The Study On The Radiosensitizing Effect Of Artesunate On Nude Mouse Transplanted With HeLa And Siha Cells Of Cervical Cancer

Objective Investigate the radiosensitizing effect of Artesunate on nude mouse transplanted with HeLa and Siha cells with different p53 states of cervical cancer and compare the difference of radiosensitising effect in order to provide experimental evidence for Artesunate as a radiosensitizer.Methods The cervical carcinoma HeLa and Siha cells at a concentration of 1²×10⁷/ml and 1²×10⁸/ml were inoculated into the nude mice to establish tumor model. Mice were randomized into four groups as control group,artesunate group,irradiation group and artesunate+irradiation group respectively when average volume of tumor were about (5mm×5mm×5mm).During the term of treatment,the volume of tumors were measured per 2 days. After 14 days treatment,the mice were killed and tumor tissues were harvested used for flow cytometry detecting the alteration of cell cycle. Meanwhile,the pathological change of the tumor tissue was observed with HE staining method,immunohistochemical staining and TUNEL were performed for microvessel density(MVD) and apoptosis respectively,and Western Blot method was used to investigate the change of expression of cycle regulatory protein cyclinB1,cdc2 and wee1.Results (1)10-15 days were needed to establish tumor model transplanted with HeLa cells,the rate of tumor formation is 100%,and 15-20 days were needed to establish tumor model transplanted with Siha cells,the rate of tumor formation is 100%;(2)The growth of transplantation tumor of HeLa cells was significantly inhibited by artesunate combined with radiation , tumor weight(0.64±0.28)g of artesunate+radiation group was lower than irradiation group(1.31±0.44)g(P<0.05),and the tumor weight inhibition of artesunate+radiation group was 67.67%. After 14 days' treatment,the average tumor volume of artesunate+radiation group(579.18±120.36)mm³ was obviously lower than irradiation group(979.36±289.00)mm³ (P<0.05),while the volume inhibition rate 61.59% for artesunate+radiation group was higher than irradiation group 35.05%. The growth of transplantation tumor of Siha cells has no obviously inhibition by Art.(3)Compared with irradiation group,the apoptosis in artesunate+radiation group of transplantation tumor of HeLa cells is more obvious in the visual fields of microscope. But there was no change in transplantation tumor of Siha cells.(4)In transplantation tumor of HeLa cells,the apoptosis index in artesunate+radiation group (50.30±5.91)% was higher than in irradiation group (30.20±5.09)% (P<0.05),but no change between these two groups in transplantation tumor of Siha cells.(5)Immunohistochemistry test showed:Microvessle density of transplantation tumor of HeLa cells in artesunate+radiation group(9.75±2.99)was significantly lower than in irradiation group(24.25±5.74)(P<0.05),but there were no difference between artesunate+radiation group and irradiation group in transplantation tumor of Siha cells(.6)Flow cytometry results showed that:In transplantation tumor of HeLa cells,the percent of cells in G₀/G₁ phase increased (P<0.05) and G₂/M phase decreased (P<0.05) in artesunate+radiation group compared with irradiation group. In transplantation tumor of 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.(7)In transplantation tumor of HeLa cells,cyclinB1 expression was obviously increased in artesunate+radiation group than in irradiation group,whereas there was no difference in the groups of control and Art. The expression of wee1 decreased in artesunate+radiation group than in irradiation group. The expression of cdc2 has no change in the four groups. In transplantation tumor of Siha cells,expression level of cyclinB1,cdc2 and wee1 had no significant change in all groups.Conclusion (1) Artesunate combined withγ-ray irradiation can inhibit the growth of transplantation tumor of p53 mutant type HeLa cells,but had no such effect on transplantation tumor of p53 wild type Siha cells;(2) Artesunate can increase the apoptosis ratioes of transplantation tumor of p53 mutant type HeLa cells that after irradiated,but had no such effect on transplantation tumor of p53 wild type Siha cells; (3) Artesunate combined withγ-ray irradiation can decrease the microvessel density of transplantation tumor of p53 mutant type HeLa cells,but had no such effect in transplantation tumor of p53 wild type Siha cells;(4) Artesunate can decrease the cell G₂/M arrest of transplantation tumor of p53 mutant type HeLa cells induced by irradiation,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 of transplantation tumor of p53 wild type Siha cells induced by irradiation;(5) Western blot result showed that,compared with irradiation group , expression of cyclinB1 increased and wee1 decreased in transplantation tumor of p53 mutant type HeLa cells,while the expression of cyclinB1 and wee1 had no significant change in transplantation tumor of p53 wild type Siha cells;(6) The expression of cdc2 had no significant changes in both transplantation tumor of 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. (7) Artesunate may be a safe and effective radiosensitizer in cervical cancer and deserved further study.