Intercellular signaling between ameloblastoma and osteoblasts

Abstract

Ameloblastoma is an odontogenic tumor located in the bone jaw with clinical characteristics of extensive bone resorption. It is a locally invasive tumor with a high recurrence rate despite adequate surgical removal. In bone disease, tumors and other cells including osteoblasts, osteoclasts, and osteocytes in the bone microenvironment contribute to the pathogenesis of tumor growth. However, the effect of osteoblasts on ameloblastoma cells is not well-understood, and there has been limited research on interactions between them. This study investigated interactions between ameloblastoma cells and osteoblasts using a human ameloblastoma cell line (AM-3 ameloblastoma cells) and a murine pre-osteoblast cell line (MC3T3-E1 cells). We treated each cell type with the conditioned medium by the other cell type. We analyzed the effect on cytokine production by MC3T3-E1 cells and the production of MMPs by AM-3 cells. Treatment with AM-3-conditioned medium induced inflammatory cytokine production of IL-6, MCP-1, and RANTES from MC3T3-E1 cells. The use of an IL-1 receptor antagonist suppressed the production of these inflammatory cytokines by MC3T3-E1 cells stimulated with AM-3-conditioned medium. The MC3T3-E1-conditioned medium triggered the expression of MMP-2 from AM-3 cells. Furthermore, we have shown that the proliferation and migration activity of AM-3 cells were accelerated by MC3T3-E1 conditioned media. In conclusion, these intercellular signalings between ameloblastoma cells and osteoblasts may play multiple roles in the pathogenesis of ameloblastoma.

Keywords: AM-3; Ameloblastoma; Cytokine; Intercellular communication; Osteoblast; Tumor-bone microenvironment.

Fig. 1

Productions of IL-6, MCP-1, and RANTES by MC3T3-E1 cells were stimulated by AM-3 CM. After MC3T3-E1 cells were treated with D-KSFM (as a control (−)), AM-3 CM, and AM-3 CM in the presence of IL-1Ra (100 ng/mL) for 24 or 72 h, concentrations of IL-6, MCP-1, and RANTES protein in CM were quantified by the Q-Plex™ ELISA kit. Results are means ± SE from three independent experiments. Asterisks (*) indicate statistically significant results, p < 0.05.

Fig. 2

Secretion of MMP-2 from AM-3 cells stimulated by MC3T3-E1 CM. (A) The AM-3 cell-derived production of MMP-2 protein in CM was quantified by the Q-Plex™ ELISA kit. Results are means ± SE from three independent experiments. An asterisk (*) indicates statistically significant results, p < 0.05. (B) The protein expression of MMP-2 in CM of AM-3 ameloblastoma cells treated without (−) or with (+) MC3T3-E1 CM is shown by Western blot. AM-3 only, CM of AM-3 cells cultured with D-KSFM for 72 h. Control, a mixture of D-KSFM (50% (v/v)) and MC3T3-E1 CM (50% (v/v)) incubated at 37 °C for 72 h.

Fig. 3

The proliferations of AM-3 cells were stimulated by MC3T3-E1 CM. (A) Representative microscopic images. AM-3 cells were seeded and cultured in the presence of α-MEM (50% (v/v)) as a control (a, c, e) or in the presence of MC3T3-E1 CM (50% (v/v)) (b, d, f). (a and b), 1-day cultured. (c and d), 3-day cultured. (e and f), 5-day cultured. Scale bar, 100 μm. (B) Cell number growth data. Results are means ± SE from three independent experiments. An asterisk (*) indicates statistically significant results, p < 0.05. Closed circles (●) show AM-3 cells treated with MC3T3-E1 CM. Open circles (○) show AM-3 cells treated with α-MEM/FBS (as a control).

Fig. 4

Cellular motility of AM-3 cells is accelerated by MC3T3-E1 CM. (A) Representative fluorescent images. AM-3 cells migrated for 1 h (a and b) or 2 h (c and d). (a and c), AM-3 cells were seeded and cultured in the presence of α-MEM (50% (v/v)) as a control. (b and d), AM-3 cells were seeded and cultured in the presence of MC3T3-E1 CM (50% (v/v)). Scale bar, 100 μm. (B) Relative migrated cell number data. Results are means ± SE from three independent experiments. Asterisks (*) indicate statistically significant results, p < 0.05. Closed circles (●) show AM-3 cells treated with MC3T3-E1 CM. Open circles (○) show AM-3 cells treated with α-MEM/FBS (as a control).