Study Of Radiosensitization And Underlying Mechanisms Of Artemisinin On Human Cervical Cancer Cell

Among DNA damages,the most fatal one is DNA double-strand breaks(DSB).The DSB caused by X-ray will initiate cell cycle checkpoint.The cells will be arrested at G₁ or G₂ phase.The DNA will stop replication in cells of S phase.All of that is for only one purpose.It is ready to repair the damage.The cell cycle checkpoint is an important pathway for DNA to repair for self-protection.The tumor cells of p53 wild type will be arrested at G₁ phase to repair when it is damaged.The cells of p53 mutagenic will be arrested at G₂ phase to repair when it is irradiated.The tumor will have mutation with p53 when it is growing.If the tumor have that,it will be hard to be killed.We should find a method to resolve it.G₁ phase checkpoint play roles through p53 protein,so for tumor cells which lost p53 function,G₂ phase checkpoint become the most important protection mechanism of DNA repair before mitosis in damaged cells,G₂ phase arrest is essential to the survival of these cells.We can relieve the block of the G₂ phase.The result is that the tumor cell have no time to repair and die.Objective Measure the cytotoxicity of the GM0639 fibroblast,wild p53 cervical cancer cell Siha and mutant p53 cervical cancer cell HeLa of artemisinin to determine the optimized effection dose and time.Mesure radiosensitivity of GM0639,Siha,HeLa cell by MTT and microcolony formation to determin which cell is radiosensitized by artemisinin. To investigate the radiosensitizing effect of artemisinin and mechanisms.Methods(1) The GM0639 fibroblast,wild p53 cervical cancer cell Siha and mutant p53 cervical cancer cell HeLa were cultured in vitro with artemisinin.The cell proliferation was measured by MTT assay.0,27.67,55.34,110.69,221.37,442.75nmol/ml of artemisinin were chosen to be investigated.The GM,Siha and HeLa cell interact with artemisinin for 24 and 48 hours.(2)To detect the inhibition ratio that artemisinin of 55.34nmol/ml and 110.69nmol/ml be interacted with the GM,Siha and HeLa cells for 0,2,4 and 6 hours using MTT.The second step is to combine with 4Gy X-ray.(3) The radiosensitization effect on three cells by artemisinin was measured by MTT and microcolony formation.(4) Flow cytometric analysis was used to detect the cell cycle and apoptosis by artemisinin after 6 Gy X-ray irradiation.(5) Influence of artemisinin on expression level of cell cycle related proteins CyclinB1,Weel was investigated by Western-blot.Results(1)When the concentration of artemisinin was higher,the inhibition rate of the GM,Siha and HeLa cell was higher(p＜0.01).When the time intreacted with the GM, Siha and HeLa cell is longer,the inhibition rate of the GM,Siha and HeLa cells was higher(p＜0.01).(2)Artemisinin of 55.34nmol/ml and 110.69nmol/ml had no cytotoxicity to the GM,Siha and HeLa cells interacted for 0,2,4 and 6 hours.When 4Gy X-ray irradiate the cell interacted with artemisinin of 110.69nmol/ml for 6h,the survival rate of the HeLa cell obviously decreased(p＜0.01).The other results were negative.(3)Artemisinin of 110.69nmol/ml had no radiosensitivity on GM and Siha after 6h's interaction(p＞0.05),but had obvious radiosensitivity on HeLa cell.The SER is 1.17 by MTT method.Microcolony formation showed artemisinin had no radiosensitivity on GM and Siha,but have obvious radiosensitivity on HeLa,the SER is 1.12.(4)Compared with the control group,there is no influence to the cell cycle or apoptosis rate of GM and Siha,while to the HeLa,the G₁ phase decreased(p＜0.01) and the G₂ phase increase with the change of irradiation dose. Artemisinin and X-ray group could decrease the G₂ phase arrest that caused by X-ray. Compared with the control group and the irradiation group,artemisinin and X-ray group could increase the apoptosis rate.(5)X-ray or artemisinin could't change the expression of the CyclinB1 and Wee1 protein of GM and Siha,while to the HeLa cell,6Gy X-ray could decreae CyclinB1 and increase Wee1 protein.The artemisinin and X-ray group could inverse the expression of the protein.Conclusion(1)Cytotoxic effect of artemisinin in the test on the GM0639 fibroblast, wild p53 cervical cancer cell Siha and mutant p53 cervical cancer cell HeLa was obviously dose and time dependent.(2)Artemisinin had no radiosensitivity on GM and Siha,but had obvious radiosensitivity on HeLa.Radiosensitizing effect of Artemisinin on HeLa is related to decrease of repair ability to sublethal injury.(3)X-ray could increase G₂ phase of HeLa.Artemisinin could decrease the rate of G₂ phase.Test result showed that artemisinin could cause HeLa apoptosis.(4)X-ray could decreae the CyclinB1 and increase the Wee1 protein.Artemisinin could inverse that tendency.