Effects Of Epigallocatechin-3-gallate On Proliferation And Apoptosis Of Human Odontogenic Keratocyst Epithelial Cells

Objective The aim of the study was to investigate the effects of(-)-Epigallocatechin gallate(EGCG)on proliferation and apoptosis of odontogenic keratocyst(OKC)epithelial cells in vitro,and to explore the potential molecular mechanisms.Materials and Methods Keratinocytes isolated from the epithelial lining of odontogenic keratocyst by cold enzyme digestion,and the primary cells were cultured in keratinocyte serum-free medium.Immunofluorescence staining was used to identify epithelial cell marker wide-spectrum keratin(CK),keratin 10(CK10),keratin 14(CK14),and mesenchymal cell marker Vimentin on primary cultured cells.The cell proliferation was detected by CCK-8 assay after 24,48,and 72 h treatment with EGCG of 0,20,40,80,160 and 320 ?mol/L,respectively.Then cells were treated with 20,160,and 320 ?mol/L EGCG for 48 h,cell cycle distribution was detected by flow cytometry with PI staining and apoptosis was detected by flow cytometry with FITC-annexin V/PI staining.The expression of FZD3 and JNK3 proteins in Wnt/JNK signaling pathway was detected by quantitative real-time PCR and western blotting.Human oral keratinocytes(HOKs)were used as the control.Statistical analysis was performed using SPSS 22.0 software.Results The odontogenic keratocyst keratinocytes were successfully cultured in vitro and subcultured for 3-4 generations.The primary cells were polygonal and densely arranged in tile-like appearance.Epithelial cell biomarkers CK,CK10 and CK14 were positive,and mesenchymal markers Vimentin were negative.OKC epithelial cells and HOKs were treated with EGCG at different concentrations of 0,20,40,80,160,320 ?mol/L for 24,48,and 72 h,and the cell proliferation decreased in a time and dose-dependent manner(P < 0.05).After 48 h of EGCG treatment,the 50% inhibitory concentration(IC50)for OKC epithelial cells was 158.7 ?mol/L,while IC50 for HOKs was 282.1 ?mol/L.Compared with the control group(0 ?mol/L),medium(160 ?mol/L)and high(320 ?mol/L)concentrations of EGCG increased the proportion of sub-G0 and G0/G1 phases in OKC epithelial cells and decreased the proportion of S and G2/M cells(P < 0.05).Cell cycle was arrested at G1 phase.However,high concentration(320 ?mol/L)EGCG increased the proportion of sub-G0 and G0/G1 phases in HOKs,and decreased the proportion of S and G2/M cells(P < 0.05),resulting in significant G1 phase arrest.The apoptosis rate ofOKC epithelial cells in low,medium and high concentrations of EGCG(20,160,320 ?mol/L)increased in a dose-dependent manner(P < 0.05).While the apoptosis rate of HOKs in the high concentration group(320 ?mol/L)was significantly increased(P < 0.05).The expression of Wnt signaling pathway related proteins FZD3 and JNK3 in OKC epithelial cells was up-regulated compared with that in HOKs(P < 0.05).FZD3 and JNK3 proteins were significantly down-regulated in OKC epithelial cells at 160 and 320 ?mol/L groups(P < 0.05),while in HOKs,this reduction was detected at 320 ?mol/L group(P < 0.05).Conclusion EGCG inhibits proliferation,arrests cell cycle at G1 phase and induces apoptosis of OKC epithelial cells,and the molecular mechanism is possibly involved in the inhibition of FZD3 and JNK3 proteins in Wnt signaling pathway.In a certain range of concentrations,EGCG can selectively inhibit the proliferation of OKC epithelial cells but has no significant effect on the growth of normal cells.However,high concentrations of EGCG are cytotoxic to HOKs.