Nanosecond Electric Pulses Induce Early and Late Phases of DNA Damage and Cell Death in Cisplatin-Resistant Human Ovarian Cancer Cells

Abstract

Chemoresistance is a challenge for management of ovarian cancer, and therefore the response of resistant cells to nanosecond electric pulses (nsEP) was explored. Human ovarian cancer cell line COC1 and the cisplatin-resistant subline COC1/DDP were subjected to nsEP (32 ns, 10 kV/cm, 10 Hz pulse repletion frequency, and 10 min exposure duration), and then the cellular responses were followed. The percentages of dead cells and of comet-formed cells in the alkaline assay displayed two peak levels (i.e., 2 and 8 h after nsEP exposure), with the highest value noted at 8 h; the percentage of comet-formed cells in the neutral assay was increased at 8 h; the apoptotic percentage was increased at 8 h, with collapse of the mitochondrial membrane potential and the activation of caspase-3 and caspase-9. The comet assay demonstrated DNA single-strand break at 2 h and double-strand break at 8 h. nsEP resulted in lower cytotoxicity in COC1/DDP cells compared with COC1 cells. These findings indicated that nsEP induced early and late phases of DNA damage and cell death, and these two types of cell death may have distinct applications to treatments of chemoresistant ovarian cancers.

Figure 1

Percentages of dead cells (a) and of comet-formed cells in the alkaline assay (b) after nsEP treatments: the negative death fraction demonstrated proliferation of control cells; two peak levels were detected at 2 and 8 h, with the highest value at 8 h; the value in COC1 cells was higher than that in COC1/DDP cells. Percentage of comet-formed cells in the neutral assay (c): the value was increased at 8 h. Images under the alkaline assay (d): more comets were observed at 2 and 8 h in both cell lines; few comets emerged at 24 h, indicating repair. Images under the neutral assay (e): comets appeared at 8 h, demonstrating single-strand break at 2 h and double-strand break at 8 h. Data were mean ± standard deviation for 3 independent experiments. The scale bar was 50 μm; (a) versus control, p<0.05; (b) versus 2 h, p<0.05; and (c) versus COC1 at the same time point, p<0.05.

Figure 2

Apoptosis detected with flow cytometry after 2 and 8 h (a, b): Q4 represented early apoptotic cells, and Q2 represented late apoptotic cells; a higher apoptotic percentage was detected at 8 h, and the value in COC1 cells was higher than that in COC1/DDP cells. Activity of caspase-3 (c): relative light unit (RLU) reflected the enzymatic level; a higher level was noted at 8 h. HMGB1 level (d): no increase was detected. Data were mean ± standard deviation for 3 independent experiments. (a) versus control, p<0.05; (b) versus 2 h, p<0.05; (c) versus COC1 at the same time point, p<0.05.

Figure 3

Mitochondrial membrane potential detected with the JC-1 assay. Images under fluorescence microscopy (a): green fluorescence was due to the monomer of JC-1 and the number of green cells was increased at 8 h, indicating collapse of the potential. The membrane potential qualified with the ratio of fluorescence intensity (b): the potential was decreased at 8 h. Activity of caspase-9 at 2 and 8 h (c): relative light unit (RLU) reflected the enzymatic level; the enzymatic activation was detected at 8 h. Data were mean ± standard deviation for 3 independent experiments. The scale bar was 50 μm; (a) versus control, p<0.05.