The Study On The Effect And Mechanism Of Artesunate On The Radiosensitivity Of HeLa And Siha Cells Of Human Cervical Cancer

Objective This study is to investigate the effect of Artesunate(Art) on radiosensitivity of p53 mutant HeLa cells and p53 wild type Siha cells of human cervical cancer in vitro. And try to find out the mechanism of radiosensitivity by comparing and analyzing the difference of radiosensitising effect of Art on human cervical cancer cells with different p53 states.Methods Use human cervical cancer cells HeLa and Siha as the experimental cells. (1) Studide the impact of artesunate to cell survial fraction of human cervical cancer cells HeLa and Siha by using MTT, in order to determine the most appropriate drug concentration in the succedent experiment;(2) The radiosensitization effect on HeLa and Siha cells by artesunate was measured by clonogenic assay and cell apoptosis that analysed with flow cytometry;(3) The changes of cell cycle distribution effected by artesunate combined withγ-ray irradiation was detected by flow cytometry;(4) Changes of expression of cell cycle regulatory protein wee1,cyclinB1,cdc2 that caused by artesunate combined withγ-ray irradiation was investigated by Western-blot .Results (1) MTT analysis showed that:the inhibition rate of artesunat to human cervical cancer cells of HeLa and Siha was higher along with the increase of drug concentration (P<0.01). Determined the concentration of artesunate in the radiosensitization experiment:for HeLa cell is 2.0μmol/L,for Siha cell is 2.5μmol/L, the acting time is 24 h. And the inhibition effect of artesunate to the cells is low(10%) at the selected acting time and drug concentration;(2) The clonogenic assay showed that:with the same concentration of artesunate,clone formation rate decreased with the increase of radiation dose (P <0.01). As to HeLa cells,in radiation group and drug plus radiation group,the mean lethal dose (D₀) were 2.95Gy and 2.07Gy respectively,the quasi-threshold dose (D_q) was 2.01Gy and 1.24Gy respectively,the SER was 1.43.To Siha cells,the mean lethal dose (D₀) was 2.68Gy and 2.61Gy respectively,the quasi-threshold dose (D_q) was 1.91Gy and1.86Gy respectively,the SER was 1.03. (3) Annexinâ…¤/PI double staining showed:Compared with the apoptosis rate of 12.26% and 40.08% in radiation group (the dose was 2,6Gy),the apoptosis rate at the same dose in artesunate combined with radiation group rised to 22.71% and 59.92%,and the difference was significant(P<0.01). As in Siha cells,the apoptosis rates of cells in radiation group (the dose was 2,6Gy) were 12.27% and 32.25%,at the same dose in drug combined with radiation group,the apoptosis rates were 14.35% and 33.67% respectively,the difference was not significant(P>0.05). (4) Flow cytometry analysis showed that:at the dose of 2,6Gy,in HeLa cells,the percent of cells in G₀/G₁ phase increased (P<0.01) and G₂/M phase decreased (P<0.01) in artesunate combined with radiation group compared with simplicity radiation group. In Siha cells,the percent of cells in G₀/G₁ phase and G₂/M phase didn't show obvious change (P>0.05) in the groups of radiation combined with or without artesunate. (5) Western blot analysis showed that:for HeLa cells,at the radiation group (the dose was 2,6,12Gy),along with the increase of dose the expression of wee1 increased,the expression of cyclinB1 decreased while cdc2 had no significant change. At the same dose,the expression of wee1 decreased,cyclinB1 increased and cdc2 had no significant change in the drug combined with radiation groups. In Siha cells,expression level of wee1,cyclinB1 and cdc2 had no significant change in all groups.Conclusions (1) The inhibition effects of artesunate on human HeLa and Siha cells of cervical cancer in the test were in concentration-dependent;(2) Artesunate has different inhibition effect on HeLa and Siha cell,at the same drug concentration,the inhibition effect on HeLa cells was stronger than on Siha cells;(3) Artesunate can increase the radiosensitivity of human HeLa cells of cervival cancer with p53 mutant,but had no such effect on wide type p53 Siha cells .Radiosensitizing effect of artesunate on HeLa cell is related to decrease the repair ability to sublethal injury;(4) Artesunate can increase the apoptosis ratioes of HeLa cells with p53 mutant that after irradiated, but had no such effect on Siha cells with wild type p53;(5) Artesunate can decrease the cell G₂/M arrest induced by radiation,which probably drived more irradiation damaged cells to enter mitosis phase,finally the cells died cause of had no time to rapair. But artesunate had no significant inhibition effct on G₁/S arrest;(6) The exposure ofγ-ray can induce the G₂/M arrest in HeLa cells with p53 mutant by increase the expression level of wee1 and decrease the content of cyclinB1. Artesunate can play the radiosensitizing effect by inverse the tendency. Siha cells were arrested in G₁/S phase,so the expression of protein that related to G₂/M phase had no changes after drug treated with;(7) The expression of cdc2 had no significant changes in both HeLa and Siha cells that treated with drug combined with radiation,showing it is possible that artesunate can affect on cdc2 through wee1,rather than directly to cdc2.