Mechanism Of ATO Induced Drug Resistant In Neuroblast And The Effect Of Combining With Amytal

Objective:Arsenic trioxide(ATO)treatment can induce apoptosis or differentiation of tumor cells,which is the first choice for clinical treatment of promyelocytic leukemia.It has been confirmed that a variety of cancer cells are prone to develop drug resistance after ATO treatment.Therefore,the wide application of ATO in clinical practice is limited.This study aims to investigate the anti-neuroblastoma effect of ATO and the molecular mechanism of drug resistance,as well as the effect of ATO combined with other drugs in the treatment of neurocytoma.Methods:In this study,we selected neuroblastoma as the object.Firstly,the roles of necroptosis,autophagy,NF-κB,MAPKs,apoptosis and other signaling pathways were verified in ATO mediated cell killing.Then,ATO resistant neuroblastoma single clonal D cell line and sensitive P cell line were selected by ATO containing medium;Under ATO stimulated condition,the differences of mitochondrial morphology between P cells and D cells as observed by confocal fluorescence microscopy;RNA-sequencing analyses were also performed on the above two cells to compare their different genes.Based on this,PPI and cluster analysis were performed to screen the genes that most likely to be involved in D/P cell death,and the biological functional effects of these genes on D/P cells were verified.In addition,this study also verified the efficacy of ATO in combination with Amytal for neuroblastoma cell killing,and the mechanism of combined drugs on killing neurocytoma through related experiments.Results:In this study,D/P cell lines resistant to ATO(D)or sensitive to ATO(P)are successfully identified,and we demonstrated that ATO exerted an anti-neuroblastoma effect by inducing apoptosis.In D/P cells,autophagy,NF-κB,MAPKs and other signaling pathways don’t involve in ATO-induced cell death,but intrinsic apoptosis pathway was activated because of mitochondria was impaired during ATO treatment in P cells,while ATO has no effect on mitochondrial morphology of D cells.Bioinformatics analysis identified 9 genes,including BCL-2,are most likely to be involved in the process of D/P cell death.Overexpression of BCL-2 in P cells resulted in equal levels of tolerance to ATO as D cells.In the tumor bearing experiment of nude mice,we found that the tumor size of implanted P/D cells is the same,indicating that there is no difference in the proliferation of P/D cells.However,when the tumor bearing mice were treatment with ATO,we found that the tumor size of P cells was greatly decreased than that of D cells,which further confirmed that ATO had a good killing effect on sensitive cells.In addition,we also found that combination of ATO with Amytal can induce cell pyroptosis and increase the killing effect on neurocytoma by activating Caspase-3 leading to the cleavage of GSDME.Conclusion:This study reveals that ATO exerts an anti-tumor effect by inducing apoptosis in neuroblastoma.In terms of molecular mechanism,we find that the high expression of BCL-2is responsible for the drug resistance in cells to protect the integrity of mitochondria,so that cells can survive.In addition,ATO combined with Amytal can play a pyrolytic killing effect on neurocytoma,and synergistically inhibit the growth of tumor cell.In conclusion,this study shows that ATO can be a potential drug in neuroblastoma treatment,and drug resistance to ATO can be improved by combination therapy,which provides a new aspect for clinical treatment of cancers.