Kazuo Ryoke

Cancer‐associated fibroblasts and CD163‐positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance

BACKGROUND Stromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer-associated fibroblasts (CAFs) and the incidence of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion. METHODS The study included 108 patients with OSCC. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated. RESULTS The cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low- and high-grade groups on the basis of the number of CD68-positive and CD163-positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan-Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163-positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163-positive macrophage level was a significant and an independent prognostic factor (P = 0.045). CONCLUSIONS Cancer-associated fibroblasts and CD163-positive macrophages may be potential prognostic predictors of OSCC.

High expression of EZH2 is associated with tumor proliferation and prognosis in human oral squamous cell carcinomas

Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes and is important in cell cycle regulation. Overexpression of EZH2 protein has been associated with biological malignancy of prostate cancer and several other cancers. The aim of the present study was to evaluate the expression of EZH2 protein in human oral normal mucosa, dysplasia and oral squamous cell carcinoma (OSCC) with clinicopathological profiles. EZH2 expression was assessed by Western blotting and immunohistochemistry in 3 OSCC cell lines, 10 normal mucosae, 50 dysplasias and 102 OSCCs. The labeling indices (LIs) of EZH2, Ki-67, P53, and the apoptotic index (AI) were evaluated by immunohistochemistry and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method. Western blot analysis detected EZH2 protein as a single band at 91kDa in the 3 OSCC cell lines, but it was almost absent in non-tumoral oral mucosae. The LI of EZH2 was highest in the OSCCs, followed by the dysplasias (p<0.05) and normal mucosae (p<0.05) with significant difference. The LI of EZH2 correlated with the clinical stage, tumor size, lymph node metastasis and LIs of Ki-67 and P53, but not with the AI in OSCCs, and inversely correlated with the histological differentiation of OSCCs. The survival rate calculated by the Kaplan-Meier method revealed that OSCC patients with higher EZH2 expression showed poorer prognosis than those with a lower EZH2 expression (p<0.01). These results suggest that overexpression of EZH2 is correlated with malignant potential and poor prognosis in OSCCs. EZH2 might serve as a novel biomarker for predicting prognosis in patients with OSCCs.

Usefulness as guided bone regeneration membrane of the alginate membrane

Alginate membrane is a new bioabsorbable, guided bone regeneration (GBR) membrane, which is placed directly on the surface of the bone defect. It is designed to drop a calcium chloride aqueous solution into the bone defect, which is filled with sodium alginate aqueous solution. Alginate membrane is an excellent agent for this procedure due to its close assimilation to the surface of the bone. In this study, we evaluated the short-term biocompatibility of alginate membrane in the bone defects of rat tibiae. GBR membrane availability was also examined. Consequently, we found that the healing process in bone defects covered with an alginate membrane was delayed in comparison with that of controls, however, the defect was restored to nearly original condition. In contrast, in the controls, bone defect repairs exhibited partitioning as a result of connective tissue involvement. Furthermore, we observed a relation between the sodium alginate concentration and the rate of absorption of the sodium alginate membrane. Absorption of a 1.5% sodium alginate membrane was slow. As a result, the compound was not absorbed completely and bone repairs resembled an hourglass. Moreover, the inflammatory response was absent surrounding the alginate membrane. The present findings suggested that the alginate membrane functions effectively as a GBR membrane. In addition, the alginate membrane derived from 3% calcium chloride and 1% sodium alginate was most suitable as a GBR membrane.

Involvement of microRNAs in regulation of osteoblastic differentiation in mouse induced pluripotent stem cells.

Backgoround MicroRNAs (miRNAs), which regulate biological processes by annealing to the 3′-untranslated region (3′-UTR) of mRNAs to reduce protein synthesis, have been the subject of recent attention as a key regulatory factor in cell differentiation. The effects of some miRNAs during osteoblastic differentiation have been investigated in mesenchymal stem cells, however they still remains to be determined in pluripotent stem cells. Methodology/Principal Findings Bone morphogenic proteins (BMPs) are potent activators of osteoblastic differentiation. In the present study, we profiled miRNAs during osteoblastic differentiation of mouse induced pluripotent stem (iPS) cells by BMP-4, in which expression of important osteoblastic markers such as Rux2, osterix, osteopontin, osteocalcin, PTHR1 and RANKL were significantly increased. A miRNA array analysis revealed that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a were significantly downregulated. Interestingly, miR-124a and miR-181a directly target the transcription factors Dlx5 and Msx2, both of which were increased by about 80-and 30-fold, respectively. In addition, transfection of miR-124a and miR-181a into mouse osteo-progenitor MC3T3-E1 cells significantly reduced expression of Dlx5, Runx2, osteocalcin and ALP, and Msx2 and osteocalcin, respectively. Finally, transfection of the anti-miRNAs of these six miRNAs, which are predicted to target Dlx5 and Msx2, into mouse iPS cells resulted in a significant increase in several osteoblastic differentiation markers such as Rux2, Msx2 and osteopontin. Conclusions/Significance In the present study, we demonstrate that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a miRNAs, especially miR-124a and miR-181a, are important regulatory factors in osteoblastic differentiation of mouse iPS cells.

Expression of Minichromosome Maintenance 2 (MCM2), Ki-67, and Cell-Cycle-Related Molecules, and Apoptosis in the Normal-Dysplasia-Carcinoma Sequence of the Oral Mucosa

Objective: We examined cell cycle and cell death biomarker trends with the normal-dysplasia-carcinoma sequence of the oral epithelia analyzing the pathological significance of a new biomarker, minichromosome maintenance 2 (MCM2). Methods: This study analyzed 12 patients with normal oral epithelia, 69 with dysplasia, and 35 with squamous cell carcinoma (SCC); in 13 patients, SCCs were preceded by dysplasia. The sections were immunostained for MCM2, Ki-67, P53, P27Kip1 and P21CIP1/WAF1, and conducted by TUNEL methods. Western blot analysis of MCM2 was performed in the 4 human cultured oral SCCs, all of which showed the expression. Results: Significantly higher labeling indices (LI; %) of MCM2, Ki-67, and P53, as well as lower LI of TUNEL indices (TI; %), P27, and P21 were noted in the SCCs than in the dysplasias. The 13 dysplasias developed SCC with significantly higher LI of MCM2 and P53, and lower LI of P21 than the other dysplasias (each p < 0.05). The LI of MCM2, P21 and the TI were not correlated with P53 expression. Conclusions: Oral dysplasia was characterized by lower cell proliferation and a higher frequency of cell death compared to SCCs. The higher LI of MCM2 and P53 and the lower LI of P21 might predict malignant transformation of oral dysplasia. MCM2 is regulated via a P53-independent pathway, and a useful biomarker of proliferating cells.

Minichromosome maintenance-7 and geminin are reliable prognostic markers in patients with oral squamous cell carcinoma: immunohistochemical study

BACKGROUND Minichromosome maintenance (MCM) proteins act as the origin licensing machinery that regulates initiation of DNA replication. Geminin is a licensing repressor and prevents reinitiation of DNA replication by blocking reloading of MCM proteins at replication origins. Recent studies have proposed that MCM7 and geminin may be sensitive proliferative and prognostic markers of various malignant tumors. This study aimed to analyze the expression of MCM7 and geminin to clarify pathobiological significance in human oral squamous cell carcinomas (OSCCs). METHODS We performed immunohistochemical analysis on 10 specimens of normal oral epithelia, 50 lesions with dysplasia and 113 OSCCs. Labeling indices (LIs) for MCM7, geminin and Ki-67 were evaluated, comparing with clinicopathological profiles. RESULTS The mean LIs for MCM7 were 29.2% for normal epithelia, 32.2% for dysplasias, and 51.1% for OSCCs; the value was significantly higher in the last than in the former two (P < 0.01). The mean LIs for geminin were 6.8% for normal epithelia, 9.2% for dysplasias, and 21.3% for OSCCs; the value was significantly higher in the OSCCs (P < 0.01). The MCM7 LIs were correlated with the histological grade of OSCCs, in which the highest LIs were noted in the poorly differentiated type (P < 0.01). The survival rate was significantly lower in patients with a higher MCM7 LI (>49.5%) than in those with a lower LI (P < 0.05) at stage III-IV. However, the survival rate in the patients with a higher geminin LI (>19.5%) was significantly higher than in those with a lower LI (P < 0.05) at stage IV.

Zoledronic acid, a third-generation bisphosphonate, inhibits cellular growth and induces apoptosis in oral carcinoma cell lines

Bisphosphonates (BPs) inhibit bone resorption by preventing osteoclast maturation and apoptosis induction. Recently, BPs have also been shown to have antitumor effects against various types of carcinomas in vitro and in vivo. In this study, we investigated the antitumor effect of zoledronic acid (ZOL), a third generation bisphosphonate, on proliferation, cell cycle and apoptosis of oral cancer cells. Direct antitumor effects of ZOL against four oral carcinoma cell lines (squamous cell carcinoma, HSC3, HSC4, SCCKN; salivary adenocarcinoma, HSY) were measured by WST assay. Apoptosis-related molecules were analyzed by Western blot analysis and cell cycle was analyzed by flow cytometry. ZOL had a dose-dependent antitumor effect in the four oral cancer cell lines. ZOL activated caspase-3, -8 and -9 and induced cellular apoptosis. Western blot analysis showed that ZOL increased cleaved anti-human poly(ADP-ribose) polymerase expression and decreased Bcl-2 and Bid expression. Treatment with ZOL increased the number of cells in apoptosis, sub G1 phase and S phase, and reduced the number of cells in the G0/G1 and G2/M phase in a concentration-dependent manner. ZOL inhibits cell proliferation and induces apoptosis of oral cancer cells in vitro. These findings suggest that ZOL might be beneficial in the treatment of oral carcinoma patients.

Mechanism of action and therapeutic indication of prosthetic mandibular advancement in obstructive sleep apnea syndrome.

Abstract Prosthetic mandibular advancement (PMA) was applied to nine patients with obstructive sleep apnea syndrome (OSAS) and its therapeutic usefulness, mechanism of action, and clinical indication were discussed based on polysomnographic findings and serial examination of upper airway before and during PMA treatment. Apnea hypopnea index significantly decreased during PMA treatment compared with the value before treatment (P < 0.01) and the rate of the treatment responder counted 78.1%. Cephalometric variables indicated forward and inferior advancement of mandible in our subjects. Magnetic resonance imaging of the upper airway during sleep revealed a marked improvement of velopharyngeal obstruction in most subjects. In addition, intraesophageal negative pressure during sleep decreased significantly. Our results confirmed the high therapeutic efficacy of PMA for OSAS and indicated forward advancement of the mandible and decrease of negative pressure loading on upper airway with PMA might suppress velopharyngeal collapse. Thus, PMA was regarded as one of the treatments of choice for OSAS occurring based on with velopharyngeal narrowing.

Reduction of Osteoclasts in a Critical Embryonic Period Is Essential for Inhibition of Mouse Tooth Eruption

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast‐deficient Csfmop homozygous (op/op) mice, which lack functional macrophage colony‐stimulating factor (M‐CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c‐Fms, a receptor for M‐CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate‐resistant acid phosphatase‐positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1‐day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP‐positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.

Clinical Investigation on Pulmonary Metastasis of Head and Neck Carcinomas

Objective: To establish a treatment strategy for pulmonary metastases, we clinically investigated the characteristics of distant metastases from head and neck carcinomas. Methods: In 636 head and neck carcinomas, the pathophysiology of distant metastases was investigated by charts, roentgenographies, computed tomographies and scintigraphies. Results: Of the squamous cell carcinomas, oropharyngeal tumors were most highly metastatic, followed by lower gingiva, floor of the mouth, maxillary sinus, and tongue. In distant metastases, 30 (4.7%), 5 (0.8%), and 7 (1.1%) metastasized to the lungs only, lungs and other organs, and organs excluding the lungs, respectively. In pulmonary metastases, the right, left and both lungs were involved in 18, 5, and 8 patients, respectively, although details were not obtained for 4 patients. Pulmonary metastases consisted of 1, 2, and 3 or more tumors in 18, 4, and 6 patients, respectively. Diffuse cancer cell infiltration was observed in 3 patients. Of the 42 patients with distant metastases, 12 patients died of progressive pulmonary metastases, and 5 of these patients manifested only 1 pulmonary lesion throughout life. However, the metastatic pulmonary tumors were controlled surgically or conservatively in 3 patients. Conclusion: These results indicate that distant metastases from head and neck carcinomas involve the lungs most frequently and that chemoimmunotherapy and surgical removal of the metastatic tumors are recommended when indicated.