The Mechanisms Of Clioquinol Induced-pyroptosis In Acute Myeloid Leukemia

Background and aims:Acute myeloid leukemia(AML)is a clonal disorder of bone marrow hematopoietic stem cells,and is one of the most common hematological malignancies.The treatment of AML largely depends on chemotherapy that induces AML cell apoptosis or differetiation contributed by individual agents.Pyroptosis is a novel type of cell death and it has been proposed that induction of pyroptosis might be a promising strategy for the treatment of AML.Clioquinol(CLQ),an antifungal and antiparasitic drug,has been shown to display potent anti-AML activities by inducing AML cell apoptosis and autophagy.Recently,we found that CLQ also induces AML cell pyroptosis but the underlying mechanism is unclear.Therefore,the aim of this dissertation is to undiscover and clarify the role and mechanism of CLQ in inducing pyroptosis of AML cells.It will help for understanding a novel type of cell death and it may provide a theoretical basis for the further application of CLQ-induced pyroptosis in the treatment of AML.Methods:(1)HEK293FT,HeLa,leukemia and myeloma cell lines were collected to detect the expression profile of pyroptotic related proteins,and OCI-AML2,OCI-MY5,NB4,and HEL cell lines were selected for further study.The pyroptotic levels of the CLQ treated cells were analyzed by Western blot(WB),Lactate dehydrogenase(LDH)release analysis,cell morphology photography,and PI staining.The Pan-Caspase inhibitor Z-VAD-FMK(zVAD)and the Caspase-3 inhibitor Z-DEVD-FMK(zDEVD)were used,and the effects of C aspase-3 on CLQ-induced cell pyroptosis were detected by both Western blot and LDH release assays.(2)To investigate the mechanism of CLQ-induced pyroptosis,primary blood samples from AML patients and healthy volunteers were collected,and the gene expression profiling was analyzed by cDNA microarray assay.Also,the differentially expressed genes between CLQ-treated cells and control counterparts were also measured by cDNA microarray assay.The induction role of CLQ on the protein and transcription levels of candidate genes was verified by WB,reverse transcription-PCR(RT-PCR)and quantitative real-time PCR(qRT-PCR).(3)OCI-AML2 and NB4 cells were treated with increasing concentrations of CLQ,and the protein levels of phosphorylated and total proteins of the STAT family were detected by WB.Furthermore,OCI-AML2 and NB4 cells were treated with fludarabine(FLUD),a specific STAT1 activation inhibitor,followed by the determination of the phosphorylation and the total protein levels of STAT 1,as well as the protein levels of candidate genes by WB.(4)Lentiviral over-expressing plasmids of IFIT1 and IFIT3 were constructed,and HEK293FT cells were used to generate lentiviruses,that were used to infect AML cells.AML cells with overexpressing IFIT genes were treated with CLQ to determinate the effects of IFIT genes on CLQ in the induction of AML cells pyroptosis.(5)Lentivirus was used to infect AML cells.After overexpression of IFIT genes,cell migration ability was analyzed by Transwell assay,and cell cycle related indicators were analyzed by Western blot and qRT-PCR.Results:(1)The expression profile analysis showed that GSDME and GSDMD was generally low expressed in myeloma and leukemia cells,specifically,GSDME/GSDMD is high in OCI-AML2 and OCI-MY5,low in HEL,and GSDME/GSDMD lacked in NB4.These cell lines were selected for further studies.LDH release analysis,cell image photography and PI staining analysis showed that CLQ induced AML cells pyroptosis in a concentration-dependent manner,and WB analysis demonstrated that CLQ induced the cleavage of GSDME but not GSDMD in a pytoptotic manner.Futher,WB and LDH release analysis showed that the effects of cell pyroptosis levels induced by CLQ were significantly decreased after treatment of zVAD-FMK and zDEVD-FMK,indicating that CLQ induced cell pyroptosis through the Caspase-3/GSDME axis.(2)Through cDNA microarray analysis,we found that IFIT1,IFIT3,IFI27 and IFI44L were low expressed in primary AML cells,but they were significantly induced by CLQ.Western blot,RT-PCR and qRT-PCR analyses further confirmed the induction effect of CLQ on these IFN inducible genes.(3)Through the detection of potential upstream proteins of the selected ISGs,WB showed that CLQ could significantly induce the expression of STAT1 and phosphoSTAT1(Tyr701),but had no significant effect on other STAT family proteins.WB results showed that STAT1 and its phosphorylated protein played an important role in the expression of IFIT1 and IFIT3 induced by CLQ.(4)WB and LDH release analysis showed that overexpression of IFIT1 and IFIT3 facilitate the role of CLQ to induce cell pyroptosis,and overexpressed IFIT1 and IFIT3 by lentivirus infection significantly reduced the pyroptotic levels induced by CLQ,indicating that IFIT1 and IFIT3 might play a critical role in CLQ-induced pyroptosis.(5)The Transwell assay showed that IFIT1 and IFIT3 suppressed cell migration of AML cells.Also,the IB analysis showed that cell cycle related protein phospho-Rb was downregulated and p21 was upregulated after overexpression of IFIT1 and IFIT3.Meanwhile,qRT-PCR analysis showed that exogenous IFIT1 and IFIT3 increase p21 transcription,indicating that IFIT1 and IFIT3 inhibited cell cycle progression.Conclusion:CLQ significantly induces Caspase-3/GSDME-dependent AML cell pyroptosis and the mechanistic analyses revealed that this effect may be achieved by the induction of the transcription levels of interferon inducible genes IFIT1,IFIT3,IFI27 and IFI44L.These ISGs were lowly expressed in primary AML cells and they are markedly induced by CLQ treatment.CLQ induces pyroptosis of AML cells in a concentration-dependent manner,and CLQ-induced AML cell pyroptosis was partly enhanced by the overexpression of IFIT1 or IFIT3.Furthermore,overexpressed IFIT1 and IFIT3 inhibit the cell cycle progression and suppress the cell migration ability of AML cells.