title: [In vitro study of BRD4 inhibitor GSK525762A against primary adult common B-cell acute lymphoblastic leukemia cells in vitro].
abstract: OBJECTIVE
  To investigate the effects of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of primary common B-cell acute lymphoblastic leukemia (common B-ALL) cells from adult patients, then to further explore the possible mechanisms.
  
  
  METHODS
  Purified leukemia cells from 14 common B-ALL adult patients (4 Ph⁺ and 10 Ph⁻ cases) were obtained by flow cytometry sorting, and maintained in a mimic bone marrow microenvironment culture system for short-term culture. Leukemia cells were treated with various concentrations of GSK525762A. The inhibitory effects of BRD4 inhibitor on common B-ALL leukemia cells were measured by CCK-8 assay and the apoptosis of those cells was determined by AnnexinⅤ/7-AAD staining using flow cytometry. The transcripts of c-MYC, CDK6 and Bcl-2 were detected by quantitative RT-PCR, and the expression of c-MYC, CDK6 and Bcl-2 proteins were detected via Western blot.
  
  
  RESULTS
  GSK525762A could inhibit the proliferation of leukemia cells from all 14 common B-ALL patients in a dose-dependent manner, the median value of IC50 was 256.25 (90.64-1 378.39)nmol/L. GSK525762A could promote cells apoptosis of B-ALL leukemia cells in a dose-dependent manner, the median apoptosis rates respectively were 45.17%(9.38%-70.91%), 66.02% (24.36%-96.34%) and 89.29% (39.29%-99.37%) after treated by 500, 1 000 and 2 500 nmol/L GSK525762A. GSK525762A has a similar effect on Ph⁺ ALL and Ph⁻ B-ALL, but the effect of proliferation inhibition and apoptosis enhancement on Ph+ B-ALL is weaker than that on Ph⁻ B-ALL. Compared with vehicle control group, the levels of c-MYC, Bcl-2 and CDK6 transcripts in leukemic cells were reduced after treatment for 24 h and 48 h by 1 000 nmol/L GSK525762A, and there are no significant differences in the downregulation of c-MYC and CDK6 mRNA between Ph⁺ and Ph⁻ B-ALL; however, the inhibitory effect on Bcl-2 transcription was weaker in Ph⁺ B-ALL cells than that in Ph⁻ B-ALL cells. Moreover, c-MYC, Bcl-2 and CDK6 protein levels decreased in GSK525762A treated group.
  
  
  CONCLUSIONS
  GSK525762A could strongly inhibit the proliferation of common B-ALL and trigger apoptosis; meanwhile it has certain effects against Ph⁺ ALL in vitro. The effect may be achieved by down-regulation of c-MYC, CDK6 and Bcl-2 expression.
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Sha
authors: S
authors: Ma
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Chong
authors: C
authors: Chen
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Junfeng
authors: J
authors: Zhu
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Yuping
authors: Y
authors: Li
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Xue
authors: X
authors: Wang
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Xuguang
authors: X
authors: Song
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Jiang
authors: J
authors: Cao
authors: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors: Kailin
authors: K
authors: Xu
mesh_terms: D017209:Apoptosis; D001569:Benzodiazepines / Q000494:pharmacology*; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor / Q000187:drug effects; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism; D015536:Down-Regulation; D005434:Flow Cytometry; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D000092468:Bromodomain Containing Proteins
chemical_list: D001569:Benzodiazepines; D018797:Cell Cycle Proteins; D051361:Cyclin-Dependent Kinase 6; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; C000595707:BCL2 protein, human; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C554645:molibresib; C495911:CDK6 protein, human