Targeting FOXM1 By Thiostrepton Sensitizes Esophageal Squamous Cell Carcinoma To Radiotherapy In Vivo And In Vitro

Objective: To determine the radio-enhancing property of thiostrepton,FOXM1 inhibitor,in Esophageal Squamous Cell Carcinoma and the underlying mechanism.Methods: Human ESCC cell lines ECA-109 and TE-13 were separately devided into four groups:Control(C)group,Thiostrepton treatment(TST)group,Iron radiation(IR)group and Thiostrepton plus IR(TST+IR)group.Western blotting were used to detect the regulatory effect of TST on protein levels of FOXM1.Cell viability was detected by CCK-8 assay.Radiosensitization was evaluated by clonogenic survival assay.Analyses of the distribution of cell cycle at different stages and cell apoptosis were carried out through flow cytometry.The levels of phospho-γH2AX foci at 1h,6h,and 24 h after 8 Gy irradiation were determined by immunofluorescence to investigate the impact of TST on X-ray-induced DSB repair kinetics.And the expression levels of XIAP、Bax、Bcl-2、Cleaved-PARP、Cleaved-caspase 3、cyclin B1、Cdc2、phosphoCdc 2 were determined by Western blotting.Finally,the radioenhancing effect of thiostrepton in vivo were determined by rude xenografts model.Results: Western-blotting results showed that FOXM1 protein levels in ECA-109 and TE-13 cells with TST treatment decreased in a dose-dependent manner.Of note,the levels of FOXM1 protein in the 4μM,6μM,and 8μM thiostrepton treated groups were significantly lower than the control group(P<0.05).In addition,in ECA-109 and TE-13 cells,FOXM1 protein levels increased significantly after radiotherapy(P<0.05)and thiostrepton could effectively suppress the overexpression of FOXM1 after irradiation(P<0.05).Compared with the control group,the cell viability of ECA-109 and TE-13 cells were both dose-and time-dependently reduced with thiostrepton treatment. Compared with the irradiated group,the clone-forming capability of ECA-109 and TE-13 cells was significantly reduced in TST+IR group(P<0.05)and the SER were 1.87 and 1.35,respectively.Flow cytometry results showed that compared with the control group,G2/M phase cells of the two cell in IR or TST group were significantly increased(P<0.05),and cells in S phase were significantly decreased(P<0.05).In ECA-109 cells,G2/M phase cells in TST+IR group were significantly increased compared with the IR group,with a significant reduction in G0/G1 phase cells(P <0.05).However,in TE-13 cells,G2/M phase cells in TST+IR group were significantly higher than the IR group(P <0.05),while no significant changes in S phase and G0/G1 phase cells(P> 0.05).Apoptosis analysis showed that compared with the control group,the apoptotic rate of TST group in ECA-109 and TE-13 cells showed no significant change(P>0.05).However,the apoptosis rate in TST+TR group were significantly increased(P<0.05).Western-blotting results showed that there was no significant difference in the expression levels of Bax,Bcl-2,XIAP,Cleaved-PARP,and Cleaved-caspase 3 proteins between the TST group and the control group(P>0.05).Compared with the IR group,the expression levels of Bax,Bcl-2,Cleaved-PARP,and Cleaved-caspase 3 protein were significantly increased in TST+IR group(P<0.05),while the expression level of XIAP showed no obvious difference(P>0.05).In ECA-109 and TE-13 cells,the number of γH2AX focal was significantly increased at 1 h,6 h,and 24 h after irradiation(P<0.05)and the number of γH2AX focal increased significantly at 1h,6h and 24 h after radiation treatment(P<0.05)than that in IR group.Finally,in vivo xenografts experiments in nude mice showed that the volume and weight of xenografts tumors in TST+IR group were significantly lower and tumor inhibition rate(T/C %)was significantly higher than those in the IR group(P<0.05).Conclusion: enhancement of the radiosensitivity of ESCC cells induced by thiostrepton was demonstrated and the underlying mechanism may consist of induction of radiosensitive G2/M arrest and suppression of DNA damage repair,pointing to FOXM1 as a potential target for future treatment strategies.