Takao Fuchigami

Ryk is essential for Wnt-5a-dependent invasiveness in human glioma

Glioblastoma is characterized by marked invasiveness, but little is known about the mechanism of invasion in glioblastoma cells. Wnts are secreted ligands that regulate cell proliferation, differentiation, motility and fate at various developmental stages. In adults, misregulation of the Wnt pathway is associated with several diseases. Recently, we reported that Wnt-5a was overexpressed and correlated with cell motility and infiltrative activity through the regulation of matrix metalloproteinase (MMP)-2 in glioma-derived cells. Although several receptors for Wnt-5a were identified, the receptors of Wnt-5a that mediate cellular responses of glioma were not clearly identified. Knockdown of receptor-like tyrosine kinase (Ryk) but not that of Ror2 suppressed the activity of MMP-2 and Wnt-5a-dependent invasive activity in glioma cells. These results suggest that Ryk is important for the Wnt-5a-dependent induction of MMP-2 and invasive activity in glioma-derived cells and that Ryk might have a novel patho-physiological function in adult cancer invasion. Furthermore, not only the expression of Wnt-5a but also that of Frizzled (Fz)-2 and Ryk was correlated with the WHO histological grade in 38 human glioma tissues. Taking these findings together, Fz-2 and Ryk could be therapeutic or pharmacological target molecules for the control of Wnt-5a-dependent invasion of human glioma in the near future.

A novel ameloblastoma cell line (AM-3) secretes MMP-9 in response to Wnt-3a and induces osteoclastogenesis

OBJECTIVE Ameloblastoma has a high risk of bone invasion and local recurrence. However, the mechanisms of bone invasion in ameloblastoma remain unclear. In this study, we established an experimental model for matrix metalloproteinase (MMP) induction and osteoclastogenesis using ameloblastoma-derived cells. STUDY DESIGN We established an ameloblastoma-derived cell line without viral genes and analyzed the expression of all Wnt and Frizzled members and MMPs by real-time reverse transcription-polymerase chain reaction, and analyzed the activity of MMP-2 and MMP-9 by the in-gel-gelatinase assay. RESULTS AM-3, newly established ameloblastoma-derived cells retained the morphology of primary-cultured ameloblastoma cells. AM-3 cells overexpressed the messenger RNA of Wnt-5a, Frizzled-2, MMP-2, and MMP-9 and showed the potential of osteoclastogenesis. In addition, Wnt-3a-treatment induced expression and activation of MMP-9 in AM-3 cells. CONCLUSIONS Our study suggests that AM-3 cells retained the characteristics of ameloblastoma, without acquiring typical features of cancer cells. Furthermore, Wnt signaling induced MMP-9 in ameloblastoma cells.

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave interactively via these cytokines to create a microenvironment that leads to the extension of ameloblastomas.

Short-Term Molding Effects on the Upper Alveolar Arch Following Unilateral Cleft Lip Repair With/Without Nasal Vestibular Expansion

Objective To elucidate the various effects on maxillary growth following different procedures for vestibular expansion at the time of primary lip repair for unilateral cleft lip and palate (UCLP). Participants Thirty patients with complete UCLP who underwent primary lip repair using a triangular-flap technique with nasal vestibular expansion (NVE; the NVE group) and 30 patients who underwent the same lip repair with closure of the nasal floor (non-NVE group) were enrolled in this study. Interventions Serial dental casts on lip and palatal repair were scanned with a laser scanner. The three-dimensional coordinates of seven anatomical landmarks and their growth changes, the curvature radius rate between major/minor segments, and the collapse rates were compared between the two groups. Results At the time of lip repair, the incisal point was located slightly anteriorly in the non-NVE group. At the time of palatal repair, the cleft edge of the alveolar process in the minor segment was located significantly anteriorly and laterally in the NVE group, showing the significantly forward change of the minor segment. The minor segment collapsed in the non-NVE group. The collapse rate of the NVE group (3.3%) was significantly lower than that of the non-NVE group (40.0%). Conclusions NVE following simultaneous advancement of nasolabial components on the affected side at the time of primary lip repair for UCLP facilitates the forward molding of the maxilla, resulting in a more symmetrical alveolar arch form.

Examination of the early wound healing process under different wound dressing conditions

OBJECTIVE Various types of wound-healing dressings have been used to assist in the healing of surgical wounds. We analyzed the wound-healing process in an animal model using different existing wound dressings. STUDY DESIGN Full-thickness defects were created using a biopsy punch on the backs of 7-week-old rats. The wounded areas were covered with NEOVEIL (polyglycolic acid [PGA]) or TERUDERMIS (collagen sponge [CS]) affixed using a rat jacket. The wound area, neo-epithelium length, and α-smooth muscle actin (α-SMA) expression were evaluated and compared among the control, PGA, and CS groups. RESULTS The wound areas in the control group on days 4 and 7 were significantly smaller than those in the PGA and CS groups. The expression of α-SMA in granulation tissue peaked on day 4 for all groups. The expression of α-SMA in the control group on days 4 and 7 after injury was greater than in the PGA and CS groups. However, there was no significant difference in the expression of α-SMA between the PGA and CS groups. CONCLUSIONS In this study, PGA and CS suppressed wound contracture and reduced expression of α-SMA in wound areas. However, PGA and CS did not affect the neo-epithelium length at the wound site.

Fibroblasts promote the collective invasion of ameloblastoma tumor cells in a 3D coculture model

Ameloblastoma is a benign tumor of the odontogenic epithelium with several histological subtypes. All subtypes of ameloblastoma contain abundant stroma; the tumor cells invade collectively into the surrounding tissues without losing intratumor cell attachments. However, the molecular mechanisms mediating ameloblastoma invasion remain unclear. Here, we evaluated the functional significance of the interactions between ameloblastoma tumor cells and stromal fibroblasts on collective cellular invasion using a three‐dimensional cultivation method, double‐layered collagen gel hemisphere (DL‐CGH) culture. The AM‐1 plexiform and AM‐3 follicular human ameloblastoma cell lines and HFF‐2 human fibroblasts were labeled with GFP and DsRed, respectively. Collective cellular invasion of ameloblastoma cells was assessed in the presence or absence of fibroblasts. Notably, without fibroblasts, AM‐1 cells formed sharp, plexiform‐like invasive processes, whereas AM‐3 cells formed a series of blunt processes often observed during collective migration. In comparison, under the cocultures with HFF‐2 fibroblasts, AM‐3 cells formed tuft‐like invasive processes and collectively invaded into outer layer more than that observed with AM‐1 cells. Moreover, HFF‐2 fibroblasts localized to the tips of the invasive tumor processes. These findings suggest that tumor‐associated cells assist tumor cell invasion. Microscopic analysis of sectioned three‐dimensional cultures revealed that AM‐3/HFF‐2 hemispheres were histologically similar to follicular ameloblastoma tumor samples. Therefore, our findings suggest that ameloblastoma subtypes exhibit distinct invasion patterns and that fibroblasts promote collective tumor invasion in follicular ameloblastoma.