Effects Of 8-bromo-7-methoxy Chrysin On The Activities Of DNMT1 And Characteristics Of Live Cancer Stem Cells Derived From SMMC-7721 Cell Line

Objective: To interpret a new molecular mechanisms that chrysin(ChR)and its novel synthetic analogue 8-bromo-7-methoxy chrysin(BrMC)attenuates or eliminates the characteristics of SMMC-7721-derived liver cancer stem-like cells(LCSLCs)by inhibiting DNMT1 activity.Methods: SMMC-7721 cell line was cultured in vitro.The second generation sphere cells obtained from suspended culture with stem cell-conditioned medium were used as LCSLCs.The CD133 positive population was determined by flow cytometry(FCM).The sphere formation assay,wound-healing assay and transwell invasion assay were respectively used to determine the ability of self-renewal,migration and invasion of SMMC-7721 cell line and LCSLCs,and the mRNA expressions of stem cell markers CD133,CD44,ALDH1,ABCG2 and multifunction transcription factors Bmi1,OCT4,Sox2,Nanog were analyzed by qPCR analysis.Subsequently,DNA methyltransferase activity assay kit was used to determine the activity of DNMT(DNMT1,DNMT3 a and DNMT3b)in embryonic liver LO-2 cells,SMMC-7721 cells and LCSCLs;western blot and qPCR were analyzed DNMT1 expression of LO-2 cells,SMMC-7721 Cell and LCSCLs.Next,the effect of DNMT1 activity and characteristics of LCSLCs with ChR(40?M)treatment or BrMC(5,10 ?M)treatment for 72 h was determined above method.In addition,the effect of DNMT1 activity and characteristics of LCSLCs with 5'-aza-2'-deoxycytidine(Aza-dC)(1,2.5,5 ?M)treatment for 72 h was detected above method.Next,the effect ofDNMT1 activity and characteristics of LCSLCs with BrMC(10 ?M)or Aza-dC(5 ?M)or combination treatment for 72 h was tested above method.Finally,the effect of DNMT1 activity and characteristics of LCSLCs with ChR(40 ?M)or Aza-dC(5 ?M)or combination treatment for 72 h was examined above method.Result:1.The second generation spheres derived from SMMC-7721 cell line had higher CD133 positive population and showed stroner ability of self-renewal,migration and invasion,which over-expressed stem cell surface markers CD133,CD44,ALDH1,ABCG2 and multifunction transcription factors Bmi1,OCT4,Sox2,Nanog,as compared with SMMC-7721 cells,2.Compared with embryonic liver LO-2 cells,SMMC-7721 cells,LCSLCs DNMT1 activity was increased and DNMT1 activity,and was significantly higher than DNMT3 a and DNMT3 b.3.Compared with 1% DMSO treated LCSLCs,ChR(40.0 ?M)and different concentrations of BrMC(5.0 and 10.0 ?M)significantly inhibited DNMT1 activity and characteristics of LCSLCs in a dose-denpendent manner(P < 0.05),and the potency of BrMC was higher than the lead compound ChR.4.Compared with 1% DMSO treated LCSLCs,different concentrations of Aza-dC(2.5,5.0,and 10.0 ?M)significantly inhibited DNMT1 activity and characteristics of LCSLCs in a concentration-dependent manner(P<0.05).5.Compared with 1% DMSO treated LCSLCs,BrMC(5.0 ?M)or Aza-dC(5.0 ?M)significantly inhibited DNMT1 activity and characteristics of SMMC-7721-derived LCSLCs;and BrMC can enhance the inhibition of the DNMT1 activity and characteristics of LCSLCs by Aza-dC.6.Compared with 1% DMSO treated LCSLCs,ChR(40.0 ?M)and Aza-dC(5.0 ?M)alone inhibited DNMT1 activity and characteristics of LCSLCs.ChR enhanced the inhibition of the DNMT1 activity and characteristics of SMMC-7721-derived LCSLCs by Aza-dC.Conclusion:1.ChR and BrMC cooperatively increases the inhibitory effects of the characteristics of LCSLCs and DNMT1 activity.2.ChR and BrMC inhibit the characteristics of LCSLCs is associated with inhibition of the DNMT1 activity in SMMC-7721-derived LCSLCs.