Experimental Investigation Of Radiosensitivity Enhancement Of Sodium Valproate On Uterine Cervix Cancer Cell Line Hela

Objective Radiotherapy conducting as a traditional therapy plays an important role in the treatment of malignancies,and it is involved in about 70%of total malignancies in our country.The outcome of radiation therapy depends on the radiosensitivity of tumor cells and the tolerance of the tissue bed to radiation.So developing satisfactory radiosensitizer is an important way to enhance the radiation effect,and it also becomes a front topic of radiation oncology.There are a lot of researches supporting that the histone deacetylase inhibitors(HDACIs)not only have antitumor effect through many ways,such as inhibiting tumor cell proliferation, inducing apoptosis,inhibiting tumor angiogenesis and metastasis,cutting down the tumor invasiveness and so on,but also enhance the radiosensitivity of tumor combined with radiation.The mechanisms of radiosensitivity enhancement of the histone deacetylase inhibitors are complicated and it is not very clear so far.Sodium valproate (VPA)has been used to treat epilepsy for many years.Recently it is found that it can inhibite the histone deacetylase,because it has many good qualities such as long half life,high bioavailability,low valid blood medicine level and few side effects,it shows good perspective.In this study,we choose VPA as the representative of HDACIs to illuminate the mechanisms of enhancing radiosensitivity futher,and to guide for the clinical medication.Methods MTT assay was used to measure the proliferation inhibition of the histone deacetylase inhibitor(valproic acid,VPA)in different concentrations (0.25mmol/L,0.5 mmol/L,1 mmol/L,2 mmol/L,4 mmol/L)and durations(1d,2d,3d,4d,5d)on uterine cervix tumor cell line Hela.Morphologic changes of Hela were observed through inverted contrast microscope after it incubated with various concentration VPA for 2 days.Hela cells incubater with 0.25mmol/L VPA were exposed to different doses X-ray irradiation(IR)(0,2,4,6,8cGy).Then the cells were assayed for clonogenic survival to determine the radiosensitizing effect of VPA.Flow cytometry was used to determine the effects of various concentration VPA on apoptosis and cell cycle distribution.RT-PCR and Western Blot were used to detect the expression of caspase-3 and Bcl-2 to find the pathway of VPA to induce Hela to apoptosis.Results The changes of the growth curve showed VPA inhibited the proliferation of Hela cells significantly with time and dose dependent;After a 2 days culture with various concentration VPA,the cells exhibited deflate,the cell membrane shrinkage and bad adherence.It showed obvious apoptosis tendency.The HDAC inhibitor VPA at the concentration of 0.25mmol/L enhanced the killing effect of IR on Hela cells,and the cloning efficiency decreased obviously(P＜0.05).The sensitization enhancement ratio(SER)was 1.35,1.29 according to D_q,D₀.VPA not only induced apoptosis and caused cell cycle arrest at G₂/M phase,but also inhibited the proliferation of Hela.The expression of caspase-3 mRNA and protein increased after treatment with VPA at a dose-dependent manner,and cleaved caspase-3 protein was also observed,whereas down regulation was detected with Bcl-2.Conclusions These findings indicated that the histone deacetylase inhibitor VPA inhibited the growth of uterine cervix cancer cell line Hela at a dose-and time-dependent manner.VPA enhanced the radiosensitivity of Hela cells,and the mechanisms for this action might include inhibition of sublethal injury repair,direct cellular proliferation inhibition,increasing the sensitivity of Hela cell to X-ray irradiation-induced apoptosis and cell cycle arrest.Both of up-regulating the expression of caspase-3 and down-regulating the expression of Bcl-2 might play an imporment role in the sensitization enhancement of VPA to irradiation-induced apoptosis.