The Radiosensitization Effect Of PARP Inhibitors On Hypoxic Esophageal Squamous Cell Carcinoma Cells

Objective:Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma(ESCC).However,the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells,particularly those under hypoxic condition,exhibit radioresistance.Poly(ADP-ribose)polymerase(PARP)is a nuclear chromatin-associated protein and plays an important role in DNA damage repair.Ionizing irradiation exerts its therapeutic effect mainly by causing DNA double-strand breaks(DSB)in cancer cells.Therefore,the manipulation of DSB repair may influence the efficacy of radiotherapy treatment for cancer.The aim of this study was to determine whether or not AZD2281,a PARP inhibitor,could enhance the radiation sensitivity of hypoxic ESCC cell lines,and to explore the causes.Methods:Cultrued human ESCC cells Eca109 and TE-13 cells in the logarithmic phase were used in this study.Thiazolyl blue(MTT)assay was performed to assess the growth of ESCC cell lines.Clonogenic assay was performed to investigate the potential radiosensitization activity of AZD2281 on ESCC cell lines.AnnexinV-PI staining was performed to quantify apoptosis after treatment of ESCC cell lines with irradiation and/or AZD2281.DSB repair assay was performed by counting phospho-γH2AX foci.We also assessed the foci assembly of HR protein RAD51 in ESCC under normoxic and hypoxic conditions at indicated time points.Rad51 expression in ESCC under normoxic and hypoxic conditions at indicated time points was shown by western blots.PARP、RAD51、HIF-1α proteins expression was also assessed by western blots.Results:1.MTT assay:After 24-h treatment,AZD2281 showed no significant effect on cell growth.After 48-h treatment,however,limited inhibitory effects were observed in both cell lines.The cell viability at 50 μmol/L was 0.72±0.10 for Eca109 cells and 0.76±0.10 for TE-13 cells.2.Clonogenic assay:Under hypoxia,AZD2281 potentiated the cytotoxicity of radiation treatment,in which the SERs at 5 μmol/L were 1.50 and 1.68 for Eca109 and TE-13 cells,respectively.Under normoxia,AZD2281 also showed radiosensitization activity with SERs of 1.38 and 1.57 for Eca109 and TE-13 cells,respectively.These data demonstrate that AZD2281 promotes radiosensitivity of normoxic and hypoxic ESCC cells.3.Apoptosis assay:The results showed that the combined treatment of irradiation and AZD2281 largely increased apoptotic cells in these cell lines under hypoxia.The difference between groups of combined treatment and radiation alone treatment was statistically significant(P<0.01)under hypoxia.4.yH2AX foci:The combination of AZD2281 treatment with 4 Gy X-ray increased the formation of phospho-7H2AX foci at 1 h.At 24 h after irradiation,AZD2281-treated cells showed a significantly slower decay of γH2AX foci.RAD51 foci:Unlike RAD51 foci in normoxic cells,RAD51 foci in hypoxic Eca109 cells failed to assemble.5.Western blot:RAD51 expression was downregulated after the cells were exposed to hypoxic condition for 48 h compared with that under normoxia.PARP inhibitor AZD2281 did not alter PARP expression under either normoxic or hypoxic conditions.Under hypoxia,AZD2281 did not alter HIF-1α expression.Conclusion:AZD2281 potently sensitizes hypoxic ESCC cells to X-ray irradiation.However,AZD2281 does not alter PARP and HIF-1α expression under hypoxic conditions.HR-defective hypoxic cells are sensitive to PARP inhibitor which contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition.