ANTP-SmacN7 Fusion Peptide-induced Radiosensitization In Lung Cancer Cells And Its Potential Mechanisms

Background and objectiveRadioresistance in tumors limits the curative effect of the radiotherapy.The overexpression of apoptosis inhibitor protein IAPs(Inhibitors of Apoptosis Proteins,IAPs)in tumor cells has been proved to be closely related to tumor radiation tolerance.Its high expression can inhibit apoptosis and reduce tumor radiation sensitivity.Smac(Second mitochondria-derived activator of caspase),can be directly combined with IAP,remove its apoptosis inhibition function,so as to improve the radiosensitivity of tumor.As one pro-apoptotic protein,Smac protein mimetics,when affected by apoptosis-inducing factors,together with other mitochondrial proteins such as cytochrome-c,can be released through the mitochondrial membrane to participate in the regulation of apoptosis,which can significantly increase the apoptosis of tumor cells induced by radiation.Mimetic compounds of Smac are potential new tumor radiation-sensitizing drugs because they can increase radiation-induced tumor cell apoptosis.The Smac N7 is the smallest active functional unit of the Smac polypeptide.It can increase radiosensitization by inhibiting the effect of IAPs;however,Smac N7 cannot enter cells.Research shows that ANTP is the smallest protein region with transduction functions,which is independent of the receptor,channel,energy and endocytosis.ANTP can act directly on the lipid bilayer to complete transmembrane movement and enter cells.Therefore,we used the ANTP protein as the guiding peptide to synthesize the ANTP-Smac N7 fusion peptide and guide Smac N7 into cells to radiosensitize the cells.Here,we observed the radiosensitization effect of a new Smac mimetic antennapedia protein ANTP-Smac N7 fusion peptide in A549 cells and investigated the underlying mechanisms behind the effects of this protein on tumor cells.Another objective of the experiment was to investigate the relationship between the protein expression of Smac and IAP family in lung cancer cell line A549 and H460 cells and their radiosensitivity.Contents:Part ?: The ANTP-Smac N7 fusion peptide was synthesized and linked with fluorescein isothiocyanate to observe the protein's ability to penetrate cells.Part ?: A549 cells were divided into the control,radiation-only,ANTP-Smac N7-only and ANTP-Smac N7+radiation groups.The cells were exposed to 0,2,4 and 6 Gy,with 20 ?mol/L of ANTP-Smac N7.The radiation-sensitizing effects of the ANTP-Smac N7 fusion proteins were observed via clonogenic assay.Apoptosis was detected Annexin V/PI double staining Method using flow cytometry.A comet assay was used to assess DNA damage.The levels and degrees of cytochrome-c,PARP,caspase-8,3,9 activation and ?-H2 AX were detected via western blot assay.The change of radiation sensitization,expression of ?-H2 AX,H2AX and PARP were compared after adding the caspase inhibitor,Z-VAD.Part ?: The radiosensitivity of two lung cancer cell lines,A549 and H460,and the expression levels of Smac,XIAP,c IAP1 and c IAP2 were compared to analyze the potential mechanism.Results:Part ?: The ANTP-Smac N7 fusion protein successfully entered and accumulated in the cells,enabling the proapoptotic effect of the fusion peptide in the cellsPart ?: The ANTP-Smac N7 promoted caspase activation,increased the rate of radiation-induced apoptosis,augmented radiation-induced double-stranded DNA rupture in the A549 cells and increased DNA damage.Part ?:The radiation tolerance of A549 was better than that of H460 cells,with the lower expression of Smac and high expression of XIAP in A549 cells Conclusions:The ANTP-Smac N7 fusion peptide exerted a remarkable radiosensitization effect on A549 cells.This protein may reduce tumor cell radioresistance by inducing caspase activation and may be a potential new Smac mimetic that can be applied in radiosensitization therapy.The low expression of Smac and high expression of XIAP in A549 cells was the molecular basis for its higher radiation tolerance than H460.