title: [Effect of Inhibiting and Activating Wnt Signalling Pathway on NSC67657-inducing Monocytic Differentiation of HL-60 Cells].
abstract: OBJECTIVE
  To investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism.
  
  
  METHODS
  The HL-60 cells were treated with 5, 10 and 20 µmol/L XAV-939 (inhibitor of Wnt signalling pathway) for 3 days, and with 10, 20 and 30 mmol/L LiCl (activator of Wnt signalling pathway) for 1 day; the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot, respectively; the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry, moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined. Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 µmol/L for 3 days; the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control (culture mediam) group, simple NSC67657-treated group, NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed.
  
  
  RESULTS
  20 µmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively, could significantly down- and up-regulate the expression of cyclinD1, TCF1 and c-Jun genes (P < 0.05) and proteins (P < 0.05); moreover, the number of CD10(+) HL-60 cells in these conditions was below 1%, no early apoptosis of HL-60 cells was found. In the simple NSC67657-treated groups, the expression of cyclinD1, TCF1 and c-Jun proteins was down-regulated (P < 0.05), and the percentage of CD14(+) HL-60 cells accounted for 62.13 ± 9.44; after the HL-60 cells were treated with XAV-939, the NSC67657 could more significantly down-regulate the expression of cyclinD1, TCF1 and c-Jun proteins and the percentage of CD14(+) HL-60 cell accounted for 84.17 ± 5.39%, as compared with simple NSC67657-treated group; as compared with blank controls group, the expression of cyclinD1, TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14(+) HL-60 cells decreased to 33.99 ± 8.37% in NSC67657 combined LiC1 streated group, but which were higher than those in simple NSC67657-treated group (P < 0.05).
  
  
  CONCLUSIONS
  20 µmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins. The influence of XAV-939 and LiC1 on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.
authors.affiliation: Center of Laboratorial Medicine, Sun Yat-sen University Affiliated Zhongshan Hospital, Zhongshan 528403, Guangdong Province, China.
authors.forename: Wei-Jia
authors.initials: WJ
authors.lastname: Wang
authors.affiliation: Center of Laboratorial Medicine, Sun Yat-sen University Affiliated Zhongshan Hospital, Zhongshan 528403, Guangdong Province, China. E-mail: snowtouching@hotmail.com.
authors.forename: Xiu-Ming
authors.initials: XM
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Laboratorial Medical Diagnostics of Ministry of Education of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Laboratorial Medical Diagnostics of Ministry of Education of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jin-Shu
authors.initials: JS
authors.lastname: Wang
mesh_terms: D017209:Apoptosis; D002454:Cell Differentiation; D019938:Cyclin D1 / Q000378:metabolism; D005434:Flow Cytometry; D018922:HL-60 Cells; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000378:metabolism; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology*; D009000:Monocytes / Q000166:cytology*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013256:Steroids / Q000494:pharmacology*; D060449:Wnt Signaling Pathway
chemical_list: C544140:(4-(10, 13-dimethyl-3-methylsulfonyloxy-2,3,4,5,6,7,8,9,11,12,14,15,16, 17-tetradecahydro-1H-cyclopenta(a)phenanthren-17-yl)pentyl) methanesulfonate; C530104:CCND1 protein, human; C496487:HNF1A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D018950:Lipopolysaccharide Receptors; D008698:Mesylates; D016755:Proto-Oncogene Proteins c-jun; D013256:Steroids; D019938:Cyclin D1