Effects of novel lactoferrin peptides on LPS-induced alveolar bone destruction in a rat model.

To develop novel bovine lactoferrin (bLF) peptides targeting bLF-tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding sites, we identified two peptides that could target bLF-TRAF6 binding sites using structural analysis. Moreover, another peptide that could bind to the TRAF6 dimerization area was selected from the bLF sequence. The effects of each peptide on cytokine expression in lipopolysaccharide (LPS)-stimulated osteoblasts (ST2) and on osteoclastogenesis were examined using an LPS-treated co-culture of primary bone marrow cells (BMCs) with ST2 cells and a single culture of osteoclast precursor cells (RAW-D) treated with soluble receptor activator of NF-κB ligand. Finally, the effectiveness of these peptides against LPS-induced alveolar bone destruction was assessed. Two of the three peptides significantly suppressed LPS-induced TNF-α and interleukin-1ß expression in ST2 cells. Additionally, these peptides inhibited and reversed LPS-induced receptor activator of NF-κB ligand (RANKL) upregulation and osteoprotegerin (OPG) downregulation, respectively. Furthermore, both peptides significantly reduced LPS-induced osteoclastogenesis in the BMC-ST2 co-culture and RANKL-induced osteoclastogenesis in RAW-D cells. In vivo, topical application of these peptides significantly reduced the osteoclast number by downregulating RANKL and upregulating OPG in the periodontal ligament. It is indicated that the novel bLF peptides can be used to treat periodontitis-associated bone destruction.

High-frequency low-intensity semiconductor laser irradiation enhances osteogenic differentiation of human cementoblast lineage cells.

PURPOSE: Laser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells. METHODS: HCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0-2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining. RESULTS: The laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation. CONCLUSION: High-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.

Translational research of boron neutron capture therapy for spinal cord gliomas using rat model.

Boron neutron capture therapy (BNCT) is a type of targeted particle radiation therapy with potential applications at the cellular level. Spinal cord gliomas (SCGs) present a substantial challenge owing to their poor prognosis and the lack of effective postoperative treatments. This study evaluated the efficacy of BNCT in a rat SCGs model employing the Basso, Beattie, and Bresnahan (BBB) scale to assess postoperative locomotor activity. We confirmed the presence of adequate in vitro boron concentrations in F98 rat glioma and 9L rat gliosarcoma cells exposed to boronophenylalanine (BPA) and in vivo tumor boron concentration 2.5 h after intravenous BPA administration. In vivo neutron irradiation significantly enhanced survival in the BNCT group when compared with that in the untreated group, with a minimal BBB scale reduction in all sham-operated groups. These findings highlight the potential of BNCT as a promising treatment option for SCGs.

CD40-CD40 ligand interaction between periodontal ligament cells and cementoblasts enhances periodontal tissue remodeling in response to mechanical stress.

OBJECTIVE: We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L-expressing cells under mechanical stress. Furthermore, we investigated whether CD40-CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. BACKGROUND: PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40-CD40L interaction induces by converting the force to biological functions between the cement-PDL complex, are not fully understood. METHODS: The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel-titanium closed-coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP-expressing CD40L plasmid vector was transfected into HCEM (CD40L-HCEM). CD40L-HCEM was co-cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co-culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation- and extracellular matrix (ECM)-related genes was analyzed by real-time PCR. RESULTS: Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro-stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co-culturing CD40L-HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co-culturing CD40L-HCEM with hPDS upregulated cytodifferentiation- and ECM-related genes but did not affect cell viability and proliferation. CONCLUSION: We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis.

Porphyromonas gingivalis-odontogenic infection is the potential risk for progression of nonalcoholic steatohepatitis-related neoplastic nodule formation.

Porphyromonas gingivalis (P.g.), a major periodontal pathogen is a known risk factor for various systemic diseases. However, the relationship between P.g. and nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is unclear. Thus, we aimed to elucidate whether P.g.-odontogenic infection promotes NASH-related HCC development/progression and to clarify its mechanism. Using high-fat diet (HFD)-induced NASH mouse model, P.g. was infected odontogenically. After 60 weeks of infection, tumor profiles were examined. Chow diet (CD) groups were also prepared at 60 weeks. Nodule formation was only seen in HFD-mice. P.g.-odontogenic infection significantly increased the mean nodule area (P = 0.0188) and tended to promote histological progression score after 60 weeks (P = 0.0956). Interestingly, P.g. was detected in the liver. HFD-P.g. (+) showed numerous TNF-α positive hepatic crown-like structures and 8-OHdG expression in the non-neoplastic liver. In P.g.-infected hepatocytes, phosphorylation of integrin ß1 signaling molecules (FAK/ERK/AKT) was upregulated in vitro. In fact, total AKT in the liver of HFD-P.g. (+) was higher than that of HFD-P.g. (-). P.g.-infected hepatocytes showed increased cell proliferation and migration, and decreased doxorubicin-mediated apoptosis. Integrin ß1 knockdown inhibited these phenotypic changes. P.g.-odontogenic infection may promote the progression of neoplastic nodule formation in an HFD-induced NASH mouse model via integrin signaling and TNF-α induced oxidative DNA damage.

Molecular Mechanisms of Inhibitory Effects of Bovine Lactoferrin on Invasion of Oral Squamous Cell Carcinoma.

Lactoferrin (LF), an iron-binding glycoprotein, has been reported to have anticancer properties. However, the molecular mechanisms behind its anticancer effects on oral squamous cell carcinoma (OSCC) have not yet been elucidated. Therefore, we aimed to clarify the effects of LF on invasion of OSCC, and its underlying molecular mechanism. OSCC cell lines, HSC2 and HOC313, were treated with bovine LF (bLF). The effects of bLF on cell invasion were examined by a chamber migration assay, wound healing assay, and Boyden chamber method with a basement-membrane-analogue. Expression levels of MMP-1, MMP-3, and AP-1 were examined using RT-PCR, qRT-PCR, and western blotting. Roles of LRP1, a receptor of bLF, on cell invasion were analyzed using siLRP1 knockdown cells. Furthermore, to clarify the importance of LRP1 in invasion, the effects of bLF on tPA-induced invasion of OSCC cells were examined. The invasion assays showed that bLF suppressed invasion of the OSCC cells. Moreover, bLF down-regulated AP-1, and resulted in reductions of MMP-1 and MMP-3. With SiLRP1 knockdown, OSCC cells failed to induce their invasion, and bLF was not able to exert its effects on invasion. Furthermore, bLF remarkably inhibited tPA-induced cell invasion. These findings suggest the importance of LRP1 in bLF-suppressed invasion of OSCC cells via the reduction of AP-1 and MMP production.

Bovine Lactoferrin Suppresses Tumor Angiogenesis through NF-κB Pathway Inhibition by Binding to TRAF6.

Tumor angiogenesis is essential for tumor progression. The inhibition of tumor angiogenesis is a promising therapy for tumors. Bovine lactoferrin (bLF) has been reported as an anti-tumor agent. However, bLF effects on tumor angiogenesis are not well demonstrated. This study evaluated the inhibitory effects of bLF on tumor angiogenesis in vivo and in vitro. Herein, tumor endothelial cells (TECs) and normal endothelial cells (NECs) were used. Proliferation, migration, tube formation assays, RT-PCR, flow cytometry, Western blotting, siRNA experiments and immunoprecipitation were conducted to clarify the mechanisms of bLF-induced effects. CD-31 immunoexpression was examined in tumor tissues of oral squamous cell carcinoma mouse models with or without Liposomal bLF (LbLF)-administration. We confirmed that bLF inhibited proliferation/migration/tube formation and increased apoptosis in TECs but not NECs. TNF receptor-associated factor 6 (TRAF6), p-p65, hypoxia inducible factor-α (HIF-1α) and vascular endothelial growth factor (VEGF) were highly expressed in TECs. In TECs, bLF markedly downregulated VEGF-A, VEGF receptor (VEGFR) and HIF-1α via the inhibition of p-p65 through binding with TRAF6. Since NECs slightly expressed p-p65, bLF-TRAF-6 binding could not induce detectable changes. Moreover, orally administrated LbLF decreased CD31-positive microvascular density only in TECs. Hence, bLF specifically suppressed tumor angiogenesis through p-p65 inhibition by binding to TRAF6 and suppressing HIF-1α activation followed by VEGF/VEGFR down-regulation. Collectively, bLF can be an anti-angiogenic agent for tumors.

Salivary gland polymorphous adenocarcinoma: Clinicopathological features and gene alterations in 36 Japanese patients.

BACKGROUND: Polymorphous adenocarcinoma is a common intraoral minor salivary gland carcinoma in Western countries but is extremely rare in Japan. The current study aimed to characterize the clinicopathological features and status of molecular alterations of polymorphous adenocarcinoma-associated genes, such as PRKD1/2/3, ARID1A, and DDX3X, in a large cohort of Japanese patients with polymorphous adenocarcinoma. METHODS: We examined the cases of 36 Japanese patients with salivary gland polymorphous adenocarcinoma and 26 cases involving histopathological mimics. To detect gene splits, fluorescence in situ hybridization was carried out for polymorphous adenocarcinoma-associated genes. Additionally, we applied a SNaPshot multiplex assay to identify PRKD1 hotspot mutations. RESULTS: This study revealed the indolent clinical course of polymorphous adenocarcinoma with a high 10-year overall survival rate (92.9%), accompanied by occasional local recurrences and cervical lymph node metastasis (23.3%). Twenty cases (55.6%) of polymorphous adenocarcinoma (but none of the mimics) exhibited alterations in at least one polymorphous adenocarcinoma-associated gene. Rearrangement of polymorphous adenocarcinoma-associated genes and PRKD1 E710D were identified in 17 (47.2%) and 4 (11.1%) cases, respectively; one case showed coexisting PRKD3 split and PRKD1 E710D. In the multivariate analysis, high clinical stage (p = 0.0005), the presence of prominent nucleoli (p = 0.0003), and ARID1A split positivity (p = 0.004) were independent risk factors for disease-free survival. CONCLUSION: Japanese patients with polymorphous adenocarcinoma showed clinicopathological features similar to those reported in Western countries. This study disclosed that polymorphous adenocarcinoma-associated genetic alterations were common and specific findings in polymorphous adenocarcinomas. The diagnostic role and possible prognostic significance of polymorphous adenocarcinoma-associated genetic alterations in polymorphous adenocarcinomas were suggested.

Oral administration of bovine lactoferrin suppresses the progression of rheumatoid arthritis in an SKG mouse model.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory bone destruction in which tumor necrosis factor alpha (TNF-α) plays a key role. Bovine lactoferrin (bLF) is a multifunctional protein with anti-inflammatory and immunomodulatory properties. This study aimed to clarify the inhibitory effects of bLF on the pathological progression of RA. The mannan-induced arthritis model in SKG mice (genetic RA model) was used. Orally applied liposomal bLF (LbLF) markedly reduced ankle joint swelling and bone destruction. Histologically, pannus formation and osteoclastic bone destruction were prevented in the LbLF-treated animals. Moreover, orally administered LbLF improved the balance between Th17 cells and regulatory T cells isolated from the spleen of mannan-treated SKG mice. In an in vitro study, the anti-inflammatory effects of bLF on TNF-α-induced TNF-α production and downstream signaling pathways were analyzed in human synovial fibroblasts from RA patients (RASFs). bLF suppressed TNF-α production from RASFs by inhibiting the nuclear factor kappa B and mitogen-activated protein kinase pathways. The intracellular accumulation of bLF in RASFs increased in an applied bLF dose-dependent manner. Knockdown of the lipoprotein receptor-related protein-1 (LRP1) siRNA gene reduced bLF expression in RASFs, indicating that exogenously applied bLF was mainly internalized through LRP-1. Immunoprecipitated proteins with anti-TNF receptor-associated factor 2 (TRAF2; an adapter protein/ubiquitin ligase) included bLF, indicating that bLF binds directly to the TRAF2-TRADD-RIP complex. This indicates that LbLF may effectively prevent the pathological progression of RA by suppressing TNF-α production by binding to the TRAF2-TRADD-RIP complex from the RASFs in the pannus. Therefore, supplemental administration of LbLF may have a beneficial effect on preventive/therapeutic reagents for RA.

Elimination of Porphyromonas gingivalis inhibits liver fibrosis and inflammation in NASH.

AIM: Non-alcoholic steatohepatitis (NASH) is a critical liver disease showing potential progression to liver cirrhosis/cancer. Previously, we had reported that odontogenic infection of Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, exacerbates fibrosis in NASH through the production of fibrosis mediators such as transforming growth factor-ß1 (TGF-ß1) and galectin-3. In this study, we determined the effects of therapeutic interventions using antibiotics on NASH progression induced by P. gingivalis odontogenic infection. MATERIALS AND METHODS: To eliminate P. gingivalis infection, the macrolide antibiotic [azithromycin (AZM)] was applied locally and/or systemically to a high-fat-diet-induced NASH mouse model with P. gingivalis odontogenic infection. After treatment with AZM, liver and periodontal tissues were analysed with focus on inflammation markers such as tumour necrosis factor-α (TNF-α)/Tnf-α and interleukin-1ß (IL-1ß)/Il-1ß, and fibrosis markers such as galectin-3, phosphorylated Smad2 (pSmad2; key signalling molecule of TGF-ß1), and the number of hepatic crown-like structures (hCLSs). Further, Non-alcoholic Fatty Liver Disease Activity Score (NAS), a common histological scoring system, and fibrosis area were evaluated. RESULTS: P. gingivalis odontogenic infection significantly increased the expression of Tnf-α, Il-1ß, galectin-3, and pSmad2, the number of hCLSs, and NAS score, whereas the elimination of P. gingivalis odontogenic infection, especially local with or without systemic application, significantly inhibited them. CONCLUSION: This study suggests that elimination of P. gingivalis odontogenic infection inhibited NASH progression induced by the infection.

FK866 Protects Human Dental Pulp Cells against Oxidative Stress-Induced Cellular Senescence.

FK866 possesses various functional properties, such as anti-angiogenic, anti-cancer, and anti-inflammatory activities. We previously demonstrated that premature senescence of human dental pulp cells (hDPCs) was induced by hydrogen peroxide (H2O2). The present study aimed to investigate whether H2O2-induced premature senescence of hDPCs is affected by treatment with FK866. We found that FK866 markedly inhibited the senescent characteristics of hDPCs after exposure to H2O2, as revealed by an increase in the number of senescence-associated ß-galactosidase (SA-ß-gal)-positive hDPCs and the upregulation of the p21 and p53 proteins, which acts as molecular indicators of cellular senescence. Moreover, the stimulatory effects of H2O2 on cellular senescence are associated with oxidative stress induction, such as excessive ROS production and NADPH consumption, telomere DNA damage induction, and upregulation of senescence-associated secretory phenotype factors (IL-1ß, IL-6, IL-8, COX-2, and TNF-α) as well as NF-κB activation, which were all blocked by FK866. Thus, FK866 might antagonize H2O2-induced premature senescence of hDPCs, acting as a potential therapeutic antioxidant by attenuating oxidative stress-induced pathologies in dental pulp, including inflammation and cellular senescence.

Identification of regulatory mRNA and microRNA for differentiation into cementoblasts and periodontal ligament cells.

OBJECTIVE: Periodontitis causes periodontal tissue destruction and results in physiological tooth dysfunction. Therefore, periodontal regeneration is ideal therapy for periodontitis. Mesenchymal stem cells (MSCs) are useful for periodontal regenerative therapy as they can differentiate into periodontal cells; however, the underlying regulatory mechanism is unclear. In this study, we attempted to identify regulatory genes involved in periodontal cell differentiation and clarify the differentiation mechanism for effective periodontal regenerative therapy. BACKGROUND: The cementum and periodontal ligament play important roles in physiological tooth function. Therefore, cementum and periodontal ligament regeneration are critical for periodontal regenerative therapy. Mesenchymal stem cell transplantation can be a common periodontal regenerative therapy because these cells have multipotency and self-renewal ability, which induces new cementum or periodontal ligament formation. Moreover, MSCs can differentiate into cementoblasts. Cementoblast- or periodontal ligament cell-specific proteins have been reported; however, it is unclear how these proteins are regulated. MicroRNA (miRNA) can also act as a key regulator of MSC function. Therefore, in this study, we identified regulatory genes involved in cementoblast or periodontal cell differentiation and commitment. METHODS: Human MSCs (hMSCs), cementoblasts (HCEM), and periodontal ligament cells (HPL cells) were cultured, and mRNA or miRNA expression was evaluated. Additionally, cementoblast-specific genes were overexpressed or suppressed in hMSCs and their expression levels were investigated. RESULTS: HCEM and HPL cells expressed characteristic genes, of which we focused on ets variant 1 (ETV1), miR-628-5p, and miR-383 because ETV1 is a differentiation-related transcription factor, miR-628-5p was the second-highest expressed gene in HCEM and lowest expressed gene in HPL cells, and miR-383 was the highest expressed gene in HCEM. miR-628-5p and miR-383 overexpression in hMSCs regulated ETV1 mRNA expression, and miR-383 overexpression downregulated miR-628-5p expression. Moreover, miR-383 suppression decreased miR-383 expression and enhanced ETV1 mRNA expression, but miR-383 suppression also decreased miR-628-5p. Furthermore, silencing of ETV1 expression in hMSCs regulated miR-628-5p and miR-383 expression. Concerning periodontal cell commitment, miR-628-5p, miR-383, and ETV1 regulated the expression of HCEM- or HPL cell-related genes by adjusting the expression of these miRNAs. CONCLUSION: HCEM and HPL cells show characteristic mRNA and miRNA profiles. In particular, these cells have specific miR-383, miR-628-5p, and ETV1 expression patterns, and these genes interact with each other. Therefore, miR-383, miR-628-5p, and ETV1 are key genes involved in cementogenesis or HPL cell differentiation.

Porphyromonas gingivalis, a cause of preterm birth in mice, induces an inflammatory response in human amnion mesenchymal cells but not epithelial cells.

INTRODUCTION: Inflammation and infection, including dental infectious diseases, are factors that can induce preterm birth. We previously reported that mice with dental Porphyromonas gingivalis infection could be used as a model of preterm birth. In this model, cyclooxygenase (COX)-2 and interleukin (IL)-1ß levels are increased, and P. gingivalis colonies are observed in the fetal membrane. However, the mechanism underlying fetal membrane inflammation remains unknown. Therefore, we investigated the immune responses of human amnion to P. gingivalis in vitro. METHODS: Epithelial and mesenchymal cells were isolated from human amnion using trypsin and collagenase, and primary cell cultures were obtained. Confluent cells were stimulated with P. gingivalis lipopolysaccharide (P.g-LPS) or P. gingivalis. mRNA expressions of IL-1ß, IL-8, IL-6 and COX-2, protein expressions of nuclear factor (NF)-κB pathway components and culture medium levels of prostaglandin E2 were evaluated. RESULTS: Following stimulation with 1 µg/mL P.g-LPS, the mRNA expression levels of IL-1ß, IL-8, IL-6 and COX-2 in mesenchymal cells were increased 5.9-, 3.3-, 4.2- and 3.1-fold, respectively. Similarly, the expression levels of IL-1ß, IL-8, IL-6 and COX-2 in mesenchymal cells were increased by 7.6-, 8.2-, 13.4- and 9.3-fold, respectively, after coculture with P. gingivalis. Additionally, stimulation with P.g-LPS or P. gingivalis resulted in the activation of NF-κB signaling and increased production of IL-1ß and prostaglandin E2. In contrast, no significant changes were observed in epithelial cells. DISCUSSION: Our findings suggest that mesenchymal cells might mediate the inflammatory responses to P. gingivalis and P.g-LPS, thereby producing inflammation that contributes to the induction of preterm birth.

Odontogenic infection by Porphyromonas gingivalis exacerbates fibrosis in NASH via hepatic stellate cell activation.

Odontogenic infection of Porphyromonas gingivalis (P.g.), a major periodontal pathogen, exacerbates pathological progression of non-alcoholic steatohepatitis (NASH). In this study, we aimed to clarify the detailed mechanism in which P.g. induced hepatic stellate cells (HSCs; key effector cells in liver fibrosis) activation. In the liver of high fat diet-induced NASH mouse model with P.g. odontogenic infection, immunolocalization of P.g. was detected. The number of hepatic crown-like structure, which was macrophage aggregation and related to liver fibrosis, was drastically increased and fibrosis area was also increased through upregulating immunoexpression of Phosphorylated Smad2 (key signaling molecule of TGF-ß1) and Galectin-3. P.g.-secreted trypsin-like enzyme [gingipain; an activator of protease-activated receptor 2 (PAR2)] stimulated HSC proliferation and differentiation through Smad and ERK signaling induced by TGF-ß1 produced from HSCs with P.g.-infection. Further, Galectin-3 produced from HSCs with P.g. infection and P.g.-derived LPS/lipoprotein stimulation stabilized TGFß-receptor II resulting in increasing sensitivity for TGF-ß1, finally leading to HSC differentiation via activating Smad and ERK signaling. In addition to them, hepatocytes (main component cells of liver) contributed to HSC activation through TGF-ß1 and Galectin-3 production in paracrine manner. Collectively, P.g.-odontogenic infection exacerbates fibrosis of NASH by HSC activation through TGF-ß1 and Gal-3 production from HSCs and hepatocytes.

The transition of tissue inhibitor of metalloproteinases from -4 to -1 induces aggressive behavior and poor patient survival in dedifferentiated liposarcoma via YAP/TAZ activation.

Liposarcoma (LS) is the most common soft-tissue sarcoma. Dedifferentiated liposarcoma (DDLS) shows more aggressive biological behavior than that of well-differentiated liposarcoma (WDLS), so advanced therapeutic agents based on molecular mechanism are urgently needed. Here we show that tissue inhibitors of metalloproteinases (TIMPs) from TIMP-1 to TIMP-4 are differently expressed and regulate yes-associated protein (YAP)/transcriptional co-activator with PDZ binding motif (TAZ) in LS. Database analysis showed high TIMP-1 expression in DDLS patients correlating with poor prognosis, but high TIMP-4 expression in WDLS patients with better prognosis. Stable TIMP-1 knockdown inactivated YAP/TAZ and inhibited proliferation, colony formation and migration in DDLS cells, which was rescued by a constitutive active YAP. However, stable overexpression of TIMP-1 showed the opposite in WDLS cells. Stable TIMP-4 knockdown activated YAP/TAZ and promoted proliferation and migration in WDLS cells, which was suppressed by YAP/TAZ inhibitor (verteporfin) or knockdown of YAP/TAZ. Recombinant TIMP-4 showed opposite results in DDLS cells. These results indicate that dedifferentiation in LS shifts the expression of TIMPs from type 4 to type 1, inducing more aggressive behavior and poor prognosis through YAP/TAZ activation, which can be prognostic markers and therapeutic targets for LS patients.

Molecular mechanisms underlying the inhibitory effects of bovine lactoferrin on osteosarcoma.

Osteosarcoma (OS) is one the most common primary malignancies of the bone in children and young adults with high metastasis. The use of non-toxic naturally derived compounds is one of present strategies in OS therapy to reduce secondary effects and chemo-resistance. Lactoferrin (LF), a transferrin protein derived from milk, currently appears to be an anticancer agent. However, its suppressive effects on OS have not been fully investigated. Therefore, we aimed to examine the molecular mechanism underlying the inhibitory effects of bovine LF (bLF) on OS. OS cell lines (NOS1, U2OS, MG63, and 143B) and an osteoblastic (ST2) were treated with bLF. Effects of bLF on OS-cell proliferation and migration were examined by proliferation and wound-healing assays. Expression levels of low-density-lipoprotein-receptor-related protein 1 (LRP1) and cytokines including interleukin-1 beta (IL-1ß), IL-6, and receptor-activator of nuclear factor kappa-Β ligand (RANKL) were measured using western blotting. Osteoclast formation was examined by co-culture of 143B, ST2, and bone marrow cells. We found that bLF down-regulated IL-1ß, IL-6, and RANKL expression and suppressed phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 in 143B cells; bLF also drastically suppressed 143B-activated RANKL production in ST2 cells. This may have contributed to the reduction in the number of differentiated osteoclasts. Taken together, these data reveal that bLF down-regulates NF-κB to attenuate proliferation, migration, and bone resorption in OS and the OS-microenvironment. This study provides new findings and the precise underlying mechanisms of the inhibitory effects of bLF on OS. bLF can be a possible therapeutic agent for OS patients.

Fetal Membrane Inflammation Induces Preterm Birth Via Toll-Like Receptor 2 in Mice With Chronic Gingivitis.

Inflammation is associated with preterm birth. We previously described a mouse model of chronic inflammation-induced preterm birth after dental Porphyromonas gingivalis infection. The aim of this study was to employ this model system to investigate the mechanisms through which enhanced uterine contractility induces preterm birth. Messenger RNA (mRNA) encoding contraction-associated proteins, such as oxytocin receptors, was measured at various gestational time points by real-time polymerase chain reaction (PCR). Spontaneous and oxytocin-induced uterine contractile activity at gestational day 18 was assessed using a tissue organ bath. The expression levels of Toll-like receptor 2 (TLR2), TLR4, cyclooxygenase (COX)-2, nuclear factor-kappa B (NF-κB) p65, and p38 mitogen-activated protein kinase (MAPK) on gestational day 18 were also determined by real-time PCR or Western blotting. Messenger RNA encoding contraction-associated proteins was increased at gestational day 18, and the spontaneous contractile activity (1.6-fold greater area under the contraction curve) and sensitivity to oxytocin (EC50: 8.8 nM vs 2.2 nM) were enhanced in the P gingivalis group compared to those in the control group. In the P gingivalis group, COX-2 mRNA expression was not elevated in the placenta or myometrium but was upregulated 2.3-fold in the fetal membrane. The TLR2 mRNA levels in the fetal membrane were 2.7-fold higher in the P gingivalis group, whereas TLR4 levels were not elevated. Activation of the NF-κB p65 and p38 MAPK pathways was enhanced in the fetal membrane of the P gingivalis group. Thus, in mice with chronic dental P gingivalis infection, TLR2-induced inflammation in the fetal membrane leads to upregulation of uterine contractility, leading to preterm birth.

Detection of MAPK/ERK pathway proteins and KRAS mutations in adenomatoid odontogenic tumors.

OBJECTIVE: This study aimed to assess the frequency of KRAS mutation and its association with the presence of the MAPK/ERK signaling pathway proteins in adenomatoid odontogenic tumors. STUDY DESIGN: Paraffin-embedded tissue samples from nine cases of adenomatoid odontogenic tumor were used. Genomic DNA was extracted from each sample; in one case, genetic mutations in 50 cancer-associated genes were examined by next-generation sequencing. Hotspot mutations in the RAS family were analyzed by Luminex assay using the remaining eight cases. Subsequently, immunohistochemistry for KRAS, CRAF, BRAF, EGFR, ERK, MEK, and BRAFV600E was performed. RESULTS: A KRAS G12D missense mutation was detected in the DNA sequence of the tumor cells, but it was not detected in the stromal tissue. KRAS G12V and KRAS G12R mutations were detected in two and four cases, respectively. For immunohistochemistry, all the cases were EGFR, KRAS, BRAF, CRAF positive, one case was ERK negative,and one case was MEK and ERK negative, all the other remaining cases were MEK and ERK positive. CONCLUSION: KRAS mutation at codon 12 and the presence of MAPK/ERK pathway proteins were detected suggesting their association with tumorigenesis of adenomatoid odontogenic tumors.

Bovine lactoferrin reverses programming of epithelial-to-mesenchymal transition to mesenchymal-to-epithelial transition in oral squamous cell carcinoma.

Epithelial-to-mesenchymal transition (EMT) is a biological process of invasion and metastasis in cancers, including in oral squamous cell carcinoma (OSCC). However, an effective anticancer drug that directly targets EMT has not yet been discovered. Therefore, we aimed to investigate the repressive effects of bovine lactoferrin (bLF) on EMT to achieve mesenchymal-to-epithelial transition (MET) in OSCC. OSCC cell lines, HOC313 (EMT-induced) and SCCVII (without EMT induction), were treated with bLF. The effects of bLF on EMT in OSCC were identified histologically by haematoxylin and eosin staining and observed morphologically and immunohistochemically using an anti-E-cadherin antibody. Expression levels of E-cadherin and vimentin were investigated using RT-PCR and western blotting. Immuno-expression of E-cadherin was examined in vivo in tumour tissues of C3H/HeN mice, transplanted with SCCVII cells, with or without bLF administration. We found that bLF changed the spindle-like mesenchymal cells to cuboidal-like epithelial cells and enhanced the affinity of membrane-bound E-cadherin in HOC313 cells. The transformation of EMT-MET in HOC313 cells was confirmed by the upregulation of E-cadherin and suppression of vimentin. Moreover, bLF suppressed TWIST expression through downregulation of ERK1/2 phosphorylation. Additionally, the inhibition tumour cell infiltration and increase in E-cadherin expression were observed in xenografts of the mice orally administered with bLF. Thus, based on the results from in vitro and in vivo studies, we concluded that bLF caused the restoration of epithelial properties through MET. Importantly, this finding is novel and is the first report indicating that bLF inhibited EMT and induced MET in OSCC, suggesting that bLF may provide a novel therapeutic strategy in OSCC.

Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer.

N-cadherin is a neural cell adhesion molecule that aberrantly occurs in head and neck cancers to promote cancer cell growth. However, the underlying mechanisms remain unclear. Here we report that N-cadherin increases cancer cell growth by inhibiting apoptosis. Apoptosis eliminates old, unnecessary, and unhealthy cells. However, tumor cells have the ability of avoiding apoptosis that increases cancer cell growth. Recent studies have found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells by reacting with four distinct cell surface receptors: TRAIL-R1 (DR-4), TRAIL-R2 (DR-5), TRAIL-R3 (DcR-1), and TRAIL-R4 (DcR-2). Among these TRAIL receptors, the death receptors DR-4 and DR-5 transmit apoptotic signals owing to the death domain in the intracellular portion. Conversely, the decoy receptors DcR-1 and DcR-2 lack a complete intracellular portion, so neither can transmit apoptotic signals. DcR-1 or DcR-2 overexpression suppresses TRAIL-induced apoptosis. In this study, N-cadherin overexpression increased DcR-2 expression and decreased DR-5 expression. In contrast, knockdown of N-cadherin expression upregulated DR-5 expression and downregulated DcR-2 expression. A significantly positive relationship between N-cadherin and DcR-2 expression was also found in HNSCC specimens. Those specimens with a lower apoptotic index showed a higher expression of N-cadherin and/or DcR-2. In addition, we demonstrated that N-cadherin interacts directly with DcR-2. Notably, DcR-2 induces cancer cell survival through the cleavage of caspases and PARP by activating MAPK/ERK pathway and suppressing NF-kB/ p65 phosphorylation, which has a very important role in resistance to chemotherapy.