Bone morphogenetic protein receptor 1α promotes osteolytic lesion of oral squamous cell carcinoma by SHH-dependent osteoclastogenesis.

Oral squamous cell carcinoma (OSCC) is an aggressive tumor that usually invades the maxilla or mandible. The extent and pattern of mandibular bone invasion caused by OSCC are the most important factors determining the treatment plan and patients' prognosis. Yet, the process of mandibular invasion is not fully understood. The following study explores the molecular mechanism that regulates the mandibular invasion of OSCC by focusing on bone morphogenetic protein receptor 1α (BMPR1α) and Sonic hedgehog (SHH) signals. We found that BMPR1α was positively correlated to bone defect of OSCC patients. Mechanistically, BMPR1α signaling regulated the differentiation and resorption activity of osteoclasts through the interaction of OSCC cells and osteoclast progenitors, and this process was mediated by SHH secreted by tumor cells. The inhibition of SHH protected bone from tumor-induced osteolytic activity. These results provide a potential new treatment strategy for controlling OSCC from invading the jawbones.

pERK-mediated IL8 secretion can enhance the migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells.

BACKGROUND: Cancer stem cells (CSCs) drive tumor initiation and progression and participate in tumor chemoresistance. We recently discovered that oral squamous cell carcinoma (OSCC) cells that highly express CD10 (CD10H cells) present cancer stem cells (CSC)-associated characteristics, which, in turn, affect the tumor growth, epithelial-mesenchymal transition (EMT), and resistance to cisplatin. In this study, we further investigated this mechanism in vitro and in vivo. We hypothesized that IL8 might regulate migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells through the ERK pathway. METHODS: CD10 MicroBead Kit was used to select HN6 cells with high and low expression of CD10. The target protein IL8 was screened via protein chip assay. Lentiviral transduction and specific inhibitor were applied to investigate the signaling pathway. Real-time PCR, Western blot, and immunohistochemistry were used to analyze the mRNA and protein expression; transwell assay, spheroid formation assay, and cell viability assay were used to study the cell biological behavior in vitro; xenograft animal model was used to evaluate the tumor formation rate in vivo. RESULTS: Overexpression of CD10 promoted CSC-related genes expression and enhanced migration, invasion, spheroid formation, and chemoresistance in HN6 cells. Moreover, the overexpression of IL8 was detected in OSCC tumor tissue and cell lines (HN6 and CAL27) overexpressing CD10. IL8 secreted by CD10H HN6 promoted migration and invasion and restored tumor chemosensitivity via the p-ERK signaling pathway, while the inhibition of IL8 increased the chemosensitivity to cisplatin. CONCLUSIONS: IL8 secretion by CD10 positive cells promotes migration, invasion, and cisplatin resistance of OSCC via the p-ERK signaling pathway.

Cancer stemness of CD10-positive cells regulated by Hedgehog pathway promotes the resistance to cisplatin in oral squamous cell carcinoma.

OBJECTIVE: To explore the role of CD10 in cisplatin resistance of oral squamous cell carcinoma (OSCC) and its association with the Hedgehog (Hh) signaling pathway and cancer stem cells (CSCs). METHODS: The correlation between cell viability and CD10 expression was analyzed in different OSCC cell lines after the cisplatin treatment. Genes related to chemotherapy resistance, cancer stem cells and the epithelial-mesenchymal transition were detected by quantitative real-time PCR (qPCR) in CD10high and CD10low OSCC cells. Mouse xenograft model and venous metastasis model were used to explore the potential regulatory mechanism of the resistance effect of CD10 on cisplatin. RESULTS: The higher expression of CD10 gene in different cell lines displayed enhanced cisplatin resistance ability. The expression of genes related to chemotherapy resistance, cell stemness, and the epithelial-mesenchymal transition was significantly higher in CD10high cells compared with CD10low cells. Moreover, the combination of cisplatin and Hh pathway inhibitors significantly reduced the resistance of CD10 to cisplatin in the xenograft model and venous metastasis models. CONCLUSION: CD10-positive cells are implicated in developing cisplatin resistance of OSCC, which could be related to its cancer stem cell characteristics regulated by the Hedgehog pathway.

Adiponectin promotes human jaw bone marrow mesenchymal stem cell chemotaxis via CXCL1 and CXCL8.

Adiponectin (APN) is known to promote the osteogenic differentiation of human jaw bone marrow mesenchymal stem cells (h-JBMMSCs). However, the underlying mechanism has not been fully elucidated. Previously, we showed that APN could promote h-JBMMSC osteogenesis via APPL1-p38 by up-regulating osteogenesis-related genes. Here, we aimed to determine whether APN could promote h-JBMMSC chemotaxis through CXCL1/CXCL8. The CCK-8, wound healing and transwell assays were used to evaluate the proliferation, migration and chemotaxis of h-JBMMSCs with or without APN treatment. Chemotaxis-related genes were screened using RNA-seq, and the results were validated using real-time PCR and ELISA. We also performed Western blot using the AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, to identify the signalling pathway involved. We found that APN could promote h-JBMMSC chemotaxis in the co-culture transwell system. CXCL1 and CXCL8 were screened and confirmed as the up-regulated target genes. The APN-induced CXCL1/8 up-regulation to promote chemotaxis could be blocked by CXCR2 inhibitor SB225002. Western blot revealed that the phosphorylation of AMPK and p38 MAPK increased in a time-dependent manner with APN treatment. Additionally, WZ4003 and SB203580 could suppress the APN-induced overexpression of CXCL1 and CXCL8. The results of the transwell chemotaxis assay also supported the above results. Our data suggest that APN can promote h-JBMMSC chemotaxis by up-regulating CXCL1 and CXCL8.

The Osteogenesis Effect and Underlying Mechanisms of Local Delivery of gAPN in Extraction Sockets of Beagle Dogs.

A plastic and biodegradable bone substitute consists of poly (L-lactic-co-glycolic) acid and 30 wt % ß-tricalcium phosphate has been previously fabricated, but its osteogenic capability required further improvement. We investigated the use of globular adiponectin (gAPN) as an anabolic agent for tissue-engineered bone using this scaffold. A qualitative analysis of the bone regeneration process was carried out using µCT and histological analysis 12 weeks after implantation. CBCT (Cone Beam Computed Tomography) superimposition was used to characterise the effect of the different treatments on bone formation. In this study, we also explored adiponectin's (APN) influence on primary cultured human jaw bone marrow mesenchymal stem cells gene expressions involved in the osteogenesis. We found OPEN ACCESS Int. J. Mol. Sci. 2015, 16 24947 that composite scaffolds loaded with gAPN or bone morphogenetic protein 2 (BMP2) exhibited significantly increased bone formation and mineralisation following 12 weeks in the extraction sockets of beagle dogs, as well as enhanced expression of osteogenic markers. In vitro investigation revealed that APN also promoted osteoblast differentiation of primary cultured human jaw bone marrow mesenchymal stem cells (h-JBMMSCs), accompanied by increased activity of alkaline phosphatase, greater mineralisation, and production of the osteoblast-differentiated genes osteocalcin, bone sialoprotein and collagen type I, which was reversed by APPL1 siRNA. Therefore, the composite scaffold loaded with APN exhibited superior activity for guided bone regeneration compared with blank control or Bio-Oss® (a commercially available product). The composite scaffold with APN has significant potential for clinical applications in bone tissue engineering.

Melanoma differentiation-associated gene-7/interleukin-24 as a potential prognostic biomarker and second primary malignancy indicator in head and neck squamous cell carcinoma patients.

The significance of melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) expression in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study was designed to investigate and evaluate the clinical significance of MDA-7/IL-24 expression in HNSCC by detecting expression by immunostaining in 131 HNSCC specimens. The function of MDA-7/IL-24 was investigated by real-time polymerase chain reaction (PCR) and Western blot in Ad5.mda-7-infected HNSCC cell lines. Our results showed that MDA-7/IL-24 was mainly expressed in the cytoplasm of HNSCC cells. MDA-7/IL-24 high patients presented with a favorable postoperative prognosis compared with MDA-7/IL-24 low patients, and high expression of MDA-7/IL-24 was significantly correlated with a lower incidence of second primary malignancies (SPMs) in the head and neck regions. In vitro assays showed that high expression of MDA-7/IL-24 could upregulate the expression of the epithelial terminal differentiation markers cytokeratin (KRT) 1, KRT4, KRT13, phosphorylated endoplasmic reticulum stress protein (p)-EIF2a, and the apoptosis-related protein cleaved caspase-3. It also downregulated the epithelial proliferative markers KRT5, KRT14, Integrin ß4, and anti-apoptosis protein Bcl-2, which might be partially involved in the underlying mechanisms of Ad.mda-7-mediated HNSCC differentiation and apoptosis. Our results indicate that MDA-7/IL-24 can be a prognostic biomarker and an indicator of second primary malignancies (SPM) in HNSCC.

Prognostic factors associated with pulp status in patients with cracked teeth treated with occlusal veneer: A 6- through 24-month prospective clinical study.

BACKGROUND: The authors evaluated the prognostic factors associated with pulp status in patients with cracked teeth (CT) treated with occlusal veneer. METHODS: An analysis of 80 CT (71 patients) with 1 or more crack lines (CLs) and normal pulp vitality or reversible pulpitis was performed. All patients received occlusal veneer and their demographic and clinical data were recorded. Pulp status and clinical features were recorded at 1 week and posttreatment at 1, 2, 3, 6, 12, 18, and 24 months. RESULTS: Maxillary first molars were commonly involved (30 [38%]). The number of CLs on the finish line ranged from 1 through 7 and most had 3 CLs (24 [30%]). The number of CLs through preparation on the finish line ranged from 0 through 4, and 2 CLs (42 [53%]) were the most prevalent. During follow-up, 5 of 80 CT progressed to pulp disease, resulting in a success rate of 93.8%. Results of the Cox model and Kaplan-Meier analysis showed that probing depth greater than 6 mm, widening periodontal ligament of apical area, more than 4 CLs on finish line, and more than 2 CLs through preparation on the finish line were risk factors associated with pulp status (P < .05). CONCLUSIONS: Occlusal veneer can protect CT without preventive root canal therapy. PRACTICAL IMPLICATIONS: The success rate and risk factors of pulp disease in CT restored with occlusal veneer are reported.

Management and prognosis of a vital cracked tooth by occlusal veneer for 14 months: A case report.

Key Clinical Message: An occlusal veneer is an ultrathin restoration method and a minimally invasive approach that can preserve more dental tissue and provide better aesthetic outcomes, thus increasing patient satisfaction. Abstract: An occlusal veneer is an ultrathin restoration method and a minimally invasive approach that can preserve more dental tissue and provide better aesthetic outcomes, thus increasing patient satisfaction; however, no previous studies reported on treating cracked teeth using occlusal veneer. Accordingly, we described the diagnosis and treatment process of a cracked tooth using occlusal veneer in a single case. A 29-year-old male presented at our dental clinic complaining of biting pain in the mandibular molar on the right-hand side. A routine oral examination with radiography was performed to evaluate the oral condition and treatment planning. The #16 tooth had a crack line surrounding the whole distal-lingual cusp from the occlusal surface. After discussing various therapeutic options with the patient, an occlusal veneer was performed. One week after treatment with occlusal veneer, the patient had no complaints. A 14-month follow-up showed promising clinical and radiographic outcomes. Occlusal veneer is an alternative treatment option for a cracked tooth, as it can preserve more dental tissue and potentially save pulp vitality.

Biting and thermal sensitivity relief of cracked tooth restored by occlusal veneer: A 12-to 24 months prospective clinical study.

OBJECTIVES: To analyze the relief time and risk factors of biting/thermal sensitivity in cracked tooth (CT) restored using occlusal veneer. METHODS: 63 CT were analyzed, and their demographic and clinical data and medical history were collected. Patients were followed-up to examine the relief of thermal/biting sensitivity. RESULTS: The maxillary first molar was the most prevalent (N = 25, 40%). The number of crack lines on the finish line ranged from 1 to 6 while the number of crack lines through preparation on the finish line from 0 to 4. Pain relief achieved steadily to 52% for thermal and 62% for biting at 1 week to over 90% for each by 3 months and was completely resolved (no pain) for each by 12 months. Painful of lateral percussion was related to a long period of thermal sensitivity (≥1 month) after restoration with occlusal veneer. The number of crack lines through preparation on the finish line >2 was correlated with biting sensitivity (≥1 month) post-treatment. CONCLUSIONS: Most patients (>90%) became asymptomatic of biting and thermal sensitivity within 3 months of CT restored by occlusal veneer. Lateral percussion and the number of crack lines through preparation on the finish line could be significant factors affecting postoperative symptoms. CLINICAL SIGNIFICANCE: Occlusal veneer is an ultrathin restoration and had no need for restricting clinical crown height, which could protect and relief the biting/thermal sensitivity of CT without preventive root canal therapy.

Adiponectin inhibits lipoplysaccharide-induced inflammation and promotes osteogenesis in hPDLCs.

Periodontal diseases are infections of the structures that surround and support the teeth; they are characterized by local inflammation and alveolar bone loss. Most treatments focus on only one aspect, inhibiting inflammation, or promoting osteoblasts. We set out to develop a new method that would intervene in the two aspects simultaneously. Adiponectin (APN), secreted by adipocytes, inhibits the inflammatory response and promotes osteogenesis. However, its role in human periodontal ligament cells (hPDLCs) is unclear. Therefore, we aim to investigate whether APN could suppress lipopolysaccharide (LPS)-induced inflammation and promote osteogenesis in hPDLCs. In the present study, we stimulated hPDLCs with LPS in the presence or absence of APN. Real-time PCR and Western blotting results demonstrated that APN partially inhibited the activation of the classical nuclear factor κ-B (NF-κB) pathway. These results were confirmed by a change of expressions of NF-κB downstream inflammatory genes, such as decreased cyclooxygenase (COX)-2 and tumor necrosis factor α (TNF-α), along with increased interleukin (IL)-10. As for the role of APN in osteogenesis, Alizarin Red S staining showed that APN treatment induced more calcium deposition nodules than controls. We also found that APN enhanced the expression of osteoblast-related genes (osteopontin (OPN), collagen 1, osteocalcin, alkaline phosphatase, runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein 2) in hPDLCs via the APPL1 (the adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1)/p38 signal transduction pathway. Therefore, APN inhibits LPS-induced inflammation and promotes osteogenesis in hPDLCs and may have potential therapeutic value in treating periodontitis by inhibiting the inflammatory lesions and contributing to bone tissue regeneration.

Porphyromonas, Treponema, and Mogibacterium promote IL8/IFNγ/TNFα-based pro-inflammation in patients with medication-related osteonecrosis of the jaw.

Objective: Refractory infection is an important factor affecting the progression of medication-related osteonecrosis of the jaw (MRONJ) from clinical stage I to stage II/III. The aim of this study was to explore the distribution of bacteria and their association with the inflammatory pathway of stage II/III MRONJ. Materials and Methods: Nine specimens of fresh inflammation tissue, located next to the necrotic bone or sequestrum, were collected from MRONJ patients. Nine specimens from normal oral mucosa were collected from healthy patients. The 16S rRNA gene sequencing method was used to determine the distribution characteristics of the bacterial colony. The protein microarray analysis was used to detect the expression of inflammatory cytokines. Results: The average relative abundance of Bacteroidetes, Spirochaetes, Synergistetes, and Tenericutes was higher, while Proteobacteria and Actinobacteria were lower in the MRONJ group. Most pro-inflammatory cytokines were up-regulated in the MRONJ group; yet, only IFNγ, TNFα, and IL8 showed statistical differences (P < 0.05). Porphyromonas and Treponema were positively correlated with IL8, and Mogibacterium was positively correlated with IFNγ and TNFα. Conclusions: IL8/IFNγ/TNFα pro-inflammatory effect caused by Porphyromonas, Treponema, and Mogibacterium may be the leading cause of advancing MRONJ and thus may be used as a new target for infection control.

Influence on proliferation and adhesion of human gingival fibroblasts from different titanium surface decontamination treatments: An in vitro study.

OBJECTIVES: To investigate the effects of different decontamination treatments on microstructure of titanium (Ti) surface as well as proliferation and adhesion of human gingival fibroblasts (HGFs). MATERIAL AND METHODS: Ti discs with machined (M) and sand blasted, acid etched (SAE) surfaces were treated with five different decontamination treatments: (1) stainless steel curette (SSC), ultrasonic system with (2) straight carbon fiber tip (UCF) or (3) metal tip (UM), (4) rotating Ti brush (RTB), and (5) Er:YAG laser (30 mJ/pulse at 30 Hz). Surface roughness was analyzed under optical interferometry. HGFs were cultured on each disc. Proliferation and adhesive strength were analyzed. qRT-PCR and ELISA were performed to detect the RNA and protein expression of FAK, ITGB1, COL1A1, and FN1 respectively from different Ti surfaces. RESULTS: Surface roughness increased on M surface. Proliferation, adhesive strength and gene expression were higher on M surface than SAE surface. Decontamination treatments affected surface parameters significantly (P < 0.001), making M surface less smooth while SAE surface became less rough. SSC, UCF, UM and RTB decreased proliferation on M surfaces significantly (P < 0.05). UCF, RTB and laser increased proliferation on SAE surface significantly (P < 0.05). UM decreased adhesive strength on M surface significantly and laser increased adhesive strength on SAE surface significantly (P < 0.05). Gene expression increased with time and was altered by decontamination treatments significantly (P < 0.001). CONCLUSIONS: Decontamination treatments influence surface roughness and cell behavior of HGFs. Laser might be an optimal decontamination treatment which has the least negative effect on M surface and the most positive effect on SAE surface.

Bevacizumab Regulates Cancer Cell Migration by Activation of STAT3.

There are numerous clinical cases indicating that long-term use of bevacizumab may increase the invasiveness of tumors. However, to date, little is known about underlying molecular mechanisms. Therefore, the purpose of our study was to investigate effects of bevacizumab in four cancer cells lines (WSU-HN6, CAL27, Tca83, and HeLa). It was found to promote migration and invasion in the WSU-HN6 and Tca83 cases, while exerting inhibitory effects in CAL27 and HeLa cells. The signal transducer and activator of transcription (STAT) 3 inhibitors niclosamide and S3I-201 inhibited the STAT3 signal pathway, which is activated by bevacizumab. These inhibitors also substantially blocked bevacizumab-induced migration of WSU-HN6 and Tca83 cells. Bevacizumab upregulated interleukin (IL)-6 and phosphorylated (p)-STAT3 expression time-dependently. Therefore, we propose that bevacizumab has differential effects on the migration of different cancer cell lines and promotes migration via the IL-6/STAT3 signaling pathway.

High MMP-21 expression in metastatic lymph nodes predicts unfavorable overall survival for oral squamous cell carcinoma patients with lymphatic metastasis.

The aim of the present study was to examine the clinical significance of lymph node metastatic (LNM) foci in predicting the overall survival of oral squamous cell carcinoma (OSCC) patients with LNM. MMP-21 was screened based on the LNM animal model of OSCC. Then four proteins, matrix metalloproteinase (MMP)-2, MMP-21, vascular endothelial growth factor (VEGF)-C and VEGF receptor (VEGFR)-3 were examined by immunohistochemistry in 63 OSCC specimens, including the primary tumors (PTs) and the corresponding LNM foci. The expression levels between the PTs and LNM foci were compared by Wilcoxon paired test. Relationships between expression of the four proteins and patient overall survival were assessed by Kaplan-Meier based on the median of the labeling index. The Cox proportional hazards model was used to assess the relative hazard factors. MMP-21 and VEGF-C expression levels were higher in the LNM foci than levels in the PTs. Results showed that MMP-2 and VEGF-C expression levels in the PTs and MMP-2, MMP-21 and VEGF-C expression in the LNM foci correlated with the overall survival of the OSCC patients with lymphatic metastasis. MMP-21 expression level in the LNM foci was the most reliable predictor among all the tested factors. These results suggest that high MMP-21 expression in LNM foci can be used to predict survival in OSCC patients with LNM. Characteristics of LNM foci may be more reliable than PT characteristics in predicting the overall survival of OSCC patients with lymphatic metastasis.

Generation of homologous cell pairs using the oral lymphatic system.

The purpose of this study is to establish in vivo and in vitro models for studying lymphatic metastasis of squamous cell carcinoma (SCC). Three cell lines CAL-27, Tca-83, and HeLa were injected into the tongue of nude mice. Forty days after injection, we could isolate cells of 2 homologous cell lines LN-CAL-27 and LN-HeLa from lymph node metastasis lesions. Then, the homologous cell pairs were compared by the CCK-8 assay, wound healing assay, real-time PCR, western blot, and animal experiments. The results showed that all the three cell lines could be used to establish lymphatic metastasis animal models, and the lymphatic metastasis process was observed clearly. In addition, the homologous cell pairs performed differently from parent lines with respect to biological behavior and lymphatic metastasis-related gene and protein expression. In conclusion, CAL-27, Tca-83, and HeLa cells could be used to simulate the lymphatic metastasis process of oral cancer in vivo. Furthermore, the homologous cell pairs (CAL-27 and LN-CAL-27; HeLa and LN-HeLa) are potential tools for in vitro investigation of the mechanisms underlying metastasis.

Molecular characteristics of homologous salivary adenoid cystic carcinoma cell lines with different lung metastasis ability.

Although the homologous salivary adenoid cystic carcinoma (SACC) cell lines SACC-83 and SACC-LM have already been used as SACC models to investigate the underlying mechanisms of metastasis, the molecular features of these SACC cell lines remain unclear. We screened 136 genes related to metastasis in order to investigate the biological and molecular properties of these two cell lines by short tandem repeat (STR) profiling, immunostaining, transwell invasion assay, real-time PCR and western blotting. STR and immunostaining results showed that SACC-83 and SACC-LM are homologous cancer cell lines, derived from adenoepithelial cells and, to date, are not contaminated by each other or other cancer cell lines. Transwell invasion assay results showed that SACC-LM had increased invasion ability compared to SACC-83. 29 of the 136 differentially expressed genes including EREG, S100P, cyclooxygenase (COX)-2, phospho-Akt (p-Akt), matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14 were found following gene screening in SACC-83 and SACC-LM cells. Compared with SACC-83, SACC-LM presents higher expression of COX-2, S100P and lower expression of MMP-2, p-Akt, which could be candidates for identifying the homologous pair cell lines.