The Effect Of Apoptosis Inhibitory Protein Targeting Agents LCL161 And YM155 On Radiosensitivity Of Esophageal Cancer And Its Mechanism

Part 1 Smac-mimetic compound LCL161 sensitizes esophageal carcinoma cells to radiotherapy by targeting c-IAP1 and activating caspase-8Purpose: Currently,treatment of unresectable esophageal squamous cell carcinoma?ESCC?is primarily relied on chemoradiotherapy.However,the outcome has not improved significantly due to the intrinsic radioresistance of cancer cells.The present study was designed to determine in ESCC cells the radiosensitizing effect of LCL161,a novel second mitochondrial derived activator of caspase?Smac?mimetic targeting inhibitor of apoptosis proteins.Methods: ESCC cell lines were treated with LCL161 or radiation,alone or in combination.Cell proliferation was detected by MTT assay.Radiosensitization was evaluated by clonogenic survival assay.Cell apoptosis was detected by flow cytometry.The levels of phospho-?H2AX foci at 30 min,2 h,8 h,and 24 h after 4 Gy irradiation were determined by immunofluorescence to investigate the impact of LCL161 on X-ray-induced DSB repair kinetics.Western blot was used to detect the expression of IAP family members in ESCC cells and in NIH3T3 cells as normal control.And at last,the expression and secretion of TNF? were detected by RT-PCR and ELISA,respectively.Results: We found that LCL161 effectively decreased the level of c-IAP1 that is over expressed in the tested ESCC lines in a dose dependent manner.Treatment of Eca109 cells with 5 ?M LCL161 8 h prior to radiation led to a significant survival curve shift compared to untreated Eca109 cells with an SER of 1.42.LCL161 also induced a remarkable radiosensitization in TE13 and KYSE150 cells,with a SER of 2.0 and 1.64,respectively.The effect was accompanied by apoptosis enhancement which was abrogated by a pan-caspase inhibitor z-VAD-FMK,indicting an involvement of caspase activation.Further mechanistic studies suggested that LCL161 radiosensitization in ESCC cells was mainly mediated by NF-?B activation and TNF? production,followed by cleavage of caspase-8,leading to enhanced apoptosis.In addition,The combination of LCL161 treatment with 4 Gy X-ray increased the formation of phospho-?H2AX foci at 30 min by 1.7-fold compared to X-radiation alone.And LCL161-treated cells showed a slower decay of ?H2AX foci after irradiation which indicated that LCL161 could increase radiation induced DNA double stranded breaks?DSBs?and attenuate the efficiency of DSB repair.Conclusion: LCL161 acts as a strong radiosensitizer in human esophageal cancer cells with high levels of IAPs and exhibits great potential for an application in improving clinical outcome in ESCC radiotherapy.Part 2 Survivin inhibitor YM155 enhances radiosensitization in esophageal squamous cell carcinoma by the abrogation of G₂ checkpoint and suppression of homologous recombination repairPurpose: Survivin is preferentially expressed in cancer cells and plays a crucial role in cell division and apoptosis evasion.YM155,a small-molecule inhibitor of survivin,has been reported to enhance the cytotoxicity of various DNA-damaging agents.Here,we evaluated the radiosensitizaion potential of YM155 with especial emphasis on its effect on G₂ checkpoint control in human esophageal squamous cell carcinoma?ESCC?cells.Methods: Cell viability was determined using CCK8 assay.The radiosensitization effect of YM155 was evaluated on the basis of clonogenic survival and progression of tumor xenograft.Cell cycle progression was determined by flow cytometric analysis.Radiation-induced DNA double strand break?DSB?and homologous recombination repair?HRR?were detected by immunofluorescence staining of ?-H2 AX and RAD51,individually.Expression of survivin and biochemical markers indicative of the G₂/M transition was demonstrated by use of immunoblotting.Results: A low dose of YM155 induced radiosensitization of reproductive cell death in cells of the ESCC cell lines Eca109 and TE13.When the concentration of YM155 reached 10 nmol/L,the SER?sensitization enhancement ratio?of Eca109 and TE13 cells was 1.51 and 1.73,respectively.Flow cytometry demonstrated that radiation-induced G₂/M arrest was abrogated by 10 nmol/L YM155?34.7% for Eca109 and 36.4% for TE13?,with a concomitant rise in G1 and S phases.The addition of nocodazole?0.4 ?g/m L?successfully prevented irradiated cells exposed to YM155 from cell cycle progression.Histone H1 kinase activity of Cdc2 was suppressed in irradiated Eca109 cells.However,YM155 resulted in significant activation of Cdc2 kinase in irradiated cells.Moreover,YM155 evidently abrogated X-ray induced Cdc2 hyperphosphorylation and cyclin B1 accumulation without affecting total Cdc2 and phospho-Cdc25 C protein levels.YM155 treatment resulted in a significant prolongation of ?-H2 AX signal at least 24 h post irradiation?47.7 ± 3.0%?compared with radiation alone?7 ± 2.8%;P < 0.001?.On the other hand,YM155 significantly suppressed the assembly of Rad51 foci in response to radiation,which was in contrast with kinetics of ?-H2 AX.In apoptosis analysis,the induction of early apoptotic events?Annexin V positive?was most evident when cells were treated with 8 Gy plus YM155.And higher levels of cleaved PARP and caspase-3 were observed in Eca109 cells treated with 8 Gy radiation plus 10 nmol/L YM155.Taken together,radiosensitization by YM155 was associated with abrogation of the radiation induced G₂/M checkpoint which leads to impaired Rad51 focus formation,a key step in HRR,and a prolongation of ?-H2 AX signaling in irradiated ESCC cells.Finally,the combination of YM155 plus irradiation delayed the growth of ESCC tumor xenografts to a greater extent compared with either treatment modality alone.Conclusion: Our findings suggest that G₂ checkpoint abrogation and resulting HRR inhibition contribute to radiosensitization by YM155 in ESCC cells.