Apoptosis repressor with caspase recruitment domain (ARC) promotes bone regeneration of bone marrow-derived mesenchymal stem cells by activating Fgf-2/PI3K/Akt signaling.

OBJECTIVES: This study aims to investigate whether apoptosis repressor with caspase recruitment domain (ARC) could promote survival and enhance osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: The lentivirus transfection method was used to establish ARC-overexpressing BMSCs. The CCK-8 method was used to detect cell proliferation. The BD Pharmingen™ APC Annexin V Apoptosis Detection kit was used to detect cell apoptosis. The osteogenic capacity was investigated by OCN immunofluorescence staining, ALP analysis, ARS assays, and RT-PCR analysis. Cells were seeded into calcium phosphate cement (CPC) scaffolds and then inserted subcutaneously into nude mice and the defect area of the rat calvarium. Histological analysis was conducted to evaluate the in vivo cell apoptosis and new bone formation of the ARC-overexpressing BMSCs. RNA-seq was used to detect the possible mechanism of the effect of ARC on BMSCs. RESULTS: ARC promoted BMSC proliferation and inhibited cell apoptosis. ARC enhanced BMSC osteogenic differentiation in vitro. An in vivo study revealed that ARC can inhibit BMSC apoptosis and increase new bone formation. ARC regulates BMSCs mainly by activating the Fgf-2/PI3K/Akt pathway. CONCLUSIONS: The present study suggests that ARC is a powerful agent for promoting bone regeneration of BMSCs and provides a promising method for bone tissue engineering.

In vivo and in vitro study of osteogenic potency of endothelin-1 on bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a major source of osteoblasts and are crucial for bone remolding and repair and thus they are widely used for tissue engineering applications. Tissue engineering in combination with gene therapy is considered as a promising approach in new bone regeneration. Endothelin-1（EDN-1）is produced by vascular endothelial cells which plays an important role during bone development. However, its role in BMSCs remains largely unknown. We established EDN-1 overexpressed BMSCs, proliferation ability and osteogenesis differentiation were detected respectively. Transduced BMSCs were then combined with CPC-scaffold to repair calvarial defects in rats to evaluate the in-vivo osteogenic potential of EDN-1. EDN-1 overexpressed BMSCs showed increased proliferation and significantly increased osteogenesis potential ability than vector transfected control. The in-vivo data also revealed more new bone formation with higher bone mineral density and number of trabeculae in EDN-1 overexpressed BMSCs. These findings have demonstrated the influence of EDN-1 on differentiation potential of BMSCs, which suggest that EDN-1 may be a new promising agent for bone tissue engineering.

Apoptosis repressor with caspase recruitment domain enhances survival and promotes osteogenic differentiation of human osteoblast cells under Zoledronate treatment.

Zoledronate is one of the most potent nitrogen-containing bisphosphonates which has been demonstrated to result in osteoblast apoptosis and impact osteogenic differentiation in vitro. This effect of Zoledronate on osteoblasts may partially explain bisphosphonate­associated osteonecrosis of the jaw, a serious complication associated with treatment with bisphosphonates. Apoptosis repressor with caspase recruitment domain (ARC) is a multifunctional inhibitor of apoptosis that is physiologically expressed predominantly in post­mitotic cells such as cardiomyocytes, neurons and skeletal muscle cells. However, its effect on human osteoblasts remains unclear. The current study aimed to investigate the effects of ARC on human osteoblasts under the treatment of high concentrations of Zoledronate. ARC­overexpressed human osteoblasts were established and were exposed to Zoledronate with different concentrations (0, 1 and 5 µM) in vitro. Cell numbers were detected using the MTT assay, and flow cytometry was used to identity cell apoptosis. Alkaline phosphatase staining, quantitative analysis and ectopic osteogenesis in nude mice were used to evaluate the osteogenic differentiation of ARC­overexpressed osteoblasts. It was observed that ARC is able to reverse the inhibitory effect of Zoldronate on osteoblasts. ARC is additionally able to promote osteogenic differentiation of osteoblasts and inhibit their apoptosis. These observations suggest a critical role for ARC in the regulation of human osteoblasts under Zoledronate treatment.

In Vitro and in Vivo Evaluation of Silicate-Coated Polyetheretherketone Fabricated by Electron Beam Evaporation.

Intrinsic bioinertness severely hampers the application of polyetheretherketone (PEEK), although in the field of dentistry it is considered to be an ideal titanium substitute implanting material. In this study, a bioactive silicate coating was successfully introduced onto PEEK surface by using electron beam evaporation (EBE) technology to improve its bioactivity and osseointegration of PEEK. Through controlling the duration of EBE, the incorporated amounts of silicon (Si) could be exquisitely adjusted to obtain proper biofunctionality, as assessed by cell adhesion, proliferation, osteogenic gene expression, and protein detection. In vivo, the samples were then tested in a femur implantation model to assay osseointegration effects in ovariectomized (OVX) rats. Remarkable enhancement of adhesion, spreading, osteogenesis, and differentiation of bone marrow stem cells (rBMSCs-OVX) were noted on silicate-coated samples. In particular, the group that was processed for 5 min with EBE (EBE-5 min) showed the most improvements in ALP activity and osteogenic-related gene expression compared to the remaining groups. Better osseointegration of the group that was processed for 8 min with EBE (EBE-8 min) was observed in vivo, as indicated by micro-CT test, fluorescent labeling, and histological and histomorphometric analyses. Collectively, the outcomes of the above experiments demonstrate that the present work is a meaningful attempt to promote osseointegration under osteoporotic conditions with only Si element incorporated to PEEK surface by the application of EBE technique. To the best of our knowledge, this work is the first demonstration of tuning the surface properties of PEEK via the adoption of an EBE-fabricated silicate coating to address an osteoporotic problem both in vitro and in vivo.

LATS2 induced by TNF-alpha and inhibited cell proliferation and invasion by phosphorylating YAP in oral squamous cell carcinoma.

OBJECTIVE: Many reports indicated LATS2 was a component of the Hippo pathway, could phosphorylate and inactivate YAP, acted as a tumor suppressor in human cancers. But few studies investigated the role of LATS2 in oral squamous cell carcinoma (OSCC) and clarified the mechanisms of regulation of LATS2 expression. DESIGN: The expressions of LATS2 and phosphorylated YAP were detected by Western blotting in HN6 cells treated with TNF-α in different time and different dose. Luciferase reporter assays were performed to detect whether YAP can be phosphorylated by LATS2 in HN6 cells. Cell proliferation, anchorage independent growth in soft agar, transwell cell invasion assay, and nu mice in vivo xenografts growth were performed to study the effects of overexpression of LATS2 on OSCC cells. RESULTS: In this study, we confirmed that YAP can be phosphorylated by LATS2. LATS2 can be dose- and time-dependently induced by TNF-α in HN6 cells. Overexpression of LATS2 inhibited cell proliferation, colony formation, cell invasion, and in vivo xenografts growth in OSCC cells. CONCLUSION: LATS2 could be induced by TNF-alpha and inhibited cell proliferation and invasion by phosphorylating YAP in OSCC cells. LATS2 might play a role in the tumorigenesis of OSCC and might be a potential therapeutic target in OSCC treatment.

Strontium delivery on topographical titanium to enhance bioactivity and osseointegration in osteoporotic rats.

Osseointegration remains a major clinical challenge in osteoporotic patients. Strontium (Sr) has been shown to be a significant therapy to favor bone growth by both increasing new bone formation and reducing bone resorption. In this study, we attempt to chemically functionalize Ti implants by micro-arc oxidation, alkali treatment and ion exchange. This functionalized Ti surface possessed a hierarchical topography with Sr incorporation, which can release Sr ions at a slow rate. To our knowledge, this work is the first to use this type of Sr-doped Ti surface to address osteoporotic bone mesenchymal stem cells (BMSCs) in the dual directions of bone regeneration, bone formation and bone resorption. The modified surface was demonstrated to remarkably enhance the adhesion, spreading, and osteogenic differentiation of BMSCs in vitro. The effect of the wash-out solution from various groups on osteoporotic BMSCs was also investigated. The Sr-doped group can improve the ALP activity and osteogenic gene expression. Moreover, the Sr-doped group and the wash-out solution show the most inhibition in osteoclast formation and maturation. Furthermore, the increased bioactivity of the hierarchical structure was also confirmed with the ovariectomized rat femur model in vivo. The outcome of fluorescence labeling, histology and histomorphometric analysis demonstrated a significant promotion of osseointegration in ovariectomized rats. Altogether, the experimental data indicate that the fabrication of a Sr-doped hierarchical Ti surface is a meaningful attempt to incorporate the Sr nutrient element into Ti-based implants, and it is expected to be exploited in developing better osseointegration for osteoporotic patients.

Overexpression of c-fos promotes cell invasion and migration via CD44 pathway in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most deadly malignant tumors with high invasive potential and frequently cervical lymph node metastasis. AP-1 plays a critical role in tumor invasion and metastasis, but there are few reports on the role of c-Fos in OSCC carcinogenesis and metastasis. METHODS: Investigate c-Fos expression in clinical samples from 58 primary patients with OSCC by immunohistochemistry. c-Fos knockdown stable cell lines were established by lentiviral infection and transwell cell invasion assay to detect the effects of c-Fos knockdown on tumor cell invasion. RESULTS: Nuclear and cytoplasmic c-Fos protein were both overexpression in cancerous tissues compared with adjacent non-malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.005). Higher level nuclear c-Fos expression was found in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (4.85 ± 1.43 vs. 3.61 ± 1.28, P = 0.002). Higher level of c-Fos expression was also found in tumor invasive front margin than tumor center and high nuclear expression of c-Fos indicated poor survival. Knockdown of c-Fos greatly suppressed tumor cell proliferation and invasion and downregulated CD44 and CyclinD1 expression in HN6 and SCC9 cells. However, CyclinD3, c-myc, and Erk1/2 were found no changes to c-Fos depletion. CONCLUSIONS: c-Fos promoted cell invasion and migration via CD44 pathway in OSCC. c-Fos could be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.

Vacuum extraction enhances rhPDGF-BB immobilization on nanotubes to improve implant osseointegration in ovariectomized rats.

Nanotube morphology has been previously applied to improve osseointegration in osteoporosis, but the osteogenic capability of the technique requires further improvements. This study aimed to investigate the effects of vacuum extraction on the loading of rhPDGF-BB on nanotube arrays as well as its effects on the osseointegration of ovariectomized (OVX) rats. More rhPDGF-BB protein particles aggregated on the nanotube surface and into the nanotube after vacuum extraction for 10 min. The immobilized protein could be slowly released for at least 14 days and still kept its biological activity. In vitro, the immobilized rhPDGF-BB enhanced cell adhesion, proliferation and osteogenic differentiation. In vivo, more rhPDGF-BB immobilized on the nanotube surface also promoted the osseointegration. These results suggest that the enhanced immobilization of rhPDGF-BB on nanotube arrays can potentially be used in the future as an implant surface modification strategy in dental and orthopedic applications in osteoporotic patients. FROM THE CLINICAL EDITOR: This study presents convincing evidence that enhanced immobilization of recombinant human PDGF-BB protein particles on nanotubes lead to improved osteogenic differentiation in an experimental system. When used as a surface modification strategy for dental or orthopedic implants, this method was able to promote osseointegration even in an osteoporotic animal model, raising the likelihood for potential future clinical applications.

Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function.

As one of the important ions associated with bone osseointegration, magnesium was incorporated into a micro/nanostructured titanium surface using a magnesium plasma immersion ion-implantation method. Hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 30 minutes (Mg30) and hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 60 minutes (Mg60) were used as test groups. The surface morphology, chemical properties, and amount of magnesium ions released were evaluated by field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, field-emission transmission electron microscopy, and inductively coupled plasma-optical emission spectrometry. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used to evaluate cell responses, including proliferation, spreading, and osteogenic differentiation on the surface of the material or in their medium extraction. Greater increases in the spreading and proliferation ability of rBMMSCs were observed on the surfaces of magnesium-implanted micro/nanostructures compared with the control plates. Furthermore, the osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) genes were upregulated on both surfaces and in their medium extractions. The enhanced cell responses were correlated with increasing concentrations of magnesium ions, indicating that the osteoblastic differentiation of rBMMSCs was stimulated through the magnesium ion function. The magnesium ion-implanted micro/nanostructured titanium surfaces could enhance the proliferation, spreading, and osteogenic differentiation activity of rBMMSCs, suggesting they have potential application in improving bone-titanium integration.

Antibacterial property, angiogenic and osteogenic activity of Cu-incorporated TiO2 coating.

Numerous efforts have been made to modify the surface topography and chemical composition of biomedical implants in order to enhance the antibacterial ability and the osteointegration between implants and surrounding bone tissue. In the present work, copper-incorporated TiO2 coatings were fabricated by combining micro-arc oxidation and hydrothermal treatment together to functionalize the surface of Ti implants. The as-prepared surfaces exhibited a hierarchical structure comprising nanoneedles nearly perpendicular to the microrough surface of the TiO2 coating. The Cu-loaded TiO2 coating possessed strong antimicrobial ability against Gram-negative Escherichia coli. In vitro cytocompatibility evaluation suggests that no significant cytotoxicity appeared on the Cu-incorporated TiO2 coating. Furthermore, the addition of the copper element could stimulate the expression of angiogenic genes, including the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in rat bone marrow stem cells (BMSCs). Moreover, they tended to undergo osteogenic differentiation, indicated by the up-regulation expression of osteogenic markers and the higher level of alkaline phosphatase activity. This study provides insight for the surface modification of biomedical Ti-based implants. To the best of our best knowledge, this is a successful attempt for the first time to combine micro-arc oxidation and hydrothermal treatment to introduce copper nutrient element to functionalize Ti-based implant surfaces with enhanced angiogenesis potential, osteostimulation and antimicrobial properties that can better meet clinical needs.

Epigenetic activation of AP1 promotes squamous cell carcinoma metastasis.

The transcription factor AP1 (activating protein 1), a heterodimer of the JUN and FOS proteins, promotes the invasive growth and metastasis of various tumors such as squamous cell carcinoma (SCC), breast cancer, and melanoma. AP1 activity is transcriptionally induced through a positive feedback loop. We identified the histone demethylase KDM4A (lysine-specific demethylase 4A) as a key epigenetic priming factor in this positive feedback loop. KDM4A contributed to the induction of genes encoding the AP1 transcription factors and the invasive growth and metastasis of SCC. KDM4A knockdown decreased the growth factor-induced messenger RNA expression and protein abundance of AP1 family members, including JUN and FOSL1. Mechanistically, histone demethylation by KDM4A facilitated the binding of the AP1 complex to the promoters of JUN and FOSL1, thereby promoting the positive feedback loop that maintains activation of AP1. In a mouse model of SCC, KDM4A knockdown inhibited lymph node metastasis. Moreover, the abundance of KDM4A correlated with the abundance of JUN and FOSL1 in human SCC tissues, and KDM4A expression was increased in human lymph node metastases. Our studies provide insights into the epigenetic control of AP1 and tumor invasion and suggest that KDM4A could be an important therapeutic target for inhibiting invasive SCC growth and metastasis.

Upregulation of ß2-microglobulin expression in progressive human oral squamous cell carcinoma.

The aim of the present study was to investigate ß2-microglobulin (ß2-M) expression in normal oral mucosa and progressive oral squamous cell carcinoma (OSCC) and to assess the clinical significance of ß2-microglobulin expression. The study included 10 cases of normal oral mucosa epithelium specimens, 55 cases of primary OSCC specimens, and 25 cases of OSCC metastasis specimens. Immunohistochemistry was used to determine ß2-M expression, and its correlation with clinicopathological factors in progressive OSCC was evaluated. Immunohistochemistry showed that strong ß2-M expression was significantly asscociated with tumor size (T3, T4 vs. T1, T2; P=0.001), positive node status (N positive vs. N negative; P=0.000) and advanced clinical stage (Ⅲ, Ⅳ vs. Ⅰ, Ⅱ, P=0.000) in primary OSCC lesions. Compared to primary OSCC lesions, the frequency of ß2-M expression was significantly increased in metastatic OSCC lesions (P=0.02). In addition, in vitro results from Western blotting showed increased ß2-M expression in the two OSCC lines studied. Therefore, we speculate that the up-regulation of ß2-M expression may contribute to the oncogenesis of human oral mucosa, tumor invasion and metastasis.

Correlation of increased twist with lymph node metastasis in patients with oral squamous cell carcinoma.

PURPOSE: To investigate the protein expression of Twist, Snail, and Slug in oral squamous cell carcinoma (OSCC) samples and evaluate the potential correlation between the expression status and clinicopathologic features in patients with OSCC. PATIENTS AND METHODS: Twist, Snail, and Slug protein expression was assessed by immunohistochemistry in a total of 60 OSCC samples and 10 normal oral mucosal samples. The associations between the protein expression and clinicopathologic parameters were mainly detected using the χ(2) test. The survival analysis was performed using the Kaplan-Meier method, and the prognostic analysis was performed using Cox regression models. RESULTS: Immunohistochemistry stain analysis showed that positive Twist, Snail, and Slug protein expression was observed in 70%, 63.3%, and 58.3% of the cases, respectively. Twist protein expression was positively associated with lymph node metastasis, pathologic grade, and tumor stage (P = .012, P = .008, and P = .004, respectively, χ(2) test). All patients were followed up for 6 to 59 months (mean 37). A correlation between Twist protein expression and tumor recurrence was detected (log-rank test, P = .025). Nevertheless, no correlation was found between the Snail and Slug protein expression and the clinicopathologic parameters. CONCLUSIONS: Twist might serve as a useful molecular marker for lymph node metastasis and a poor prognosis in OSCC.

[Expression of galectin-1 in carcinogenesis of oral mucosal epithelium].

OBJECTIVE: To investigate the expression of galectin-1 in oral squamous cell carcinoma(OSCC) and its clinical significance. METHODS: Detection of the mRNA and protein expression of galectin-1 in the in vitro cellular carcinogenesis model of OSCC, OSCC cell lines and tissue specimens from 30 primary OSCC patients were performed using real-time polymerase chain reaction (PCR), Western blotting and immunohistochemistry, respectively. RESULTS: The value of galectin-1 mRNA and protein level in human immortalized oral epithelia cell (HIOEC) cell was 0.071 ± 0.023, 0.118 ± 0.046, Compared with the HIOEC, galectin-1 mRNA level and protein expression were increased significantly in all the cell lines (0.141 ± 0.049, 0.504 ± 0.33) (P < 0.01). The levels of mRNA and protein expression of galectin-1 were significantly higher in the cancerous tissue (0.059 ± 0.034, 1.5 ± 0.68) than in the normal adjacent tissues (0.029 ± 0.012, 0.4 ± 0.56) (P < 0.01). CONCLUSIONS: The expression of galectin-1 gene up-regulated in carcinogenesis process of OSCC significantly may be related to the tumorigenesis and development of OSCC, which illustrates its potential clinical application as tumor marker for early diagnosis.

Yes-associated protein promotes cell proliferation by activating Fos Related Activator-1 in oral squamous cell carcinoma.

In our previous study, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including a human immortalized oral epithelial cell (HIOEC) and a cancerous cell line (HB96). Microarray analysis showed that the gene encoding Yes-associated protein (YAP) was significantly increased in HB96 cells compared with HIOEC cells. But the underlying mechanism of YAP on oncogenesis, especially its downstream targets, are still not clear. YAP expression in OSCC cell lines and tissue specimens were investigated by using real-time PCR, western blotting and immunohistochemistry staining. YAP put-back plasmid with four mutation sites after YAP-siRNA interference was constructed by site-directed mutagenesis. Cell growth and colony formation were observed after YAP-siRNA interference or YAP put-back again in CAL27 cells. YAP expression was increased in the cellular carcinogenesis models and the clinical samples from primary OSCC patients. Inhibition of YAP by siRNA interference in CAL27 cells significantly inhibited cell proliferation and colony formation in soft agar, but these abilities were rescued when YAP was put-back again. At the same time, Fos Related Activator-1 (Fra-1) was down-regulated when YAP was inhibited by siRNA interference while Fra-1 was rescued when YAP was put-back again. Immunohistochemistry results also indicated that higher levels of YAP were significantly associated with Fra-1 overexpression in OSCC clinical samples. YAP could promote cell proliferation by activating transcription factor Fra-1 in oral squamous cell carcinoma.

Establishment and characterization of an HPV16 E6/E7-expressing oral squamous cell carcinoma cell line with enhanced tumorigenicity.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial region. The mechanism of carcinogenesis of OSCC is still unclear. Based on the previous cell line, HIOEC-B(a)P-96 (HB96), which we obtained by HPV16 E6/E7-immortalized human oral epithelial cells (HIOEC) and benzo(a)pyrene inducement, we prepared a new HB-second generation cancer cell line (HB-2) by continuous passage. Its characteristics such as morphology, proliferation activity, karyotype, and tumorigenesis were studied. The HB-2 cells displayed uncontrolled cell division and lost contact inhibition leading to cell overlap. Cells were polygonal and unevenly shaped, with an increased nucleus versus plasma ratio. Increased proliferative activity was confirmed by MTT assays. The tumorigenicity was confirmed by tumor growth experiments in nude mice. Therefore, the HB-2 and HB96 cell lines are useful tools to study the mechanism of carcinogenesis of OSCC in vitro for future genomic and proteomic analyses.

A novel approach to rescue immune escape in oral squamous cell carcinoma: Combined use of interferon-γ and LY294002.

Major histocompatibility complex (MHC) class I molecules have been found to be downmodulated in many tumors. The antigen-processing machinery (APM) genes, especially transporters associated with antigen processing (TAP)-1 and tapasin play important roles in the processing of class I antigens. In this study, we investigated the expression of TAP-1 and tapasin in oral squamous cell carcinoma (OSCC); the result indicated significant down-regulation in the expression of these genes. Interferon (IFN)-γ treatment was applied. After the addition of IFN-γ, unexpectedly, the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway was activated, which induced the proliferation of tumor cells. With the combined application of LY294002 (specific inhibitor of AKT signaling) and IFN-γ, tumor cell apoptosis was induced and the expression of TAP-1 and tapasin was still up-regulated. Hence, our method is a novel and efficient approach to use IFN-γ for rescuing the cells from immunosurveillance.

Redesign the α/ß fold to enhance the stability of mannanase Man23 from Bacillus subtilis.

In this work, we engineered the α/ß fold of mannanase Man23 based on its molecular structure analysis to obtain more stable variants. By introducing 31 single-site mutations in the α/ß fold and shuffling them, the incorporation of four mutations (K178R, K207R, N340R, and S354R) displayed a good balance between high activity and stability at higher temperature and broader pH. This quartet variant was characterized by an almost threefold increased activity and a sevenfold increased stability compared to native mannanase Man23. Our results suggest that such work is safe to increase our target protein stability with no loss of activity.

Decreased expression of S100A6 in oral squamous cell carcinoma.

We previously established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and other cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and S100A6 was shown as one of the significantly down-regulated proteins accompanying cellular transformation. S100A6 was further validated for its expression in the three cell lines and in the clinical samples of cancerous and paracancerous tissues from 30 primary OSCC patients. Western blot analysis and real-time PCR revealed the decreased S100A6 protein and mRNA levels in the cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry and real-time PCR also showed decreased S100A6 protein and mRNA levels in the cancerous tissues compared to the paracancerous tissues from OSCC patients. The results presented here suggest that the expression of S100A6 decreases along with the cancerization in OSCC both in vitro and in vivo.

Downregulation of tapasin expression in primary human oral squamous cell carcinoma: association with clinical outcome.

MHC class I peptide loading complex defects are frequently observed in tumor cells which facilitate tumor cells escaping from immune surveillance. Tapasin plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. The aim of this study was to investigate the expression of tapasin in primary oral squamous cell carcinoma (OSCC) and its potential clinical implication. Formalin-fixed, paraffin-embedded tumor biopsies from 67 patients with primary OSCC were analyzed for tapasin expression using immunohistochemistry. Tapasin promoter methylation status in OSCC cell lines was determined using ethylation-specific polymerase chain reaction, bisulphate genomic sequencing, and expression reactivation assay. Lack of tapasin expression was observed in 30 of 67 (43%) tumors and was significantly associated with poor pathologic differentiation grade of OSCC (P = 0.028). The cumulative 5-year survival rate was also significantly correlated with pathologic differentiation grade (P = 0.001) and tapasin expression level (P = 0.015). Decreased tapasin expression was an indicator of poor survival (P = 0.048). Tapasin promoter methylation was observed in all three OSCC cell lines examined, and the mRNA and protein levels of tapasin increased markedly after treatment with 5-aza-2'-deoxycytidine. Downregulation of tapasin is associated with a poor clinical outcome for OSCC patients and may serve as a prognostic biomarker. The promoter methylation may contribute to the tapasin downregulation in OSCC.