Role of serum amylase and salivary cytokines in oral complications during chemoradiotherapy.

OBJECTIVES: To investigate whether serum amylase can predict the recovery of salivary volume and determine the correlation of the level of cytokines, including epidermal growth factor, hepatocyte growth factor and keratinocyte growth factor, with oral mucositis during chemoradiotherapy for oral cancer. SUBJECTS AND METHODS: This study included 84 patients treated with preoperative chemoradiotherapy followed by curative surgery, following a phase II study protocol. We measured and analysed the correlation of the stimulated saliva volume, serum amylase and cytokines in resting saliva at baseline and 1 month after chemoradiotherapy with oral mucositis levels. RESULTS: We observed a negative correlation between the serum amylase level at the beginning of chemoradiotherapy and the stimulated saliva volume at 1 month after chemoradiotherapy (p = .03). Epidermal growth factor in resting saliva was significantly reduced after chemoradiotherapy (p < .01). The incidence of severe oral mucositis during chemoradiotherapy was significantly higher and negatively associated with the epidermal growth factor and keratinocyte growth factor levels (p = .04, p = .05). CONCLUSIONS: The serum amylase level at the beginning of chemoradiotherapy may be a predictor of the recovery of the saliva volume. Furthermore, cytokines such as epidermal growth factor and keratinocyte growth factor in resting saliva affect the development of oral mucositis during chemoradiotherapy.

BRD4 promotes metastatic potential in oral squamous cell carcinoma through the epigenetic regulation of the MMP2 gene.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has increased morbidity, and its high metastatic potential affects patient survival. Bromodomain containing 4 (BRD4) is a chromatin protein that associates with acetylated histone lysines and facilitates transcription. BRD4 has been implicated in cell proliferation, metastasis, and prognosis in several types of cancer. However, the role of BRD4 in OSCC remains to be elucidated. METHODS: We investigated the role of BRD4 and its potential utility as a therapeutic target in OSCC. RESULTS: JQ1, the BRD4 inhibitor, suppressed the cell proliferation, migration, and invasion in the OSCC cell lines and in vivo. JQ1 reduced the expression levels of 15 metastasis genes in OSCC, including matrix metallopeptidase 2 (MMP2). Our chromatin immunoprecipitation assay showed that JQ1 reduced the BRD4 binding to the histone H3 lysine 27 acetylation-enriched sites in the MMP2 locus. Analyses of biopsy specimens from OSCC patients revealed that the BRD4 and MMP2 expression levels were correlated in the cancerous regions, and both were highly expressed in lymph node metastasis cases, including delayed metastasis. CONCLUSIONS: BRD4 contributes to metastasis in OSCC, through the epigenetic regulation of the MMP2 gene, and thus BRD4 may represent a therapeutic target and a novel prediction indicator for metastasis.

Circulating miRNA-1290 as a potential biomarker for response to chemoradiotherapy and prognosis of patients with advanced oral squamous cell carcinoma: A single-center retrospective study.

MicroRNAs are a class of small, endogenous, noncoding 18- to 24-nucleotide-long RNAs that can regulate multiple processes related to cancer progression. However, their clinical value in patients with oral squamous cell carcinoma has not yet been fully explored. Therefore, the aim of this study was to investigate the clinical significance of circulating microRNAs in oral squamous cell carcinoma patients. The expression levels of circulating miR-1246 and miR-1290 in healthy volunteers and oral squamous cell carcinoma patients were examined by quantitative real-time polymerase chain reaction. The expression levels of both microRNAs in the radioresistant oral squamous cell carcinoma cell line (SAS-R) and the parent cell line (SAS) and in the conditioned medium obtained from these cell lines were also examined by quantitative real-time polymerase chain reaction. In addition, the correlations between circulating microRNA status and various clinicopathological features in 55 oral squamous cell carcinoma patients with locally advanced oral squamous cell carcinoma who underwent surgery following 5-fluorouracil-based chemoradiotherapy were examined. The expression level of miR-1290 was significantly lower in the plasma of oral squamous cell carcinoma patients than in that of healthy volunteers (p < 0.01). The expression levels of microRNAs in the conditioned medium and in the cells varied from cell to cell. In the clinicopathological analyses, the frequency of patients with low miR-1290 levels was significantly higher among cases with lower pathological differentiation and among those with a poor pathological response for preoperative chemoradiotherapy (p = 0.030 each). Furthermore, Cox regression analysis based on the 5-year overall survival and disease-free survival revealed that miR-1290 status was a significant prognostic factor for patients with oral squamous cell carcinoma (hazard ratio = 0.169, p = 0.008, and hazard ratio = 0.186, p = 0.008, respectively). Circulating miR-1290 status could be a valuable biomarker for predicting the clinical response to chemoradiotherapy as well as overall survival in patients with oral squamous cell carcinoma.

Loss of CYLD promotes cell invasion via ALK5 stabilization in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) has a very poor prognosis because of its highly invasive nature, and the 5-year survival rate has not changed appreciably for the past 30 years. Although cylindromatosis (CYLD), a deubiquitinating enzyme, is thought to be a potent tumour suppressor, its biological and clinical significance in OSCC is largely unknown. This study aimed to clarify the roles of CYLD in OSCC progression. Our immunohistochemical analyses revealed significantly reduced CYLD expression in invasive areas in OSCC tissues, whereas CYLD expression was conserved in normal epithelium and carcinoma in situ. Furthermore, downregulation of CYLD by siRNA led to the acquisition of mesenchymal features and increased migratory and invasive properties in OSCC cells and HaCaT keratinocytes. It is interesting that CYLD knockdown promoted transforming growth factor-ß (TGF-ß) signalling by inducing stabilization of TGF-ß receptor I (ALK5) in a cell autonomous fashion. In addition, the response to exogenous TGF-ß stimulation was enhanced by CYLD downregulation. The invasive phenotypes induced by CYLD knockdown were completely blocked by an ALK5 inhibitor. In addition, lower expression of CYLD was significantly associated with the clinical features of deep invasion and poor overall survival, and also with increased phosphorylation of Smad3, which is an indicator of activation of TGF-ß signalling in invasive OSCC. These findings suggest that downregulation of CYLD promotes invasion with mesenchymal transition via ALK5 stabilization in OSCC cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

HMGA2 Contributes to Distant Metastasis and Poor Prognosis by Promoting Angiogenesis in Oral Squamous Cell Carcinoma.

The highly malignant phenotype of oral squamous cell carcinoma (OSCC), including the presence of nodal and distant metastasis, reduces patient survival. High-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor that is involved in advanced malignant phenotypes and poor prognosis in several human cancers. However, its biological role in OSCC remains to be elucidated. The purpose of this study was to determine the clinical significance and role of HMGA2 in the malignant potential of OSCC. We first investigated the expression pattern of HMGA2 and its clinical relevance in 110 OSCC specimens using immunohistochemical staining. In addition, we examined the effects HMGA2 on the regulation of vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, which are related to angiogenesis, in vitro. High expression of HMGA2 was significantly correlated with distant metastasis and poor prognosis. Further, HMGA2 depletion in OSCC cells reduced the expression of angiogenesis genes. In OSCC tissues with high HMGA2 expression, angiogenesis genes were increased and a high proportion of blood vessels was observed. These findings suggest that HMGA2 plays a significant role in the regulation of angiogenesis and might be a potential biomarker to predict distant metastasis and prognosis in OSCC.

FBXW7 expression affects the response to chemoradiotherapy and overall survival among patients with oral squamous cell carcinoma: A single-center retrospective study.

FBXW7 (F-box and WD repeat domain containing-7) is a tumor suppressor protein that regulates the degradation of various oncoproteins in several malignancies. However, limited information is available regarding FBXW7 expression in oral squamous cell carcinoma. Therefore, this study aimed to determine the clinical significance of FBXW7 expression in oral squamous cell carcinoma. The FBXW7 expression patterns in oral squamous cell carcinoma and adjacent normal tissues from 15 patients who underwent radical resection were evaluated using quantitative real-time polymerase chain reaction and immunohistochemical staining. In addition, immunohistochemistry was performed using paraffin-embedded sections from biopsy specimens obtained from 110 patients with oral squamous cell carcinoma who underwent surgery after 5 fluorouracil-based chemoradiotherapy. The associations of FBXW7 expression with various clinicopathological features and prognosis were evaluated in these patients. As a results, in the 15 matched samples, the FBXW7 expression was significantly decreased in the oral squamous cell carcinoma tissues compared to that in the adjacent normal tissues. In the clinicopathological analysis, compared to high protein expression, low FBXW7 expression was found to significantly associate with a poor histological response to preoperative chemoradiotherapy. Kaplan-Meier curve analysis revealed that low FBXW7 expression was significantly associated with a poor prognosis, and FBXW7 expression was found to be an independent predictor of overall survival in the multivariate analysis. Our results suggest that FBXW7 may function as a tumor suppressor protein in oral squamous cell carcinoma. In addition, FBXW7 could be a potential biomarker for predicting not only the clinical response to chemoradiotherapy but also overall survival in patients with oral squamous cell carcinoma.

IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma.

BACKGROUND: In promoting tumour malignancy IL-6 signalling is considered to have an important role. However, the biological roles of IL-6 on radiosensitivity in oral squamous cell carcinoma (OSCC) remain largely unclear. The objective of this study is to determine the effects and molecular mechanisms of IL-6 on radiosensitivity in OSCC. METHODS: Two OSCC cell lines, and OSCC tissue samples with radioresistant cells were used. We examined the effects of IL-6, or tocilizumab, a humanised anti-human IL-6 receptor antibody, or both on radiosensitivity and DNA damage after X-ray irradiation in vitro. In addition, we investigated the involvement of the Nrf2-antioxidant pathway in IL-6-mediated radioresistant mechanisms using OSCC cell lines and tissues. RESULTS: Increased levels of IL-6 suppressed radiation-induced cell death, and the blockade of IL-6 signalling by tocilizumab sensitised tumour cells to radiation. The radioresistant effect of IL-6 was associated with decreased DNA damage after radiation. We also found that IL-6 promotes the activation of not only the downstream molecule STAT3 but also the Nrf2-antioxidant pathway, leading to a significant decrease in oxidative stress by upregulating Mn-SOD. CONCLUSIONS: These results indicate that the blockade of IL-6 signalling combined with conventional radiotherapy could augment the treatment response and survival rate in patients with radioresistant OSCC.

Cancer immunotherapy using novel tumor-associated antigenic peptides identified by genome-wide cDNA microarray analyses.

Recent genome-wide cDNA microarray analysis of gene expression profiles in comprehensive tumor types coupled with isolation of cancer tissues by laser-microbeam microdissection have revealed ideal tumor-associated antigens (TAAs) that are frequently overexpressed in various cancers including head and neck squamous cell cancer (HNSCC) and lung cancer, but not in most normal tissues except for testis, placenta, and fetal organs. Preclinical studies using HLA-transgenic mice and human T cells in vitro showed that TAA-derived CTL-epitope short peptides (SPs) are highly immunogenic and induce HLA-A2 or -A24-restricted CTLs. Based on the accumulated evidence, we carried out a phase II clinical trial of the TAA-SP vaccine in advanced 37 HNSCC patients. This study showed a significant induction of TAA-specific CTLs in the majority of patients without serious adverse effects. Importantly, clinical responses including a complete response were observed in this study. Another phase II clinical trial of therapeutic TAA-SP vaccine, designed to evaluate the ability of prevention of recurrence, is ongoing in HNSCC patients who have received curative operations. Further studies in human preclinical studies and in vivo studies using HLA class I transgenic mice showed TAA-derived long peptides (TAA-LPs) have the capacity to induce not only promiscuous HLA class II-restricted CD4(+) T helper type 1 cells but also tumor-specific CTLs through a cross-presentation mechanism. Moreover, we observed an augmentation of TAA-LP-specific T helper type 1 cell responses and tumor antigen-spreading in HNSCC patients vaccinated with TAA-SPs. This accumulated evidence suggests that therapeutic TAA-SPs and LPs vaccines may provide a promising cancer immunotherapy.

Identification of CDCA1-derived long peptides bearing both CD4+ and CD8+ T-cell epitopes: CDCA1-specific CD4+ T-cell immunity in cancer patients.

We recently identified a novel cancer-testis antigen, cell division cycle associated 1 (CDCA1) using genome-wide cDNA microarray analysis, and CDCA1-derived cytotoxic T lymphocyte (CTL)-epitopes. In this study, we attempted to identify CDCA1-derived long peptides (LPs) that induce both CD4+ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II-binding peptides with CDCA1-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate CDCA1-LPs encompassing both Th cell epitopes and CTL-epitopes. We studied the immunogenicity of CDCA1-LPs and the cross-priming potential of LPs bearing CTL-epitopes in both human in vitro and HLA-class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head-and-neck cancer (HNC) patients before and after vaccination with a CDCA1-derived CTL-epitope peptide using IFN-γ enzyme-linked immunospot assays. We identified two CDCA1-LPs, CDCA1(39­64)-LP and CDCA1(55­78)-LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1-specific CTLs were induced through cross-presentation of CDCA1-LPs in vitro and in vivo. In addition, CDCA1-specific Th cells enhanced induction of CDCA1-specific CTLs. Furthermore, significant frequencies of CDCA1-specific Th cell responses were detected after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1-LPs in HNC patients (CDCA1(39­64)-LP, 74%; CDCA1(55­78)-LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1-specific Th cell responses in HNC patients and underline the possible utility of CDCA1-LPs for propagation of both CDCA1-specific Th cells and CTLs.

Stromal expression of neutrophil gelatinase-associated lipocalin correlates with poor differentiation and adverse prognosis in oral squamous cell carcinoma.

AIMS: Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily. Although its overexpression in various cancers has been reported, little is known about its expression and clinical significance in oral squamous cell carcinoma (OSCC). This study aimed to elucidate the clinical significance of NGAL in OSCC. METHODS AND RESULTS: We investigated NGAL expression immunohistochemically in tumour cells and stromal cells in 96 OSCC tissues. NGAL expression in tumour cells correlated significantly with histological tumour cell differentiation, as shown by its specific distribution in the horn pearl-forming keratinized tumour cells, but not with other major clinicopathological parameters. We found NGAL(+) cells in the stroma that were predominantly myeloperoxidase-positive neutrophils. The number of such NGAL-expressing stromal cells was associated significantly with poor differentiation and reduced overall survival in OSCC. The prognostic value of stromal NGAL expression was significant in a univariate analysis, while only a trend was found in multivariate analyses. CONCLUSIONS: This study is the first to show the clinical significance of stromal NGAL expression, which may be an indicator of poor prognosis and more aggressive histological grade in OSCC. Our data suggest that NGAL expression in tumour cells and expression in stroma are associated in different ways with OSCC differentiation.

A low Dicer expression is associated with resistance to 5-FU-based chemoradiotherapy and a shorter overall survival in patients with oral squamous cell carcinoma.

BACKGROUND: The deregulation of microRNA (miRNA) is associated with multiple processes involved in cancer progression. RNase III endonucleases, Dicer and Drosha, are key enzymes for miRNA biogenesis, and it has been reported that altered expressions of these molecules affect the clinical outcomes of patients with various cancers. However, the clinical value of measuring the levels of Dicer and Drosha in oral squamous cell carcinoma (OSCC) patients is unclear. The purpose of this study was to determine the clinical significance of the expressions of Dicer and Drosha in patients with OSCC. METHODS: Oral squamous cell carcinoma specimens were obtained from 61 patients who underwent surgery following 5-fluorouracil-based chemoradiotherapy at Kumamoto University Hospital between October 2003 and January 2009. Paraffin-embedded sections obtained from biopsy specimens were immunohistochemically analyzed. The associations between Dicer, Drosha, and various clinicopathological features were examined, and the effects of Dicer and Drosha on the prognosis were evaluated. RESULTS: A low Dicer tumor expression was significantly correlated with the pathological response to chemoradiotherapy. Furthermore, a Cox regression analysis based on the overall survival revealed the Dicer expression status (hazard ratio, 0.34; P = 0.048) and pathological response to chemoradiotherapy (hazard ratio, 0.21; P = 0.014) to be significant prognostic factors in OSCC patients. On the other hand, the Drosha expression was not associated with any clinicopathological features or the prognosis. CONCLUSION: These results suggest that Dicer is a potential biomarker for predicting the clinical response to 5-FU-based chemoradiotherapy and the overall survival in patients with OSCC.

The pathological significance of Notch1 in oral squamous cell carcinoma.

Notch signaling has been reported to be involved in several types of malignant tumors; however, the role and activation mechanism of Notch signaling in oral squamous cell carcinoma (OSCC) remains poorly characterized. The purpose of this study was to elucidate the pathological significance of Notch signaling and its activation mechanism in the development and progression of OSCC. In this study, we showed that the expression of Notch1 and intracellular Notch domain (NICD) are upregulated in OSCCs. In addition, Notch1 and NICD were found to be characteristically localized at the invasive tumor front. TNF-α, a major inflammatory cytokine, significantly activated Notch signaling in vitro. In a clinicopathological analysis, Notch1 expression correlated with both the T-stage and the clinical stage. Furthermore, loss of Notch1 expression correlated with the inhibition of cell proliferation and TNF-α-dependent invasiveness in an OSCC cell line. In addition, γ-secretase inhibitor (GSI) prevented cell proliferation and TNF-α-dependent invasion of OSCC cells in vitro. These results indicate that altered expression of Notch1 is associated with increased cancer progression and that Notch1 regulates the steps involved in cell metastasis in OSCC. Moreover, inactivating Notch signaling with GSI could therefore be a useful approach for treating patients with OSCC.

Selective inhibition of nuclear factor-κB by nuclear factor-κB essential modulator-binding domain peptide suppresses the metastasis of highly metastatic oral squamous cell carcinoma.

Nuclear factor-κB (NF-κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF-κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP-expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF-κB activities and expression of NF-κB-regulated metastasis-related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor-engrafted mice were monitored. Furthermore, the effects of a selective NF-κB inhibitor, NEMO-binding domain (NBD) peptide, on metastasis in GSAS/N5-engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF-κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF-κB-regulated metastasis-related molecules, such as fibronectin, ß1 integrin, MMP-1, -2, -9, and -14, and VEGF-C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5-inoculated mice, and was accompanied by the downregulation of the NF-κB-regulated metastasis-related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF-κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC.

Interleukin-6 signalling regulates vascular endothelial growth factor-C synthesis and lymphangiogenesis in human oral squamous cell carcinoma.

Lymph node metastasis is associated with resistance to conventional therapy and poor survival of patients with oral squamous cell carcinoma (OSCC). Although lymphangiogenesis is well known to be associated with the occurrence of lymph node metastasis in various cancers, the precise mechanisms of lymphangiogenesis in OSCC are largely unknown. IL-6, a potent pro-inflammatory cytokine, has been shown to play active roles in various cancers, including OSCC. This study aimed to investigate the involvement of IL-6 signalling in lymphatic metastasis and to evaluate the efficacy of tocilizumab, a humanized anti-human IL-6 receptor antibody, as an anti-lymphangiogenic agent for OSCC. This investigation confirmed that levels of expression of IL-6 protein and VEGF-C mRNA in OSCC tissues were significantly correlated with lymph node metastasis in patients with OSCC, as assessed by immunohistochemical analysis and real-time quantitative RT-PCR. In vitro studies showed that IL-6 regulated VEGF-C mRNA expression in a human OSCC cell line, SAS cells, through the phosphoinositide 3-kinase-Akt pathway. In addition, treatment with tocilizumab led to markedly reduced VEGF-C mRNA expression and OSCC-related lymphangiogenesis in SAS xenografts. Together, these data suggest that tocilizumab acted as expected: it inhibited lymph node metastasis in OSCC by reducing tumour lymphangiogenesis.

General rules for clinical and pathological studies on oral cancer: a synopsis.

For the doctors and other medical staff treating oral cancers, it is necessary to standardize basic concepts and rules on oral cancers to progress in the treatment, research and diagnosis. Oral cancers are integrated in head and neck cancers and are applied to the general rules on head and neck cancer, but it is considered that more detailed rules based on the characteristics of oral cancers are essential. The objectives of this 'General Rules for Clinical and Pathological Studies on Oral Cancer' are to contribute to the development of the diagnosis, treatment and research of oral cancers based on the correct and useful medical information of clinical, surgical, pathological and image findings accumulated from individual patients at various institutions.

Holliday junction recognition protein as a prognostic biomarker and therapeutic target for oral cancer.

Since oral cancer (OC) is highly malignant and the efficacy of standard treatments is limited, the development of new therapeutics is urgently awaited. To identify potential molecular targets for new OC diagnosis and therapies, we screened oncoantigens by gene expression profile and focused on Holliday junction recognition protein (HJURP), a mammalian centromere­specific chaperone. HJURP was found to be highly expressed in the majority of OC cell lines and tissues as compared to normal oral epithelial cells. Tissue microarray analysis confirmed that HJURP was expressed in 103 (67.8%) of 152 OC tissue specimens, but expression in normal oral tissues was limited. Positive HJURP expression was significantly correlated with shorter overall survival (P=0.003). Depletion of HJURP by small­interfering RNAs dramatically inhibited the growth of OC cells by inhibition of cell cycle progression and induced senescence of OC cells. In addition, inhibition of the interaction between HJURP and CENP­A significantly suppressed the growth of OC cells. These results indicate that HJURP is a potential prognostic biomarker, and targeting HJURP and its molecular pathway presents a new strategy for the development of treatments against OC.

Characterization of Opa interacting protein 5 as a new biomarker and therapeutic target for oral cancer.

Oral cancer is a leading cause of cancer­related death worldwide. Current treatment for oral cancer includes surgery, radiotherapy, and chemotherapy; however, their effectiveness is still limited. To identify a new prognostic biomarker and therapeutic target for oral cancer, the Opa interacting protein 5 (OIP5), which plays an essential role in the proper segregation of chromosomes, was examined. Immunohistochemical staining using tissue microarrays indicated that OIP5 was expressed in 120 of 164 (73.2%) oral cancers but was minimally expressed in normal oral tissues. OIP5 expression was significantly associated with poor prognosis in patients with oral cancer. Overexpression of OIP5 enhanced the growth of oral cancer cells, whereas OIP5 knockdown using small interfering RNAs (siRNAs) significantly inhibited cell growth through cell cycle arrest at the G2/M phase. Suppression of OIP5 expression also induced senescence of oral cancer cells. Overall, the findings of the present study suggest that OIP5 may be a candidate prognostic biomarker and therapeutic target in oral cancer.

Nucleostemin affects the proliferation but not differentiation of oral squamous cell carcinoma cells.

Nucleostemin (NS) has been reported as essential for stem and cancer cell proliferation. To investigate the significance of NS in oral squamous cell carcinomas (OSCCs), we examined NS expression in neoplastic tissue of the tongue and in OSCC cell lines. Nucleostemin expression in the histological samples showed positive correlation with Ki-67 expression. Furthermore, NS expression was associated with cellular proliferation in OSCC cell lines using siRNA, which upregulated p27, a cyclin-dependent kinase inhibitor. Regarding OSCC differentiation, NS expression did not influence cornification or oral epithelial differentiation markers such as involucrin and cytokeratin19. Thus, NS is widely expressed in normal and neoplastic oral epithelial tissues, and is likely a marker of proliferation.

Degradation of fibrinogen and collagen by staphopains, cysteine proteases released from Staphylococcus aureus.

Staphylococcus aureus is the most frequently isolated pathogen in gram-positive sepsis often complicated by a blood clotting disorder, and is the leading cause of infective endocarditis induced by bacterial destruction of endocardial tissues. The bacterium secretes cysteine proteases referred to as staphopain A (ScpA) and staphopain B (SspB). To investigate virulence activities of staphopains pertinent to clotting disorders and tissue destruction, we examined their effects on collagen, one of the major tissue components, and on plasma clotting. Both staphopains prolonged the partial thromboplastin time of plasma in a dose- and activity-dependent manner, with SspB being threefold more potent than ScpA. Staphopains also prolonged the thrombin time of both plasma and fibrinogen, indicating that these enzymes can cause impaired plasma clotting through fibrinogen degradation. Whereas SspB cleaved the fibrinogen Aα-chain at the C-terminal region very efficiently, ScpA degraded it rather slowly. This explains the superior ability of the former enzyme to impair fibrinogen clottability. Enzymically active staphopains, at concentrations as low as 10 nM, degraded collagen with comparable efficiency. These results show novel virulence activities of staphopains in degrading fibrinogen and collagen, and suggest an involvement of staphopains in the clotting impairment and tissue destruction caused by staphylococcal infection.

Role of ribosomal protein S19-like plasma protein in blood coagulum resorption.

Western blot analyses and monocyte chemoattraction analyses of guinea pig plasma and serum indicated the presence of a plasma protein indistinguishable from ribosomal protein S19 and the cross-linked dimerization of it gaining monocyte chemotactic capacity in association with blood coagulation as in the case of human. When coagula preformed in vitro were intraperitoneally inserted into guinea pigs, they were rapidly covered by macrophages within 24h concomitant with an intra-coagulum macrophage infiltration. Differences were observed between the surface macrophages and the penetrating macrophages in ultrastructural, histochemical and immunohistochemical analyses. The inserted coagula were resorbed by day 7. When either anti-RP S19 antibodies or Gln137Asn-RP S19, a competitive inhibitor against RP S19, was premixed into the inserted coagulum, the attachment and penetration by macrophages decreased and the coagulum resorption retarded. These results indicate the role of the plasma RP S19-like molecule in coagulum resorption via macrophage recruitment.