Targeting BCL-xL Enhances Anticancer Activity Of Oncolytic Virus Ad-tBID

Objectives:To investigate whether targeting BCL-xL could enhance anticancer activity of oncolytic virus Ad-tBID in vivo and in vitro,and explore the mechanism underlying this effect preliminarily.Methods:The overexpression of tBID in tumor cells was examined by Western Blot and immunofluorescence;the discrepancy of replication of Ad-tBID in normal cells and tumor cells was qualified by qPCR analysis;cell viability assays was performed to evaluate the sensitivity of different tumor cells to Ad-tBID;The BCL-xL inhibitor which possessed the greatest combinatorial effect with Ad-tBID was determined by DAUC,meanwhile,the target protein of this inhibitor was locked,and then confirmed with cells whose expression of BCL-xL was downgraded by siRNA;The change of expression of several BCL-2 family proteins,which had been studied most was demonstrated in the result of Western Blots;and in four mid-sensitive tumor cells,the change of transcription of these proteins was explored by qPCR analysis;immunofluorescence was performed to locate and semi-quantify BAK in tumor cells treated by Ad-tBID;Crystal violet staining assay was performed to evaluated the effect of combinatorial strategy;JC-1 dye was used to indicate mitochondrial membrane potential;caspase activity was measured to monitor the activation level of mitochondrial apoptosis pathway;BALB/c-nu mice were injected with tumor cells subcutaneously to establish animal models for assessments in vivo;viral replication in tumor tissues,proliferation and apoptosis of tumor cells were detected by immunohistochemistry.Results:Ad-tBID selectively replicated in and lyses tumor cells by induce overexpression of tBID;sensitivity to Ad-tBID varied in different tumor cells;A-1331852 was determined as the BCL-xL inhibitor of greatest DAUC fold with Ad-tBID;BCL-xL was confirmed as the target protein;Combination therapy of Ad-tBID and A-1331852 may act through facilitating the oligomerization of BAK;Combination therapy of Ad-tBID and A-1331852 induced mitochondrial apoptosis;In vivo,A-1331852 enhanced anticancer effects of Ad-tBID.Conclusions : Combination strategy of Ad-tBID and BCL-xL inhibitor A-1331852 demonstrated impressive anti-tumor effect in vitro and in vivo through facilitating the oligomerization of BAK.