. nsEP is expected to become a new means of cancer treatment, which is a pure electric field treatment without drugs and hot melt. The aim of this study is to explore the temporal pattern of DNA breaks induced by nsEP in cisplatin-sensitive and -resistant human ovarian cancer cells and discuss the correlation between DNA damage and cell death caused by nsEP.Methods:A2780 (cisplatin-sensitive subline) and C30 (cisplatin-resistant subline) in the logarithmic phase of growth were made into single cell suspension at a density of 5 x 105/ml.1 ml volumes cells were transferred into one of the 24 orifice plates after mixed in the vortex mixer. There was only one of the 24 orifice plates suffered nsEP once. The voltage was 6kV and the interelectrode distance was lcm. So the strength E was 6kV/cm with a pulse repetition frequency of 10 Hz. The pulse duration was 24ns. The exposure time was 60s. The experiment groups were exposed to nanosecond electric pulses and the control groups were sham exposed in a similar, but nonenergized system.1:Cells were collected into EP tubes and mixed eventually after nsEP. Cell viability of C30 and A2780 were determined using a tetrazolium assay (WST-8) after exposed to nanosecond electric pulses at Oh,4h,8h,12h and 24h. Then calculate the percentage of dead cells. There were experiment group (exposed to nsEP), control group (sham exposed) and blank control group (with no cells, just 100ul RPMI1640) at every time point.2:DNA damage was detected with alkaline single cell gel electrophoresis (alkaline comet assays) after exposed to nanosecond electric pulses at 4h,8h,12h and 24h. The 95th percentile of tail DNA in control group was set as the threshold of comet formation and the 75th percentiles of TL (tail length), TM (tail moment) and OTM (Olive tail moment) in experiment group were calculated. The comet formed rates were calculated at the same time.Results:1:Cell viability of C30 and A2780 varied with time after exposed to nanosecond electric pulses,4h (86.46% vs 76.29%),8h (78.52% vs 65.96%),12h (84.28% vs 75.29%),24h (85.11% vs 80.87%), and the lowest cell viability appeared at 8h (P<0.05). Cell viability of C30 was higher than that of A2780 (P<0.05).2:There was no comet tail in both A2780 and C30 control groups after the alkaline single cell gel electrophoresis. Obviously the tail length of A2780 is longer than that of C30 at 4h,8h,12h and 24h. It confirmed that A2780 were more vulnerable. Percentages of cell death and comet-formed in A2780 cells were higher than those in C30 cells. It confirmed the sensitivity of A2780 on the other hand. Statistical analysis shows that the percentage of comet-formed correlated with that of cell death in either A2780 (r=0.997, P<0.05) or C30 (r=0.998, P<0.05) cells. It indicated that cell death related to DNA damages.3:The TL, TM, OTM and its 75th percentages varied with time, and the maximum value appeared at 8h, which indicated that cell damages may reached peak at 8h. Subsequent changes may resulted from the starting of DNA damage repair mechanism. TL, TM and OTM in C30 cells were less than those in A2780 cells (P<0.05).Conclusion:The DNA damages of cisplatin-sensitive and -resistant human ovarian cancer cells varied with time. DNA damages induced by nsEP in cisplatin-sensitive cells differed from that in -resistant cells. Cisplatin-sensitive human ovarian cancer cells were sensitively damaged than-resistant cells. DNA damages resulted in fraction of cell death. Cell death induced by nsEP was associated with DNA damages. |
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