Inhibitory effects of ameloblastin on epithelial cell proliferation

• Published in: Archives of oral biology 2014-08, Vol.59 (8), p.835-840
• Main Authors: Saito, Noriko, Ariyoshi, Wataru, Okinaga, Toshinori, Kamegawa, Mariko, Matsukizono, Miho, Akebiyama, Yasuo, Kitamura, Chiaki, Nishihara, Tatsuji
• Format: Article
• Language: English
• Subjects: Advanced Basic Science; Ameloblastin; Animals; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell - drug therapy; Cell cycle; Cell Proliferation - drug effects; Cell Survival - drug effects; Cells, Cultured; Chromatography, High Pressure Liquid; Dental Enamel Proteins - pharmacology; Dentistry; Epithelial cell proliferation; Epithelial Cells - metabolism; Flow Cytometry; Humans; In Vitro Techniques; Mice; Mice, Inbred BALB C; Mouth Neoplasms - drug therapy; Periodontitis; Protein purification; Recombinant Proteins - pharmacology
• ISSN: 0003-9969; 1879-1506
• DOI: 10.1016/j.archoralbio.2014.05.010

Abstract Objective Ameloblastin is an enamel matrix protein expressed in several tissues. Many potential mechanisms have been identified by which ameloblastin functions as an extracellular matrix protein. However, the biological effects of ameloblastin on gingival epithelial cells remain unclear. In the present study, we established a novel system to purify recombinant human ameloblastin and clarified its biological functions in epithelial cells in vitro. Design Recombinant human ameloblastin was isolated from COS-7 cells overexpressing HaloTag® -fused human ameloblastin by the HaloTag® system and then purified further by reverse-phase high-performance liquid chromatography. SCC-25 cells, derived from human oral squamous cell carcinoma, were treated with recombinant ameloblastin and then cell survival was assessed by a WST-1 assay. Cell cycle analysis was performed by flow cytometry. Results The novel purification system allowed effective recovery of the recombinant ameloblastin proteins at a high purity. Recombinant ameloblastin protein was found to suppress the proliferation of SCC-25 cells. Flow cytometric analysis showed that ameloblastin treatment induced cell cycle arrest G1 phase. Conclusions We developed a procedure for production of highly purified recombinant human ameloblastin. Biological analyses suggest that ameloblastin induces cell cycle arrest in epithelial cells and regulates the progression of periodontitis.