Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

Mesenchymal stem cell-derived apoptotic bodies alleviate alveolar bone destruction by regulating osteoclast differentiation and function.

Periodontitis is caused by overactive osteoclast activity that results in the loss of periodontal supporting tissue and mesenchymal stem cells (MSCs) are essential for periodontal regeneration. However, the hypoxic periodontal microenvironment during periodontitis induces the apoptosis of MSCs. Apoptotic bodies (ABs) are the major product of apoptotic cells and have been attracting increased attention as potential mediators for periodontitis treatment, thus we investigated the effects of ABs derived from MSCs on periodontitis. MSCs were derived from bone marrows of mice and were cultured under hypoxic conditions for 72 h, after which ABs were isolated from the culture supernatant using a multi-filtration system. The results demonstrate that ABs derived from MSCs inhibited osteoclast differentiation and alveolar bone resorption. miRNA array analysis showed that miR-223-3p is highly enriched in those ABs and is critical for their therapeutic effects. Targetscan and luciferase activity results confirmed that Itgb1 is targeted by miR-223-3p, which interferes with the function of osteoclasts. Additionally, DC-STAMP is a key regulator that mediates membrane infusion. ABs and pre-osteoclasts expressed high levels of DC-STAMP on their membranes, which mediates the engulfment of ABs by pre-osteoclasts. ABs with knock-down of DC-STAMP failed to be engulfed by pre-osteoclasts. Collectively, MSC-derived ABs are targeted to be engulfed by pre-osteoclasts via DC-STAMP, which rescued alveolar bone loss by transferring miR-223-3p to osteoclasts, which in turn led to the attenuation of their differentiation and bone resorption. These results suggest that MSC-derived ABs are promising therapeutic agents for the treatment of periodontitis.

ERVK13-1/miR-873-5p/GNMT Axis Promotes Metastatic Potential in Human Bladder Cancer though Sarcosine Production.

N-methyl-glycine (sarcosine) is known to promote metastatic potential in some cancers; however, its effects on bladder cancer are unclear. T24 cells derived from invasive cancer highly expressed GNMT, and S-adenosyl methionine (SAM) treatment increased sarcosine production, promoting proliferation, invasion, anti-apoptotic survival, sphere formation, and drug resistance. In contrast, RT4 cells derived from non-invasive cancers expressed low GNMT, and SAM treatment did not produce sarcosine and did not promote malignant phenotypes. In T24 cells, the expression of miR-873-5p, which suppresses GNMT expression, was suppressed, and the expression of ERVK13-1, which sponges miR-873-5p, was increased. The growth of subcutaneous tumors, lung metastasis, and intratumoral GNMT expression in SAM-treated nude mice was suppressed in T24 cells with ERVK13-1 knockdown but promoted in RT4 cells treated with miR-873-5p inhibitor. An increase in mouse urinary sarcosine levels was observed to correlate with tumor weight. Immunostaining of 86 human bladder cancer cases showed that GNMT expression was higher in cases with muscle invasion and metastasis. Additionally, urinary sarcosine concentrations increased in cases of muscle invasion. Notably, urinary sarcosine concentration may serve as a marker for muscle invasion in bladder cancer; however, further investigation is necessitated.

Macrophage-derived apoptotic bodies impair the osteogenic ability of osteoblasts in periodontitis.

OBJECTIVES: Periodontitis is induced by the imbalance between osteoblast and osteoclast activity, which leads to periodontal tissue destruction. Macrophages play a vital role in periodontitis. However, the hypoxic periodontal environment will also induce macrophage apoptosis within a short time. Apoptotic bodies (ABs) are the major products generated from apoptotic cells, but whether macrophage-derived ABs play a regulatory role as their mother cells in periodontitis remains unknown. In the present study, we aimed to investigate the effects of ABs on osteoblasts. METHOD: ABs derived from hypoxia-induced macrophages were co-cultured with osteoblasts and the impact of ABs on osteoblast differentiation in vitro was assessed. In vivo, periodontitis model was established and macrophages-derived ABs were injected into the gingival sulcus. The effects of ABs on periodontal bone resorption were determined. RESULTS: The results showed that ABs significantly inhibit osteoblast differentiation and promoted alveolar bone resorption in periodontitis. MicroRNA (miRNAs) array analysis was performed and revealed that miR-483-5p is the key miRNA in ABs. Dual luciferase reporter assays were performed and confirmed that miR-483-5p targeted Col1A1 mRNA and attenuated its expression. CONCLUSION: Macrophage-derived ABs inhibit osteoblast differentiation via the transfer of miR-483-5p, which downregulates Col1A1 expression and finally suppresses osteogenic activity.

Role of creatine shuttle in colorectal cancer cells.

The creatine shuttle translocates the energy generated by oxidative phosphorylation to the cytoplasm via mitochondrial creatine kinase (MTCK) and creatine kinase B (CKB) in the cytoplasm. It is not apparent how the creatine shuttle is related to cancer. Here, we analyzed the expression and function of CKB and MTCK in colorectal cancer (CRC) and investigated the role of the creatine shuttle in CRC. Compared with normal mucosa, 184 CRC tissues had higher levels of CKB and MTCK, and these levels were associated with histological grade, tumor invasion, and distant metastasis. CK inhibitor dinitrofluorobenzene (DNFB) on CRC cell lines HT29 and CT26 inhibited cell proliferation and stemness to less than 2/3 and 1/20 of their control levels, respectively. In this treatment, the production of reactive oxygen species increased, mitochondrial respiration decreased, and mitochondrial volume and membrane potential decreased. In a syngeneic BALB/c mouse model using CT26 cells pretreated with DNFB, peritoneal metastasis was suppressed to 70%. Phosphorylation of EGFR, AKT, and ERK1/2 was inhibited in DNFB-treated tumors. High ATP concentrations prevented EGFR phosphorylation in HT29 cells following DNFB treatment, CKB or MTCK knockdown, and cyclocreatine administration. Despite not being immunoprecipitated, CKB and EGFR were brought closer together by EGF stimulation. These findings imply that blocking the creatine shuttle decreases the energy supply, suppresses oxidative phosphorylation, and blocks ATP delivery to phosphorylation signals, preventing signal transduction. These findings highlight the critical role of the creatine shuttle in cancer cells and suggest a potential new cancer treatment target.

Lauric Acid Overcomes Hypoxia-Induced Gemcitabine Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Although gemcitabine (GEM) is widely used in chemotherapy for pancreatic ductal adenocarcinoma (PDA), drug resistance restricts its clinical effectiveness. To examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced energy production and decreased mitochondrial reactive oxygen species levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. Inhibition of hypoxia-inducible factor-1α in both cell lines did not restore the GEM sensitivity. In contrast, treatment of both cell types with lauric acid (LAA), a medium-chain fatty acid, restored GEM sensitivity. These results suggest that decreased energy production, decreased mitochondrial reactive oxygen species levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of oxidative phosphorylation by LAA could be a tool to overcome GEM resistance. Clinical verification of the effectiveness of LAA in GEM resistance is necessary in the future.

Short Telomere Lesions with Dysplastic Metaplasia Histology May Represent Precancerous Lesions of Helicobacter pylori-Positive Gastric Mucosa.

Gastric cancers are strongly associated with Helicobacter pylori infection, with intestinal metaplasia characterizing the background mucosa in most cases. However, only a subset of intestinal metaplasia cases proceed to carcinogenesis, and the characteristics of high-risk intestinal metaplasia that link it with gastric cancer are still unclear. We examined telomere reduction in five gastrectomy specimens using fluorescence in situ hybridization, and identified areas with localized telomere loss (outside of cancerous lesions), which were designated as short telomere lesions (STLs). Histological analyses indicated that STLs were characteristic of intestinal metaplasia accompanied by nuclear enlargement but lacking structural atypia, which we termed dysplastic metaplasia (DM). A review of gastric biopsy specimens from 587 H. pylori-positive patients revealed 32 cases of DM, 13 of which were classified as high-grade based on the degree of nuclear enlargement. All high-grade DM cases exhibited a telomere volume reduced to less than 60% of that of lymphocytes, increased stemness, and telomerase reverse transcriptase (TERT) expression. Two patients (15%) exhibited low levels of p53 nuclear retention. After a 10-year follow-up, 7 (54%) of the high-grade DM cases had progressed to gastric cancer. These results suggest that DM is characterized by telomere shortening, TERT expression, and stem cell proliferation, and high-grade DM is a high-grade intestinal metaplasia that likely represents a precancerous lesion of gastric cancer. High-grade DM is expected to effectively prevent progression to gastric cancer in H. pylori-positive patients.

Inflammatory mediators for predicting the risk of trauma-induced root resorption: A systematic review.

BACKGROUND/AIM: External inflammatory root resorption (EIRR) and external replacement root resorption (ERRR) are the most common adverse outcomes after luxation injuries or dental injuries. They are usually detected radiographically after considerable progression. It can be envisaged that evaluation of inflammatory mediators might serve as an objective and reliable method of predicting the risk of resorption. The aim of this systematic review was to assess the evidence regarding the risk of resorption and to provide future research directions. METHODS: An a priori protocol was prepared by a multidisciplinary expert group, as per the Cochrane handbook and PRISMA guidelines. The systematic search was conducted in six databases and grey-literature sources. Scrutiny of titles and abstracts, and later full-text articles was performed, and data were extracted. Risk of bias analysis was done by using the Joanna Briggs Institute's Critical Appraisal Checklist for analytical cross-sectional and cohort studies. RESULTS: Eight studies were included in the systematic review and could be categorized as per the source of biomarkers, namely epithelial cells from mucosa, gingival crevicular fluid and extracted teeth. The studies utilizing epithelial cells had been conducted between 2015 and 2018 in Brazil and did not find any correlation with EIRR. Two of the studies with extracted teeth found differences in the immunologic profiles of teeth with resorption. Three studies evaluating gingival crevicular fluid found increased levels of dentine sialoprotein and Interleukin-1-α. CONCLUSION: Among the three sources, the inflammatory gingival crevicular fluid appeared to be the most non-invasive source of biomarkers for predicting trauma-induced root resorption, although the evidence about this came from two studies with moderate and one study with high risk of bias. The primary studies in this systematic review showed variability in terms of the sample sizes, age of the patients, the grading/classification of trauma-induced resorption and the evaluation methods which must be addressed by future researchers.

Advances in the Therapeutic Effects of Apoptotic Bodies on Systemic Diseases.

Apoptosis plays an important role in development and in the maintenance of homeostasis. Apoptotic bodies (ApoBDs) are specifically generated from apoptotic cells and can contain a large variety of biological molecules, which are of great significance in intercellular communications and the regulation of phagocytes. Emerging evidence in recent years has shown that ApoBDs are essential for maintaining homeostasis, including systemic bone density and immune regulation as well as tissue regeneration. Moreover, studies have revealed the therapeutic effects of ApoBDs on systemic diseases, including cancer, atherosclerosis, diabetes, hepatic fibrosis, and wound healing, which can be used to treat potential targets. This review summarizes current research on the generation, application, and reconstruction of ApoBDs regarding their functions in cellular regulation and on systemic diseases, providing strong evidence and therapeutic strategies for further insights into related diseases.

Hypomethylation of CLDN4 Gene Promoter Is Associated with Malignant Phenotype in Urinary Bladder Cancer.

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2'-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin ß1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target.

Early effect of laser irradiation in signaling pathways of diabetic rat submandibular salivary glands.

Low-power laser irradiation (LPLI) is clinically used to modulate inflammation, proliferation and apoptosis. However, its molecular mechanisms are still not fully understood. This study aimed to describe the effects of LPLI upon inflammatory, apoptotic and proliferation markers in submandibular salivary glands (SMGs) in an experimental model of chronic disorder, 24h after one time irradiation. Diabetes was induced in rats by the injection of streptozotocin. After 29 days, these animals were treated with LPLI in the SMG area, and euthanized 24h after this irradiation. Treatment with LPLI significantly decreased diabetes-induced high mobility group box 1 (HMGB1) and tumor necrosis factor alpha (TNF-α) expression, while enhancing the activation of the transcriptional factor cAMP response element binding (CREB) protein. LPLI also reduced the expression of bax, a mitochondrial apoptotic marker, favoring the cell survival. These findings suggest that LPLI can hamper the state of chronic inflammation and favor homeostasis in diabetic rats SMGs.

The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer.

DEC1 (BHLHE40/Stra13/Sharp2) is a basic helix-loop-helix (bHLH) transcription factor that is involved in the regulation of apoptosis and cell proliferation and the response to hypoxia. Epithelial-mesenchymal transition (EMT) is an important step leading to invasion and migration of various tumor cells, and TGF-ß treatment has been shown to induce cancer cells to undergo EMT. However, the significance of DEC1 in TGF-ß-induced EMT remains unknown. We examined the role of DEC1 in EMT of PANC-1 cells, a human pancreatic cancer cell line. As a result, we found that DEC1 was upregulated by TGF-ß in PANC-1 cells, and regulated the expression and the levels of nuclear, cytoplasmic or membrane localization of EMT-related factors, including phosphorylated Smad3 (pSmad3), snail, claudin-4 and N-cadherin. In the presence of TGF-ß, DEC1 knockdown by siRNA inhibited morphological changes during EMT processes, while TGF-ß induced PANC-1 cells to taken on a spindle-shaped morphology. Furthermore, a combination treatment of DEC1 expression with TGF-ß was closely linked to the migration and invasion of PANC-1 cells. Immunohistochemically, DEC1 and pSmad3 were detected within pancreatic cancer tissues, whereas claudin-4 expression was weaker in the cancer tissues compared with the adjacent non-cancer pancreatic tissues. These findings suggest that DEC1 plays an important role in the regulation of these EMT-related factors in pancreatic cancer.

The basic helix-loop-helix transcription factor DEC2 inhibits TGF-ß-induced tumor progression in human pancreatic cancer BxPC-3 cells.

The basic helix loop helix (bHLH) transcription factor DEC2 is associated with the regulation of apoptosis, circadian rhythm and the response to hypoxia. However, the significance of DEC2 in pancreatic cancer remains unknown. Here, we showed for the first time that DEC2 inhibits the progression of human pancreatic cancer. Human pancreatic cancer BxPC-3 cells were treated with or without transforming growth factor-ß (TGF-ß), siRNA against DEC2, or a combination of TGF-ß and DEC2 siRNA or DEC2 overexpression. The cells were analyzed by RT-PCR, real-time PCR, western blotting, immunofluorescent staining and ChIP assay. We also performed immunohistochemical analyses of DEC2 expression in surgically-resected pancreatic cancers. The expression of DEC2 was increased in TGF-ß-treated BxPC-3 cells. In the presence of TGF-ß, DEC2 overexpression decreased the migration and invasion of BxPC-3 cells. Knockdown of DEC2 by siRNA in the presence of TGF-ß significantly increased the expression and nuclear concentration of slug. We also showed that DEC2 siRNA decreased the binding of DEC2 to the E-box of the slug promoter. Immunohistochemically, little DEC2 was detected in pancreatic cancer tissues, whereas significant amounts were detected in the adjacent non-cancerous pancreatic tissues. These results indicate that DEC2 has inhibitory effects against human pancreatic cancer that involve TGF-ß and slug.

BHLH transcription factor DEC2 regulates pro-apoptotic factor Bim in human oral cancer HSC-3 cells.

DEC1 (BHLHE40/Stra13/Sharp2) and DEC2 (BHLHE41/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in the regulation of apoptosis, cell proliferation, circadian rhythms and the response to hypoxia. We previously showed the functional effects of DEC1 and DEC2 on apoptosis in human breast cancer MCF-7 cells. However, the roles of DEC1 and DEC2 in oral cancer are poorly understood. We examined whether DEC1 and DEC2 are involved in the regulation of apoptosis in human oral cancer HSC-3 and CA9-22 cells. The expression of DEC2 was upregulated by cis-diamminedichloroplatinum (II) (cisplatin: CDDP) treatment in HSC-3 cells, whereas CDDP treatment had little effects on the expression of DEC2 in CA9-22 cells. We showed that DEC2 overexpression inhibits pro-apoptotic factor Bim and inhibits apoptosis induced by CDDP in HSC-3 cells, whereas it had little effects on apoptosis in CA9-22 cells. DEC1 overexpression had little effects on apoptosis induced by CDDP in these cells. We also found that CDDP upregulated the amounts of DEC2 in the nucleus in HSC-3 cells. These results suggest that DEC2 has anti-apoptotic effects on apoptosis induced by CDDP in HSC-3 cells.

Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-ß) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.

PERIOD1 (PER1) has anti-apoptotic effects, and PER3 has pro-apoptotic effects during cisplatin (CDDP) treatment in human gingival cancer CA9-22 cells.

PERIOD (PER) proteins are transcriptional regulators that are involved in circadian rhythms, sleep homeostasis, cell proliferation and tumour progression. We previously showed that the expression of PER1 was related to the regulation of apoptosis in human pancreatic cancer and hepatocellular carcinoma cells. However, the significance of PER in oral cancer has not been reported, and the detailed molecular mechanisms by which anti-tumour drug induces apoptosis in gingival cancer cells are not well understood. We examined whether PER1 and PER3 are involved in the regulation of apoptosis in human gingival cancer CA9-22 cells. The expression of PER1 and PER3 was upregulated and downregulated, respectively, by cis-diamminedichloroplatinum (II) (cisplatin: CDDP) treatment in CA9-22 cells, whereas CDDP treatment had little effects on the expression of PER1 and PER3 in human gingival fibroblasts (HGF-1). We found that short interference RNA (siRNA)-mediated knockdown of PER1 enhanced apoptosis of CA9-22 cells, and that PER1 regulated the amount of Bim, an apoptosis-related molecule. On the other hand, PER3 knockdown had an inhibitory effect on the apoptosis of CA9-22 cells induced by CDDP treatment. These results suggest that the alternation of expression of PER1 and PER3 was related to the apoptosis of CA9-22 cells. Furthermore, PER1 was intensely stained in the gingival cancer tissues, whereas PER3 was significantly stained in the non-tumour tissues by immunohistochemistry. These findings suggest that PER1 and PER3 have anti-apoptotic and pro-apoptotic effects in human gingival cancer CA9-22 cells, respectively. The balance of PER1 and PER3 may modulate apoptotic reactions in gingival cancer cells.

Basic helix-loop-helix transcription factors DEC1 and DEC2 regulate the paclitaxel-induced apoptotic pathway of MCF-7 human breast cancer cells.

Differentiated embryonic chondrocyte gene (DEC) 1 (BHLHE40/Stra13/Sharp2) and DEC2 (BHLHE41/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are associated with the regulation of apoptosis, cell proliferation and circadian rhythms, as well as malignancy in various cancers. However, the roles of DEC1 and DEC2 expression in breast cancer are poorly understood. In this study, we sought to examine the roles of DEC1 and DEC2 in MCF-7 human breast cancer cells that had been treated with paclitaxel. The expression of DEC1 and DEC2 was up-regulated in paclitaxel-treated MCF-7 cells. Knockdown of DEC1 by siRNA decreased the amount of cleaved poly (ADP-ribose) polymerase (PARP), after treatment with paclitaxel, whereas DEC2 knockdown increased the amount of cleaved PARP in both the presence and absence of paclitaxel. Immunofluorescent staining revealed that paclitaxel treatment increased the amount of DEC1 in the nucleus, and increased the amount of DEC2 in both the nucleus and cytoplasm. These results indicate that DEC1 has pro-apoptotic effects, whereas DEC2 has anti-apoptotic effects on the paclitaxel-induced apoptosis in human breast cancer MCF-7 cells.

Effects of high-dose major components in oral disinfectants on the cell cycle and apoptosis in primary human gingival fibroblasts in vitro.

We evaluated the effects of high-dose major components in oral disinfectants on oral cells from the standpoints of the cell cycle and apoptosis. We examined the viability and cell cycle of human gingival fibroblasts (HGFs) treated with the components of dental disinfectants, benzethonium chloride (BEC), benzalkonium chloride (BAC), and povidone iodine (PVD-I) using a cell counting kit and flow cytometry. The IC(50) inhibitory concentration value in HGF cultures at 24 hours was 1.3x10(-2) mM BEC, 6.0x10(-3) mM BAC, and 2.6x10(-1) mM PVD-I. In the cell cycle analysis, propidium iodide-stained HGFs were arrested in G(0)/G(1) of the cell cycle by all three disinfectants, and in the apoptosis assay, annexin V-FITC/PI-stained HGFs that became apoptotic at 5.0x10(-2) and 1.0x10(-1) mM BEC and 5.0x10(-2) and 1.0x10(-1) mM BAC, but not in PVD-I at concentrations as high as 5.0x10(-1) mM. Our findings describe the effects of high-dose oral disinfectants, rather than clinical concentrations. Nevertheless, appreciating the effects of high-dose disinfectants absorbed into the human body is important, where they may accumulate in specific tissues and cells.

High mobility group box-1-inducible melanoma inhibitory activity is associated with nodal metastasis and lymphangiogenesis in oral squamous cell carcinoma.

Melanoma inhibitory activity (MIA) is an 11-kDa secretory protein isolated from malignant melanoma cells that is correlated with invasion and metastasis in various human malignancies. We examined MIA expression in 62 oral squamous cell carcinomas (OSCC) by immunohistochemistry. MIA expression was significantly associated with nodal metastasis (P = 0.00018). MIA expression was also associated with expression of high mobility group box-1 (HMGB1) (P < 0.0001) and lymph vessel density (P < 0.0001). Expression levels of MIA, HMGB1, nuclear factor kB (NFkB) p65 and HMGB1-NFkB p65 binding were significantly higher in a metastatic human OSCC cell line (HSC3) than those in a non-metastatic OSCC cell line (HSC4). Treatment with receptor for advanced glycation end products (RAGE) antisense or small interfering RNA and human recombinant HMGB1 (hrHMGB1) did not affect MIA expression, whereas HMGB1 antisense or siRNA treatment decreased MIA expression in HSC3 cells. Then HMGB1 enhanced MIA expression as an NFkB cofactor but not as a RAGE ligand. MIA neutralization by MIA antibodies increased extracellular signal-related kinase 1/2 phosphorylation, but decreased p38 phosphorylation and the expression of vascular epithelial growth factor (VEGF)-C and -D. Treatment with p38 inihibitor decreased VEGF-C and -D expression in HSC3 cells. These results suggest that MIA expression is enhanced by the interaction of intracellular HMGB1 and NFkBp65 and MIA is closely involved in tumor progression and nodal metastasis by the increments of VEGF-C and VEGF-D in OSCC.

Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.

DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up-regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1alpha (HIF-1alpha) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1alpha but not with ARNT1. The binding of HIF-1alpha to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.