7-difluoromhoxyl-5,4’-di-n-octylgenistein On EMT And Invasion Of Gastric Cancer Stem-like Cells Derived From SGC-7901 Cell Line

Purpose:To investigate the effects that 7-difluoromhoxyl-5,4’-di-n-octylgenistein(DFOG) inhibits the stemness and reverses epithelial-mesenchymal transition(EMT) Phenotype of gastric cancer stem-like cells(GCSLCs) derived from human gastric SGC-7901 cell line and its mechanism.Methods:DFOG was synthesized using the methods from Chinese invention patent application(No.201210591131.2) and its chemical constitution was identified by 1H and 13 C NMR. Human gastric cancer SGC-7901 cell line cells were cultured in vitro. And the sphere forming cells (SFCs) were enriched and amplified using suspension culture with stem cell conditional medium. Compared with parental cells, sphere forming rate, cell migrational rate, cell invasion rate and the protein expression level of CD133, CD44 and ALDH1 were used to detect by sphere-forming assay, scratch method, cell transwell chambers cell invasion assay and western blot analysis, respectively, for the characteristics of SFCs acted as GCSLCs. And then we examined the effects of treatment with various concentrations(1.0、3.0、10.0 μmol/L) of DFOG on the characteristics of GCSLCs. The protein expression of epithelial cell marker E-cadherin and mesenchymal cell marker N-cadherin was analyzed using western blot, which results can be used to evaluate EMT phenotype and the effects that DFOG reverse EMT. The protein expression of carcinogenic transcription factor FoxMl and EMT-associated transcription factor Twistl in GCSLCs and parent cells was comparatively analyzed by detecting the effects of DFOG or transfection with FoxM1 siRNA or in combination on the protein expression of FoxM1 and Twistl and the characteristics of GCSLCs and EMT phenotype.Results:1. DFOG(500.0 mg) was synthesized and obtained using the methods from patent application and its chemical constitution was identified by NMR.2. Compared with parental cells, the SFCs from human gastric cancer SGC-7901 cell line had higher sphere-forming rate(P< 0.05), enhanced capacity of cell migration and cell invasion(P< 0.05) and up-regulated protein expression of CD133, CD44 and ALDH1(P<0.05).3. Compared with parental cells, the expression of N-cadherin was elevated(P<0.05) and the E-cadherin expression was reduced in SFCs(P <0.05).4. DFOG (1.0、3.0、10.0 μmol/mL) inhibited the capacity of sphere forming(P<0.05), cell migration and cell invasion(P<0.05) and down regulated the expression of CD 133, CD44 and ALDH1(P<0.05), in a dose-dependent manner.5. DFOG (1.0、3.0、10.0 μmol/mL) down-regulated N-cadherin expression(P<0.05) and increased the expression level of E-cadherin(P <0.05).6. The expression levels of FoxMl and Twistl in GCSLCs were higher than that of the parental cells (P<0.05).7. DFOG(1.0、3.0、10.0 μmol/mL) inhibited the expression of FoxMl and Twistl in GCSLCs(P<0.05).8. FoxMl siRNA transfection and DFOG coordinated down-regulated the expression of FoxM1 and Twistl(P<0.05).9. FoxM1 enhanced the effects that DFOG suppresses the capacity of self renewal(P< 0.05), cell migration and cell invasion(P< 0.05) and decreases the expression of CD133, CD44 and ALDH1(P< 0.05) and regulate N-cadherin and E-cadherin expression(P<0.05).Conclusion:1. DFOG was obtained using the methods from patent application and its chemical constitution was identified by NMR.2. SFCs derived from human gastric cancer SGC-7901 cell line have the characteristics of GCSLCs and EMT phenotype.3. DFOG inhibits the characteristics of GCSLCs and reverses EMT phenotype.4. The effect that DFOG inhibits the characteristics of GCSLCs is involved in the down-regulated expression level of FoxM1.5. The effect that DFOG reverses EMT phenotype is associated with inhibition of Twsitl expression.