Objectives: Defensins belong to a group of antimicrobial peptides which are divided into two classes, the a- (HNPs) and b-defensisns (hBDs). HNPs, at high concentration, are cytotoxic to various types of eukaryotic cells in vitro. The present study examined how HNPs and hBDs effect on squamous cell carcinoma (SCC) cell lines. Methods: Normal oral epithelial cells and six kinds of oral squamous cell carcinoma cell lines (SCC-9, SAS, Ca-9, HSC-4, KB, BSC-OF) were used. Cell proliferation was assessed by direct cell counting and XTT assay. The cells were cultured on 4-well plates with and without HNP-1, hBD-1, -2 (Peptide Institute, Osaka) or synthesized hBD-3 (0~50ug/ml) in DMEM without serum. After 24 and 48hrs, the cell nuclei adherent to the bottom of each well was quantified on pictures taken. For XTT assay, the cells were incubated, as same as the direct cell counting, in 96-well plates. To study the role of EGF receptor activation and the downstream MAP kinase signaling pathway, cells were incubated with 10 uM of the EGF receptor inhibitor tryphostin AG1478 (Alexis corp.) or 25 uM of the MAP kinase inhibitor U0126 (Promega, WI) for 1h, followed by incubation with HNP-1 or hBDs. Individual experiments were performed in triplicates. Results were shown as means S.D. The statistical significance of the difference was analyzed using Student t-test for paired samples. Results: The cell proliferations were stimulated with HNP-1 or hBDs in a dose dependent manner. KB cells incubated with 10 ug/ml HNP-1 or hBDs showed a 2.5-fold increase in cell number at 24h. In contrast, the cells incubated with 50 ug/ml HNP-1 or hBDs showed a decrease in cell number. The induced-cell proliferation was inhibited by MAP kinase inhibitor U0126. Conclusion: The results indicate that the defensins may stimulate SCC cells proliferation at low concentration via MAP kinase pathway.

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