| Background and Objectives:Acute myeloid leukemia(AML)is a malignant bone marrow disease characterized by clonal expansion and inhibition of differentiation of myeloid progenitor cells.The standard treatment for AML is chemotherapy and allogeneic stem cell transplantation.Targeted therapy has the advantages of accurate treatment,less toxicity,good compliance and so on.The targeted development of AML field is slow,and there are few therapeutic drugs.The bromine domain protein BRD4 has been shown to be critical for the development of AML.Some drugs targeting BRD4 have entered clinical trials,and the drug properties of BRD4 targets have been initially verified,showing good therapeutic prospects in hematological cancers.Literature studies have shown that the natural product Isoliquiritigenin(ISL)can bind to BRD4 and is a potential inhibitor of BRD4.This study attempted to verify that BRD4 is a new target of isoliquiritigenin against AML in vivo and in vitro,and to explore the mechanism of isoliquiritigenin regulating BRD4,in order to provide a new idea for the anti-tumor of traditional Chinese medicine.Methods:This study was conducted at the cellular level and the animal level respectively.At the cellular level,the effect of isoliquiritigenin on AML cell viability was detected by MTS assay.The changes of nuclear morphology were observed by Gimsa staining.The changes of cell cycle,apoptosis and markers of cell differentiation were detected by flow cytometry.At the animal level,the subcutaneous xenograft tumor model of AML nude mice was established to detect the effect of isoliquiritigenin on the growth of subcutaneous xenograft tumor,and the changes of BRD4-related proteins in tumor tissues were detected by Western Blot and immunohistochemistry.In terms of mechanism study,Ubiquitin proteasome pathway inhibitors and lysosomal autophagy pathway inhibitors were used to study the degradation pathway of BRD4 protein induced by isoliquiritigenin.The interaction between BRD4 and ubiquitin was further detected by Co-immunoprecipitation.Exogenous BRD4 overexpression system was constructed,and the changes of apoptosis induced by isoliquiritigenin were detected by flow cytometry.Results:AML cell lines treated with isoliquiritigenin,cell viability was inhibited and apoptosis rate increased;The cytoplasmic ratio decreased,the expression of cell dryness related markers decreased,and the expression of cell differentiation related markers increased.Ubiquitin and proteasome related inhibitors can effectively reverse the degradation of BRD4 protein mediated by isoliquiritigenin.The results of immunoprecipitation showed that isoliquiritigenin could mediate the ubiquitination of BRD4 protein.After overexpression of BRD4,the apoptosis induced by isoliquiritigenin was significantly reduced.Isoliquiritigenin can effectively inhibit the growth of subcutaneous transplanted AML tumors,and reduce the expression of BRD4 protein and downstream c-Myc protein in tumor tissues.Conclusions:Isoliquiritigenin can effectively inhibit cell proliferation,induce cell apoptosis and cell differentiation in AML cell line.Isoliquiritigenin can effectively inhibit the growth of subcutaneous AML graft tumor.Overexpression of BRD4 can reduce the effect of isoliquiritigenin on apoptosis,suggesting that isoliquiritigenin may induce apoptosis by targeting BRD4.At the same time,isoliquiritigenin mediated BRD4 degradation through the ubiquitin-proteasome pathway.In conclusion,it is suggested that isoliquiritigenin may have the function of "molecular glue".Highlights and innovations of this study:1.This study proves that BRD4 is a new target of isoliquiritigenin in the effect of AML.2.Isoliquiritigenin was found to mediate the ubiquitination of BRD4 protein for the first time,and was degraded by the ubiquitin-proteasome pathway,suggesting that isoliquiritigenin is a depressant of BRD4 protein.3.A new idea of "molecular glue" was put forward for traditional Chinese medicine anti-tumor. |
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