Experimental Research Of IRE Combined With Cell Cycle Synchronization On Hela Cells Ablation Involving G2/M Cell Cycle Arrest

Objective: To search the ablation effect of irreversible electroporation and pulse electric field combined with cell cycle synchronization on Hela cells and the correlation mechanism of G2/M cell cycle arrest by pulse electric field.Methods: 5 groups of He La cells treated with voltage amplitude 1.75 k V/cm, 2.0 k V/cm, 2.25 k V/cm, 2.5 k V/cm and the control group were included. Pulse parameters include width(100μs), repetition rate(1 Hz) and pulse number(8).Synchronization in G1 phase can be obtained with lovastatin. S and G2/M phase were synchronized with hydroxyurea and the results were detected by flow cytometry. Cell suspension(concentration 2×106 cells/ml) including cell cycle synchronized groups and unsynchronized groups after IRE processing was re-incubated for 6 hours and 12 hours. The distribution of cell cycle was analyzed by flow cytometry. After 24 hours, cell apoptosis was observed by flow cytometry and fluorescence microscope. CCK-8 was utilized to detect the cell viability after IRE processing 24 hours later. In addition,proliferation and vitality were detected by CCK-8 every 24 h for 7 days(the voltage amplitude was 2.5k V/cm). Hela cells(cell-cycle synchronized groups and unsynchronized groups) were treated with IRE(2.5k V/cm). After two weeks, cells stained with Giemsa and the clone formation rate was observed. Western blot was used to detect cyclins of G2/M in order to understand the correlation mechanism of G2/M cell cycle arrest by pulse electric field.Results:(1) After 24 h of culture with lovastatin, the most cells were synchronized in G1 with(83.39±3.91) %. Most cells(82.12±3.44) % were arrested in S phase with the fresh medium for additional 3.5 h after 24 h treatment with hydroxyurea and for additional 8.5 h resulted in cell accumulation in G2/M phase at(78.06±3.14) %.(2) Flow cytometry results showed cells in G2 phase increased compared with the control group when the field strength was 1.75-2.5k V/cm,especially the 2.5k V/cm group(P﹤0.05). The results of Flowcytometry showed a significant difference of apoptosis between synchronized G1 group and the rest groups(P﹤0.05). Apoptosis in G2/M group showed reducing trend compared with control group.(3) CCK-8 results showed cell proliferation was markedly inhibited by IRE processing after 24 hours. There was a significant difference with the control group(P﹤0.05). The results of 7 days consecutive detection of CCK-8 showed cell activity and proliferation was G2﹥S﹥Hela﹥G1. Colony formation had the same result.(4) The results of Western blot showed the expression of cyclin B1 and p-Cdk1(Tyr-15)increased and Cdk1 expression decreased.Conclusion: Cell cycle synchronization can affect the ablation of irreversible elect roporation on Hela cells,the strongest on G1 phase and the weakest on G2/M phas e. IRE can induce DNA damage and Cdc25-cyclin B1/Cdk1 pathway joined in the G2/M phase arrest. With the attenuation of the electric field, the change of cell cyc le in tumor cell and the different effect of each cell cycle may accelerate tumor re currence.