Estrogen-responsive cancer-associated fibroblasts promote invasive property of gastric cancer in a paracrine manner via CD147 production.

Estrogen signaling has been extensively studied, especially in cancers that express estrogen receptor alpha (ERα). However, little is known regarding the effect of estrogen on cancer-associated fibroblasts (CAFs). Here, we explored the role of estrogen signaling of CAFs in gastric cancer (GC) progression. We investigated the phenotypic changes in CAFs upon 17ß-estradiol (E2) treatment using ERα-negative/positive CAFs, and the conditioned media (CM) collected from these were compared with regard to cancer cell proliferation, migration, and invasion. A paracrine factor was found using a cytokine array and was confirmed using qRT-PCR, western blotting, and enzyme-linked immunosorbent assays. ERα-CD147-matrix metalloproteinase (MMP) axis was confirmed by knockdown experiments using specific siRNAs. We found that a subset of CAFs expressed ERα. ERα-positive CAFs were responsive to E2, inducing ERα expression in a dose-dependent manner. Although E2 did not induce the proliferation of ERα-positive CAFs, the CM from E2-bound ERα-positive CAFs significantly promoted cancer cell migration and invasion. Cytokine array revealed that CD147 was induced in ERα-positive CAFs upon E2 treatment; this was mediated via ERα. Increased CD147 upregulated MMP2 and MMP9 in CAFs, and also influenced cancer cells in a paracrine manner to increase MMPs and CD147 in cancer cells. High CD147 expression in tumor tissue was associated with a worse prognosis in GC patients. Our data suggest that estrogen signaling activation in CAFs and the byproduct CD147 are among the critical mediators between the interplay of CAFs and cancer cells to facilitate cancer progression.

Modeling Pancreatic Cancer with Patient-Derived Organoids Integrating Cancer-Associated Fibroblasts.

Pancreatic cancer is a devastating disease and is highly resistant to anticancer drugs because of its complex microenvironment. Cancer-associated fibroblasts (CAFs) are an important source of extracellular matrix (ECM) components, which alter the physical and chemical properties of pancreatic tissue, thus impairing effective intratumoral drug delivery and resulting in resistance to conventional chemotherapy. The objective of this study was to develop a new cancer organoid model, including a fibrous tumor microenvironment (TME) using CAFs. The CAF-integrated pancreatic cancer organoid (CIPCO) model developed in this study histologically mimicked human pancreatic cancer and included ECM production by CAFs. The cancer cell-CAF interaction in the CIPCO promoted epithelial-mesenchymal transition of cancer cells, which was reversed by CAF inhibition using all-trans retinoic acid. Deposition of newly synthesized collagen I in the CIPCO disturbed the delivery of gemcitabine to cancer cells, and treatment with collagenase increased the cytotoxic effect of gemcitabine. This model may lead to the development of next-generation cancer organoid models recapitulating the fibrous TME.

miR-4742-5p promotes invasiveness of gastric cancer via targeting Rab43: An in vitro study.

miRNA (miR)-4742-5p is a recently identified microRNA regarding progression and metastasis in gastric cancer (GC). However, the biological function of this novel miRNA is largely unknown. We identified that the miR-4742-5p expression level was variably increased in GC cell lines. Suppression of miR-4742-5p using miR-inhibitor reduced the proliferation, migration, and invasion of GC cells with high miR-4742-5p expression, whereas overexpression of miR-4742-5p-mimic enhanced the aforementioned properties in GC cells with low miR-4742-5p expression. miR-4742-5p expression induced the decreases of Zo-1 and E-cadherin expression as well as the increases of vimentin and N-cadherin expression, leading to epithelial-mesenchymal transition (EMT) of cancer cells. RNA sequencing results indicated Ras-related GTP-binding protein 43 (Rab43) as a potential target gene. We identified that the expression of Rab43 is associated with activation of AKT and nuclear factor-kappa B (NF-κB) which are key oncogenic pathways in cancer cells. Our results demonstrate a new component in GC progression, promising a potential therapeutic strategy.

Quantitation of ligand is critical for ligand-dependent MET signalling activation and determines MET-targeted therapeutic response in gastric cancer.

BACKGROUND: Despite the promising preclinical antitumor activity of MET-targeting therapies, most clinical trials have failed. We introduced a new concept of quantitation of stroma-induced hepatocyte growth factor (HGF) to assess the actual MET signalling activity in gastric cancer (GC). METHODS: We treated serially diluted HGF and conditioned media (CM) from cancer-associated fibroblasts (CAFs) on low MET-expressing cancer cells and investigated the phenotypical and signalling changes. Stromal proportion and MET expression in GC samples were assessed, and gene set enrichment analysis (GSEA) from the public database was performed. The antitumor effect of anti-MET treatment was examined, especially when cancer cells were activated in a ligand-dependent manner. RESULTS: Relatively high doses of HGF or high-concentrated CM fully activated MET signalling cascades and promoted cell proliferation/invasion. High stromal proportion denoted worse patient survival in MET-positive GCs than in MET-negative ones. GSEA showed that the gene sets regarding proliferation, migration, and CAF as well as MET pathway signature were enriched in simultaneously MET- and HGF-positive samples. Sufficient ligand-dependent MET signalling activation increased the sensitivity to crizotinib. CONCLUSIONS: We conclude that patients whose tumours have a high stromal proportion and at least low MET expression may benefit more from MET-targeted therapies.

MicroRNA signatures associated with lymph node metastasis in intramucosal gastric cancer.

Although a certain proportion of intramucosal carcinomas (IMCs) of the stomach does metastasize, the majority of patients are currently treated with endoscopic resection without lymph node dissection, and this potentially veils any existing metastasis and may put some patients in danger. In this regard, biological markers from the resected IMC that can predict metastasis are warranted. Here, we discovered unique miRNA expression profiles that consist of 21 distinct miRNAs that are specifically upregulated (miR-628-5p, miR-1587, miR-3175, miR-3620-5p, miR-4459, miR-4505, miR-4507, miR-4720-5p, miR-4742-5p, and miR-6779-5p) or downregulated (miR-106b-3p, miR-125a-5p, miR-151b, miR-181d-5p, miR-486-5p, miR-500a-3p, miR-502-3p, miR-1231, miR-3609, and miR-6831-5p) in metastatic (M)-IMC compared to nonmetastatic (N)-IMC, or nonneoplastic gastric mucosa. Intriguingly, most of these selected miRNAs showed stepwise increased or decreased expression from nonneoplastic tissue to N-IMC to M-IMC. This suggests that common oncogenic mechanisms are gradually intensified during the metastatic process. Using a machine-learning algorithm, we demonstrated that such miRNA signatures could distinguish M-IMC from N-IMC. Gene ontology and pathway analysis revealed that TGF-ß signaling was enriched from upregulated miRNAs, whereas E2F targets, apoptosis-related, hypoxia-related, and PI3K/AKT/mTOR signaling pathways, were enriched from downregulated miRNAs. Immunohistochemical staining of samples from multiple institutions indicated that PI3K/AKT/mTOR pathway components, MAPK1, phospho-p44/42 MAPK, and pS6 were highly expressed and the expression of SMAD7, a TGF-ß pathway component, was decreased in M-IMC, which could aid in distinguishing M-IMC from N-IMC. The miRNA signature discovered in this study is a valuable biological marker for identifying metastatic potential of IMCs, and provides novel insights regarding the metastatic progression of IMC.

PTPRD-inactivation-induced CXCL8 promotes angiogenesis and metastasis in gastric cancer and is inhibited by metformin.

BACKGROUND: Protein tyrosine phosphatase receptor delta (PTPRD) is frequently inactivated in various types of cancers. Here, we explored the underlying mechanism of PTPRD-loss-induced cancer metastasis and investigated an efficient treatment option for PTPRD-inactivated gastric cancers (GCs). METHODS: PTPRD expression was evaluated by immunohistochemistry. Microarray analysis was used to identify differentially expressed genes in PTPRD-inactivated cancer cells. Quantitative reverse transcription (qRT-PCR), western blotting, and/or enzyme-linked immunosorbent assays were used to investigate the PTPRD-CXCL8 axis and the expression of other related genes. An in vitro tube formation assay was performed using HUVECs. The efficacy of metformin was assessed by MTS assay. RESULTS: PTPRD was frequently downregulated in GCs and the loss of PTPRD expression was associated with advanced stage, worse overall survival, and a higher risk of distant metastasis. Microarray analysis revealed a significant increase in CXCL8 expression upon loss of PTPRD. This was validated in various GC cell lines using transient and stable PTPRD knockdown. PTPRD-loss-induced angiogenesis was mediated by CXCL8, and the increase in CXCL8 expression was mediated by both ERK and STAT3 signaling. Thus, specific inhibitors targeting ERK or STAT3 abrogated the corresponding signaling nodes and inhibited PTPRD-loss-induced angiogenesis. Additionally, metformin was found to efficiently inhibit PTPRD-loss-induced angiogenesis, decrease cell viability in PTPRD-inactivated cancers, and reverse the decrease in PTPRD expression. CONCLUSIONS: Thus, the PTPRD-CXCL8 axis may serve as a potential therapeutic target, particularly for the suppression of metastasis in PTPRD-inactivated GCs. Hence, we propose that the therapeutic efficacy of metformin in PTPRD-inactivated cancers should be further investigated.

Functional loss of ARID1A is tightly associated with high PD-L1 expression in gastric cancer.

Notwithstanding remarkable treatment success with anti-PD-1 monoclonal antibody, oncogenic mechanism of PD-L1 regulation in gastric cancer (GC) remains poorly understood. We hypothesized that ARID1A might be related to tumor PD-L1 expression in GC. We found that tumor PD-L1 positivity was associated with loss of ARID1A and showed trend toward better survival of patients with various molecular subtypes of GC (experimental set, n = 273). Considering heterogeneous ARID1A expression, we validated this using whole tissue sections (n = 159) and found that loss of ARID1A was correlated with microsatellite instability-high (MSI-H), Epstein-Barr virus (EBV), and PD-L1 positivity. Furthermore, for patients with MSI-H tumors, the degree of PD-L1 expression was significantly higher in ARID1A-deficient tumors. After ARID1A knockdown in GC cell lines, total and membranous PD-L1 protein, and PD-L1 mRNA levels were increased based on Western blot, flow cytometry, and qRT-PCR, respectively. With IFN-γ treatment, PD-L1 expression was significantly increased both in ARID1A-deficient cancer cells and controls, but the increase was not more pronounced in the former. Loss of ARID1A increased PD-L1 via activating AKT signaling, while LY294002 (PI3K inhibitor) decreased PD-L1 levels. Furthermore, we found that 3 MSI-H tumors showing highest expression of PD-L1 had simultaneous KRAS mutation and loss of ARID1A, suggesting a possible synergistic role boosting PD-L1. Our results strongly indicate that loss of ARID1A is tightly associated with high PD-L1 expression in GC. These results would increase our understanding of the oncogenic mechanism of PD-L1 regulation in GC, and also help to find the optimal candidates for immunotherapy.

Effects of Melatonin and Its Underlying Mechanism on Ethanol-Stimulated Senescence and Osteoclastic Differentiation in Human Periodontal Ligament Cells and Cementoblasts.

The present study evaluated the protective effects of melatonin in ethanol (EtOH)-induced senescence and osteoclastic differentiation in human periodontal ligament cells (HPDLCs) and cementoblasts and the underlying mechanism. EtOH increased senescence activity, levels of reactive oxygen species (ROS) and the expression of cell cycle regulators (p53, p21 and p16) and senescence-associated secretory phenotype (SASP) genes (interleukin [IL]-1ß, IL-6, IL-8 and tumor necrosis factor-α) in HPDLCs and cementoblasts. Melatonin inhibited EtOH-induced senescence and the production of ROS as well as the increased expression of cell cycle regulators and SASP genes. However, it recovered EtOH-suppressed osteoblastic/cementoblastic differentiation, as evidenced by alkaline phosphatase activity, alizarin staining and mRNA expression levels of Runt-related transcription factor 2 (Runx2) and osteoblastic and cementoblastic markers (glucose transporter 1 and cementum-derived protein-32) in HPDLCs and cementoblasts. Moreover, it inhibited EtOH-induced osteoclastic differentiation in mouse bone marrow⁻derived macrophages (BMMs). Inhibition of protein never in mitosis gene A interacting-1 (PIN1) by juglone or small interfering RNA reversed the effects of melatonin on EtOH-mediated senescence as well as osteoblastic and osteoclastic differentiation. Melatonin blocked EtOH-induced activation of mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and Nuclear factor of activated T-cells (NFAT) c-1 pathways, which was reversed by inhibition of PIN1. This is the first study to show the protective effects of melatonin on senescence-like phenotypes and osteoclastic differentiation induced by oxidative stress in HPDLCs and cementoblasts through the PIN1 pathway.

Identification of genomic aberrations associated with lymph node metastasis in diffuse-type gastric cancer.

Diffuse-type gastric cancer (DGC) is a GC subtype with heterogeneous clinical outcomes. Lymph node metastasis of DGC heralds a dismal progression, which hampers the curative treatment of patients. However, the genomic heterogeneity of DGC remains unknown. To identify genomic variations associated with lymph node metastasis in DGC, we performed whole exome sequencing on 23 cases of DGC and paired non-tumor tissues and compared the mutation profiles according to the presence (N3, n = 13) or absence (N0, n = 10) of regional lymph node metastasis. Overall, we identified 185 recurrently mutated genes in DGC, which included a significant novel mutation at CMTM2, as well as previously known mutations at CDH1, RHOA, and TP53. Noticeably, CMTM2 expression could predict the prognostic outcomes of DGC but not intestinal-type GC (IGC), indicating pivotal roles of CMTM2 in DGC progression. In addition, we identified a recurrent loss of heterozygosity (LOH) of DNA copy numbers at the 3p12-pcen locus in DGC. A comparison of N0 and N3 tumors showed that N3 tumors exhibited more frequent DNA copy number aberrations, including copy-neutral LOH and mutations of CpTpT trinucleotides, than N0 tumors (P = 0.2 × 10-3). In conclusion, DGCs have distinct profiles of somatic mutations and DNA copy numbers according to the status of lymph node metastasis, and this might be helpful in delineating the pathobiology of DGC.

Sonic Hedgehog Promotes Cementoblastic Differentiation via Activating the BMP Pathways.

Although sonic hedgehog (SHH), an essential molecule in embryogenesis and organogenesis, stimulates proliferation of human periodontal ligament (PDL) stem cells, the effects of recombinant human SHH (rh-SHH) on osteoblastic differentiation are unclear. To reveal the role of SHH in periodontal regeneration, expression of SHH in mouse periodontal tissues and its effects on the osteoblastic/cementoblastic differentiation in human cementoblasts were investigated. SHH is immunolocalized to differentiating cementoblasts, PDL cells, and osteoblasts of the developing mouse periodontium. Addition of rh-SHH increased cell growth, ALP activity, and mineralization nodule formation, and upregulated mRNA expression of osteoblastic and cementoblastic markers. The osteoblastic/cementoblastic differentiation of rh-SHH was abolished by the SHH inhibitor cyclopamine (Cy) and the BMP antagonist noggin. rh-SHH increased the expression of BMP-2 and -4 mRNA, as well as levels of phosphorylated Akt, ERK, p38, and JNK, and of MAPK and NF-κB activation, which were reversed by noggin, Cy, and BMP-2 siRNA. Collectively, this study is the first to demonstrate that SHH can promote cell growth and cell osteoblastic/cementoblastic differentiation via BMP pathway. Thus, SHH plays important roles in the development of periodontal tissue, and might represent a new therapeutic target for periodontitis and periodontal regeneration.

Effects of sodium tri- and hexameta-phosphate in vitro osteoblastic differentiation in Periodontal Ligament and Osteoblasts, and in vivo bone regeneration.

The present study was designed to assess the effects and underlying mechanism of two poly(P) compounds, sodium triphosphate (STP, Na5P3O10) and sodium hexametaphosphate (SHMP, Na15P13O40~Na20P18O40) on osteoblastic differentiation of human periodontal ligament cells (PDLCs) and osteoblasts in vitro, and bone formation in vivo. Differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and mRNA expression for marker genes. To examine the osteogenic potential to regenerate bone, the critical-sized mouse calvarial defect model was utilized. Incubation of PDLCs and osteoblasts with STP and SHMP resulted in a dose- and time-dependent increase in growth, alkaline phosphatase (ALP) activity, mineralization and mRNA expression for marker genes. STP and SHMP increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), Akt, and mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPK). Treatment with the mTOR inhibitor, rapamycin, attenuatted STP- and SHMP-induced osteoblastic differentiation. Micro-CT and histologic analysis showed that STP significantly increased new bone formation in calvarial defects, compared with SHMP and control group. Collectively, this is the first study to demonstrate that STP and SHMP promotes the osteoblastic differentiation in vitro, whereas STP only stimulated bone repair in vivo. Therefore, STP may be useful therapeutic approach for the regeneration of bone or periodontal tissue.

Protein Interacting with Never in Mitosis A-1 Induces Glutamatergic and GABAergic Neuronal Differentiation in Human Dental Pulp Stem Cells.

INTRODUCTION: The purpose of this study was to investigate the role of protein interacting with never in mitosis A-1 (PIN1) in the neuronal or glial differentiation of human dental pulp stem cells (hDPSCs) and whether PIN1 can regulate determination of neuronal sub-phenotype. METHODS: After magnetic-activated cell sorting to separate CD34(+)/c-kit(+)/STRO-1(+) hDPSCs, cells were cultured in neurogenic medium. Differentiation was measured as Nissl staining and marker protein or mRNA expression by reverse transcriptase polymerase chain reaction, immunofluorescence, and flow cytometric analysis. RESULTS: PIN1 mRNA levels were upregulated in a time-dependent fashion during neurogenic differentiation. The PIN1 inhibitor juglone suppressed neuronal differentiation but promoted glial differentiation as assessed by the number of Nissl-positive cells and mRNA expression of neuronal markers (nestin, ßIII-tubulin, and NeuN) and a glial marker (glial fibrillary acidic protein). Conversely, overexpression of PIN1 by infection with adenovirus-PIN1 increased neuronal differentiation but decreased glial differentiation. Moreover, PIN1 overexpression increased the percentage of glutamatergic and GABAergic cells but decreased that of dopaminergic cells among total NeuN-positive hDPSCs. CONCLUSIONS: This is the first study to demonstrate that PIN1 overexpression induced glutamatergic and GABAergic neuronal differentiation but suppressed glial differentiation of hDPSCs, suggesting that enhancing PIN expression is important to obtain human glutamatergic and GABAergic neurons from hDPSCs.

Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells.

BACKGROUND: Recent reports suggest that thymosin beta-4 (Tß4) is a key regulator for wound healing and anti-inflammation. However, the role of Tß4 in osteoclast differentiation remains unclear. PURPOSE: The purpose of this study was to evaluate Tß4 expression in H2O2-stimulated human periodontal ligament cells (PDLCs), the effects of Tß4 activation on inflammatory response in PDLCs and osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs), and identify the underlying mechanism. METHODS: Reverse transcription-polymerase chain reactions and Western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages (BMMs) using conditioned medium (CM) from H2O2-treated PDLCs. RESULTS: Tß4 was down-regulated in H2O2-exposed PDLCs in dose- and time-dependent manners. Tß4 activation with a Tß4 peptide attenuated the H2O2-induced production of NO and PGE2 and up-regulated iNOS, COX-2, and osteoclastogenic cytokines (TNF-α, IL-1ß, IL-6, IL-8, and IL-17) as well as reversed the effect on RANKL and OPG in PDLCs. Tß4 peptide inhibited the effects of H2O2 on the activation of ERK and JNK MAPK, and NF-κB in PDLCs. Furthermore, Tß4 peptide inhibited osteoclast differentiation, osteoclast-specific gene expression, and p38, ERK, and JNK phosphorylation and NF-κB activation in RANKL-stimulated BMMs. In addition, H2O2 up-regulated Wnt5a and its cell surface receptors, Frizzled and Ror2 in PDLCs. Wnt5a inhibition by Wnt5a siRNA enhanced the effects of Tß4 on H2O2-mediated induction of pro-inflammatory cytokines and osteoclastogenic cytokines as well as helping osteoclastic differentiation whereas Wnt5a activation by Wnt5a peptide reversed it. CONCLUSION: In conclusion, this study demonstrated, for the first time, that Tß4 was down-regulated in ROS-stimulated PDLCs as well as Tß4 activation exhibited anti-inflammatory effects and anti-osteoclastogenesis in vitro. Thus, Tß4 activation might be a therapeutic target for inflammatory osteolytic disease, such as periodontitis.

Cudraxanthone H Induces Growth Inhibition and Apoptosis in Oral Cancer Cells via NF-κB and PIN1 Pathways.

Cudraxanthone H (CH) is a natural compound isolated from a methanol extract of the root bark of Cudrania tricuspidata, a herbal plant also known as Moraceae. However, the effect of CH on human cancer cells has not been reported previously. The aim of this study was to investigate the anticancer effects and mechanism of action of CH on oral squamous cell carcinoma (OSCC) cells. CH exerted significant antiproliferative effects on OSCC cells in dose- and time-dependent manners. CH also induced apoptosis in OSCC cells, as evidenced by an increased percentage of cells in the sub-G1 phase of the cell cycle, annexin V-positive/propidium iodide-negative cells, and nuclear morphology. This antiproliferative effect of CH was associated with a marked reduction in the expression of cyclin D1 and cyclin E, with a concomitant induction of cyclin-dependent kinase inhibitor (CDKI) expression (p21 and p27). CH inhibited the phosphorylation and degradation of IκB-α and the nuclear translocation of NF-κB p65. Furthermore, CH treatment down-regulated PIN1 mRNA and protein expression in a dose-dependent manner. PIN1 overexpression by infection with adenovirus-PIN1 (Ad-PIN1) attenuated the CH-induced growth-inhibiting and apoptosis-inducing effects, blocked CH-enhanced CDKI expression and restored cyclin levels. In contrast, inhibiting PIN1 expression via juglone exerted the opposite effects. The present study is the first to demonstrate antiproliferative and apoptosis-inducing effects of CH, which exerts its effects by inhibiting NF-κB and PIN1. These data suggest that it might be a novel alternative chemotherapeutic agent for use in the treatment of oral cancer.

Expression of Phospholipase D in Periodontitis and Its Role in the Inflammatory and Osteoclastic Response by Nicotine- and Lipopolysaccharide-Stimulated Human Periodontal Ligament Cells.

BACKGROUND: The aim of the present study is to investigate the expression of phospholipase D (PLD) 1 and PLD2 in periodontal patients and in human periodontal ligament cells (HPDLCs) exposed to nicotine plus lipopolysaccharide (LPS) from Porphyromonas gingivalis (Toll-like receptor 2 ligand). Furthermore, the effects of PLD isoform inhibition on the inflammatory response and osteoclast differentiation and its mechanisms were determined. METHODS: Proinflammatory mediators were examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To silence the gene expression of the PLD isoforms, cells were transfected with small interfering RNA (siRNA) targeting PLD1 or PLD2. Mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursor cells for in vitro osteoclastogenesis. Western blot analysis and immunofluorescence were used to assess signaling pathways. RESULTS: Chronic smokers with periodontitis exhibited significantly higher PLD1 and PLD2 messenger RNA (mRNA) expression than non-smokers with periodontitis and healthy controls. Nicotine and LPS upregulated PLD1 and PLD2 mRNA expression in a dose-dependent manner in HPDLCs. Pharmacologic and siRNA-mediated inhibition of PLD1 and PLD2 attenuated the nicotine- and LPS-induced upregulation of inducible nitric oxide (NO) synthase and cyclooxygenase-2, production of NO, and prostaglandin E2, and mRNA expression and secretion of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8. The conditioned media from HPDLCs treated with PLD isoform inhibitors or siRNA against PLD inhibited receptor activator of nuclear factor-κB (NF-κB) ligand-mediated osteoclast differentiation, as well as protein expression of nuclear factor of activated T cells c1 and c-Fos, in BMMs. In addition, PLD isoform inhibitors and siRNA inhibited the nicotine- and LPS-induced activation of phosphoinositide 3-kinase, protein kinase C, p38, extracellular signal-regulated kinase, c-Jun N-terminal protein kinase, mitogen-activated protein kinase, and NF-κB. CONCLUSION: To the best of the authors' knowledge, this study is the first to demonstrate that PLD isoform inhibition has anti-inflammatory and antiosteoclastogenic effects and thus may be a therapeutic target for the treatment of periodontitis.

Milk activates the expression of cytokines via Nrf2/HO-1 pathway in human periodontal ligament cells.

BACKGROUND: Milk is known as a suitable storage medium for avulsed teeth during emergency situations, but its potential toxicity on human periodontal ligament (PDL) cells has not been reported. The purpose of this study was to investigate the milk-induced gene profiles of PDL cells in vitro by microarray analysis after storage in milk. We additionally determined whether milk activates the cytoprotective defense mechanisms via the NF-E2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) pathway. MATERIAL AND METHODS: Gene induction in cultured human PDL cells after exposure to milk for 1 and 3 h as compared with non-treated PDL cells was analyzed by microarray analysis and subsequent RT-PCR. Reactive oxygen species (ROS) and Western blot analysis were used to determine whether milk activates the cytoprotective defense mechanisms using the Nrf2 and HO-1 pathway. RESULT: Microarray data analysis identified 868 (1 h per control) and 1782 (3 h per control) differentially expressed genes related to the duration of storage in milk. Exposure to milk for 3 and 1 h resulted in the upregulation of specific inflammatory cytokines, chemokines, and MMPs concomitant with downregulation of extracellular matrix-related genes. Exposure to milk increased the expression of peroxiredoxin-1, thioredoxin-1 and heme oxygenase (HO)-1 and stimulated the nuclear translocation of Nrf2. HO-1 inhibitor and Nrf2 siRNA blocked the milk-induced inflammatory response such as production of ROS, expression of cytokines, chemokines, and MMPs. CONCLUSION: Within the limit of this study, this study demonstrates that exposure of PDL cells to milk is associated with an upregulated expression of several pro-inflammatory proteins and key antioxidant proteins via the activation of Nrf2/ARE pathway in PDL cells.

Odontoblastic Differentiation, Inflammatory Response, and Angiogenic Potential of 4 Calcium Silicate-based Cements: Micromega MTA, ProRoot MTA, RetroMTA, and Experimental Calcium Silicate Cement.

INTRODUCTION: The aim of this study was to analyze the effects of different calcium silicate-based cements (CSCs) for pulp capping materials including MicroMega MTA (MMTA; MicroMega, Besanchon, France), RetroMTA (RMTA; BioMTA, Seoul, Korea), ProRoot MTA (PMTA; Dentsply, Tulsa, OK), and experimental CSC (ECSC) on odontoblastic differentiation, in vitro angiogenesis, and the inflammatory response in human dental pulp cells. METHODS: Differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and reverse-transcriptase polymerase chain reaction (RT-PCR) for the marker genes. The levels of inflammatory mediators and cytokines were measured by RT-PCR and an enzyme-linked immunosorbent assay. In vitro angiogenesis was assessed by RT-PCR for angiogenic genes and an endothelial tube formation assay. RESULTS: PMTA, MMTA, and ECSC increased the alkaline phosphatase activity and mineralization nodule formation and up-regulated messenger RNA (mRNA) expression of odontoblastic markers compared with RMTA. In addition, PMTA, MMTA, and ECSC up-regulated the mRNA of angiogenic genes in human dental pulp cells and increased the capillary tube formation of endothelial cells compared with RMTA. However, all CSCs showed similar expression levels of inducible nitric oxide synthase and cyclooxygenase-2 protein as well as proinflammatory mediators such as nitric oxide, prostaglandin E2, tumor necrosis factor alpha, interleukin (IL)-1ß, IL-6, and IL-8 mRNA. CONCLUSIONS: Taken together, our experimental results suggest that all CSCs are favorable materials for pulp capping, but PMTA, MMTA, and ECSC may be recommended over RMTA.

Effects of sodium tri- and hexametaphosphate on proliferation, differentiation, and angiogenic potential of human dental pulp cells.

INTRODUCTION: This study aimed to investigate the effects of 2 inorganic polyphosphates (poly[P]) are linear polymers of orthophosphate (Pi) residues linked by energy-rich phosphoanhydride poly(P) compounds, sodium triphosphate (STP, Na5P3O10) and sodium hexametaphosphate (SHMP, Na15P13O40 âˆ¼ Na20P18O40) on the proliferation, odontoblastic differentiation, and angiogenic potential of human dental pulp cells (HDPCs). METHODS: Differentiation was measured by alkaline phosphatase activity, calcified nodule formation by alizarin red staining, and marker messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction. In vitro angiogenesis was quantified by migration, mRNA levels of angiogenic genes, and endothelial tube formation. RESULTS: STP and SHMP dose dependently increased the proliferation and ALP activity and enhanced mineralized nodule formation and odontoblast marker mRNAs of HDPCs. STP and SHMP resulted in the up-regulation of angiogenic genes in HDPCs. Endothelial cells treated with conditioned medium collected from STP- and SHMP-exposed HDPCs showed an increase in migration and capillary tube formation. Knockdown of the expression of the genes encoding of inorganic pyrophosphate by small interfering RNA attenuated the STP- and SHMP-induced odontogenic differentiation and angiogenic potential. CONCLUSIONS: This study showed that STP and SHMP promote the growth, differentiation, and angiogenic potential of HDPCs. These results suggest that STP and SHMP may be candidates for dental pulp tissue engineering and regenerative endodontics.

HIF-2 Inhibition Supresses Inflammatory Responses and Osteoclastic Differentiation in Human Periodontal Ligament Cells.

Recent reports suggest that hypoxia inducible factor-2α (HIF-2α) is a key regulator of osteoarthritis cartilage destruction. However, the precise role of HIF-2α in the inflammatory response and osteoclast differentiation remains unclear. The purpose of this study was to investigate the effect of HIF-2α on inflammatory cytokines, extracellular matrix (ECM) destruction enzymes, and osteoclastic differentiation in nicotine and lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (PDLCs). HIF-2α was upregulated in chronically inflamed PDLCs of periodontitis patients, and in nicotine- and LPS-exposed PDLC in dose- and time-dependent manners. HIF-2α inhibitor and HIF-2α siRNA attenuated the nicotine- and LPS- induced production of NO and PGE2 , upregulation of iNOS, COX-2, pro-inflammatory cytokines (IL-1ß, TNF-α, IL-1ß, IL-6, IL-8, IL-10, IL-11, and IL-17), and matrix metalloproteinases (MMPs; MMP-1, -8, -13, -2 and -9), and reversed the effect on TIMPs (TIMP-1 and -2) in PDLCs. The conditioned medium produced by nicotine and LPS-treated PDLCs increased the number of TRAP-stained osteoclasts, TRAP activity and osteoclast-specific genes, which has been blocked by HIF-2α inhibition and silencing. HIF-2α inhibitor and HIF-2α siRNA inhibited the effects of nicotine and LPS on the activation of Akt, JAK2 and STAT3, ERK and JNK MAPK, nuclear factor-κB, c-Jun, and c-Fos. Taken together, this study is the first to demonstrate that HIF-2α inhibition exhibits anti-inflammatory activity through the inhibition of inflammatory cytokines and impairment of ECM destruction, as well as blocking of osteoclastic differentiation in a nicotine- and periodontopathogen-stimulated PDLCs model. Thus, HIF-2α inhibition may be a novel molecular target for therapeutic approaches in periodontitis.

Effects of bioactive cements incorporating zinc-bioglass nanoparticles on odontogenic and angiogenic potential of human dental pulp cells.

BACKGROUND: The objective of this study was to investigate the effects of bioactive calcium phosphate cements (CPC, α-tricalcium phosphate-based) incorporating zinc-bioglass (ZnBG) on the odontogenic differentiation and angiogenesis of human dental pulp cells (HDPCs). METHODS: BGs with varying concentrations of Zn (0, 2.5 and 5%) were produced via a sol-gel process. The proliferation of HDPCs on CPC/BGs was determined by MTS assay. Alizarin red staining, RT-PCR, and ALP activity were used to assess odontogenic differentiation, and western blot analysis was used to asses signaling pathways. In vitro angiogenesis was examined via mRNA expression of angiogenic genes and tubule formation. RESULTS: All cement formulations showed no cytotoxicity. The CPCs with ZnBG showed increased ALP activity, enhanced formation of mineralized nodules, and upregulated mRNA expression of DMP-1, DSPP, Runx2, and osterix in a time- and dose-dependent manner, relative to CPCs without Zn. ZnBG upregulated integrins α1, α2, ß1, and ß3 and activated integrin downstream signal pathways, such as p-FAK, p-Akt, p-paxillin, RhoA, MAPK, and NF-κB, as well as canonical and non-canonical Wnt signaling. In addition, ZnBG upregulated VEGF mRNA in HDPCs and increased the tubular structure in endothelial cells. CONCLUSIONS: Our results demonstrate that ZnBG incorporated within CPCs activates odontogenic differentiation and promotes angiogenesis in vitro through integrin, Wnt, MAPK, and NF-κB pathways. Thus, CPCs incorporating ZnBG are promising matrices in tissue engineering to stimulate endodontic regeneration.