Toshiro Kibe

Wnt‐5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP‐2

Wnts are secreted ligands that consist of 19 members in humans, regulate cell proliferation, differentiation, motility and fate in many stages including the embryonic stage and tumorigenesis. Wnts bind to cell surface receptors named Frizzleds and LRPs, and transduce their signals through β‐catenin‐dependent and ‐independent intracellular pathways. Gliomas are one of the most common intracranial tumors. Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. However, the molecular mechanisms that stimulate the invasion of glioma cells are not fully understood. We established two cell lines from human glioma cases and analyzed the expression of all Wnt and Frizzled members in these cell lines and other well‐known glioma cell lines by real‐time PCR study. The mRNA of Wnt‐5a and ‐7b and Frizzled‐2, ‐6 and ‐7 were overexpressed in glioma cells. The elevation of Wnt‐5a expression was most remarkable. Although Wnt‐5a is reported to have oncogenic and antioncogenic activity in several cancers, the role of Wnt‐5a signaling in human glioma cells remains unclear. Immunohistochemical study also revealed high expression of Wnt‐5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt‐5a expression was correlated with the clinical grade. Knockdown of Wnt‐5a expression suppressed migration, invasion and expression of matrix metalloproteinase‐2 of glioma cells. Reciprocally, treatment with purified Wnt‐5a ligand resulted in stimulation of cell migration and invasion. MMP‐2 inhibitor suppressed the Wnt‐5a‐dependent invasion of U251 cells. These results suggested that Wnt‐5a is not only a prognostic factor but also a therapeutic target molecule in gliomas for preventing tumor cell infiltration. (Cancer Sci 2011; 102: 540–548)

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.

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth.

Afatinib radiosensitizes head and neck squamous cell carcinoma cells by targeting cancer stem cells

The dismal prognosis of locally advanced and metastatic squamous cell carcinoma of the head and neck (HNSCC) is primarily due to the development of resistance to chemoradiation therapy (CRT). Deregulation of Epidermal Growth Factor Receptor (EGFR) signaling is involved in HNSCC pathogenesis by regulating cell survival, cancer stem cells (CSCs), and resistance to CRT. Here we investigated the radiosensitizing activity of the pan-EGFR inhibitor afatinib in HNSCC in vitro and in vivo. Our results showed strong antiproliferative effects of afatinib in HNSCC SCC1 and SCC10B cells, compared to immortalized normal oral epithelial cells MOE1a and MOE1b. Comparative analysis revealed stronger antitumor effects with afatinib than observed with erlotinib. Furthermore, afatinib enhanced in vitro radiosensitivity of SCC1 and SCC10B cells by inducing mesenchymal to epithelial transition, G1 cell cycle arrest, and the attenuating ionizing radiation (IR)-induced activation of DNA double strand break repair (DSB) ATM/ATR/CHK2/BRCA1 pathway. Our studies also revealed the effect of afatinib on tumor sphere- and colony-forming capabilities of cancer stem cells (CSCs), and decreased IR-induced CSC population in SCC1 and SCC10B cells. Furthermore, we observed that a combination of afatinib with IR significantly reduced SCC1 xenograft tumors (median weight of 168.25 ± 20.85 mg; p = 0.05) compared to afatinib (280.07 ± 20.54 mg) or IR alone (324.91 ± 28.08 mg). Immunohistochemical analysis of SCC1 tumor xenografts demonstrated downregulation of the expression of IR-induced pEGFR1, ALDH1 and upregulation of phosphorylated γH2AX by afatinib. Overall, afatinib reduces tumorigenicity and radiosensitizes HNSCC cells. It holds promise for future clinical development as a novel radiosensitizer by improving CSC eradication.

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.

Immortalization and characterization of normal oral epithelial cells without using HPV and SV40 genes

a b s t r a c t Background: As oral neoplasm often originates from epithelium, an immortalized epithelial cell line could be useful for the research of oral carcinogenesis. Although several oral epithelial cell lines were reported, they were either derived from cancer or immortalized by human papilloma virus or simian virus 40 genes, which have the potential to induce carcinogenesis. Materials and methods: We established two immortalized cell lines from human oral epithelium by transducing mutant cyclin dependent kinase 4, cyclin D1, and human telomerase reverse transcrip- tase with or without dominant-negative p53 into primary-cultured normal oral gingival epithelial cells using recombinant lentivirus vectors and named them MOE (mouth-ordinary-epithelium) 1a and MOE1b, respectively. Results: MOE1 cells could be passaged for nine months or more, and the morphology of the cells did not change in comparison with that of fresh primary-cultured epithelial cells. MOE1 cells did not show epithelial-mesenchymal transition. MOE1b cells retain functional p53 and were considered to have less risk of genomic instabilities. Anchorage-independent growth was not observed in MOE1 cells. The expres- sions of cancer-associated genes including keratin-17 were not elevated in MOE1 cells, whereas oral cancer-derived HSC-2 cells showed overexpression of them. Furthermore, interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor-, matrix metalloproteinase (MMP)-2, and MMP-9 were induced in response to lipopolysaccharide or heat-killed bacterium in MOE1 cells. Discussion: MOE1 cells kept the characteristics of normal epithelial cells without acquiring typical features of cancer cells and they could be useful not only for the study of oral neoplasm but also for other oral

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.

Evaluation of maxillary central incisors on the noncleft and cleft sides in patients with unilateral cleft lip and palate—Part 1: Relationship between root length and orthodontic tooth movement

OBJECTIVES To measure the root lengths of maxillary central incisors (U1) and evaluate the relationship among U1 root length, tooth movement, and type of treatment appliance in patients with unilateral cleft lip and palate over a long-term follow-up period. MATERIALS AND METHODS Occlusal radiographs of 30 patients with unilateral cleft lip and palate, acquired less than 6 months before secondary alveolar bone grafting (SBG, T1) and after edgewise treatment (T2), were measured for U1 root length (R1 and R2, root lengths at T1 and T2, respectively). Frontal and lateral cephalometric radiographs acquired at eruption of U1 (T0), T1, and T2 were evaluated to determine the inclination and position of U1. RESULTS The average values of R1 and R2 on the cleft side were significantly lower than those on the noncleft side. Frontal cephalometric analysis revealed that the horizontal distance of the root apex from the median vertical line at T0 on the cleft side was significantly smaller than that on the noncleft side and was correlated with short U1 root length on the cleft side. On the other hand, R1 in patients treated with maxillary protraction appliances between T0 and T1 was significantly shorter than that in patients without maxillary protraction appliances. However, none of the changes in cephalometric measurements were correlated with root length. CONCLUSIONS In patients with unilateral cleft lip and palate, the short root length of cleft-adjacent central incisors might be associated with the horizontal position of the root apex. In addition, orthodontic treatment with a maxillary protraction appliance before secondary alveolar bone grafting might be associated with short U1 root length.

Cephalometric Analysis of the Velopharyngeal Muscular Triangle as a Possible Prognostic Factor for Velopharyngeal Closure in Submucous Cleft Palate

Objective: To elucidate the anatomical characteristics of submucous cleft palate (SMCP), we analyzed the velopharyngeal (VP) structures focusing on the relationship between the position of posterior pharyngeal wall (PPW) in the VP muscles and VP closure acquisition in SMCP patients. Methods: Cranial landmarks for cephalomatric analysis were identified in a study of two cadavers, and the area of the velopharyngeal muscular triangle (VPM-triangle), which was bordered by the origin of the levator veli palatini muscle, the origin of the palatopharyngeal muscle, and the insertion of both muscles, was defined. We then cephalometrically measured the VP structures of 14 SMCP patients (SMCP group) and the position of the PPW within the VPM-triangle. As a comparison group, 20 healthy Japanese children (control group) and 20 patients who underwent palatal repair for cleft palate (postoperative CP group) were enrolled. In addition, we analyzed the correlation between VP closure and position of the PPW within the VPM-triangle in the SMCP group. Results: The SMCP group exhibited shorter velar length, greater pharyngeal depth and greater height. In the control and postoperative CP groups, the part of the PPW within the VPM-triangle was located near to the motion vector of the levator veli palatine muscle, while it was located significantly more posteriorly in the SMCP group. The PPW of the poor VP closure subgroup of the SMCP group tended to locate more posteriorly than those of the favorable VP closure subgroup and the control group. Conclusions: The VP forms of the SMCP group were characterized by a shorter velum, a deeper and higher pharynx, and a more posterior PPW than the motion vector of palatal muscles. A positional discrepancy of the velopharynx including the PPW position relating to the direction of the motion of the VP muscles may influence the difficulty of VP closure achievement in SMCP patients.