NAT10/CEBPB/vimentin signalling axis promotes adenoid cystic carcinoma malignant phenotypes in vitro.

OBJECTIVE: To explore the biological function and mechanisms of CEBPB and NAT10-mediated N4-acetylcytidine (ac4c) modification in salivary adenoid cystic carcinoma (SACC). MATERIALS AND METHODS: CEBPB and NAT10 were knocked down in SACC-LM cells by siRNA transfection and overexpressed in SACC-83 cells by plasmid transfection. Malignant phenotypes were evaluated using CCK-8, Transwell migration and colony formation assays. Real-time PCR, western blotting, ChIP and acRIP were used to investigate the molecular mechanisms involved. RESULTS: We found that CEBPB was highly expressed in SACC tissues and correlated with lung metastasis and unfavourable prognosis. Gain- and loss-of-function experiments revealed that CEBPB promoted SACC malignant phenotypes. Mechanistically, CEBPB exerted its oncogenic effect by binding to the vimentin gene promoter region to enhance its expression. Moreover, NAT10-mediated ac4c modification led to stabilization and overexpression of CEBPB in SACC cells. We also found that NAT10, the only known human enzyme responsible for ac4C modification, promoted SACC cell migration, proliferation and colony formation. Moreover, CEBPB overexpression restored the inhibitory effect of NAT10 knockdown on malignant phenotypes. CONCLUSIONS: Our study reveals the critical role of the newly identified NAT10/CEBPB/vimentin axis in SACC malignant progression, and the findings may be applied to improve treatment for SACC.

Long non-coding RNA MRPL23-AS1 suppresses anoikis in salivary adenoid cystic carcinoma in vitro.

Distant lung metastasis is the main factor that affects the survival rate of patients with salivary adenoid cystic carcinoma (SACC). Anoikis resistance is a feature of tumor cells that easily metastasize. The long non-coding RNA (lncRNA) MRPL23 antisense RNA 1 (MPRL23-AS1) is related to lung metastasis in SACC, but its role in anoikis resistance is unknown. After altering MPRL23-AS1 expression in SACC cells, anoikis resistance was detected by calcein AM/PI staining and annexin V/PI flow cytometry. The apoptosis marker activated caspase-3 and the bcl-2/bax ratio were detected by Western blotting. The relationship between MPRL23-AS1 and the promoter of the potential downstream target gene p19INK4D was identified by chromatin immunoprecipitation (ChIP)-PCR assay. p19INK4D expression in patient tissues was determined using qRT-PCR and immunohistochemistry. The functional experiments showed that MPRL23-AS1 could promote anoikis resistance in vitro. MRPL23-AS1 recruited the EZH2 to the promoter region of p19INK4D, inhibited p19INK4D expression, and promoted tumor cell anoikis resistance. p19INK4D overexpression did not affect anoikis in attached cells; however, it attenuated the anoikis resistance effect of MPRL23-AS1 in suspension cells. p19INK4D expression was significantly lower in SACC tissues than in normal tissues. The novel MRPL23-AS1/p19INK4D axis may be a potential SACC biomarker or therapeutic target.

Stem cells  from exfoliated  deciduous  teeth alleviate hyposalivation caused by Sjögren syndrome.

OBJECTIVES: To evaluate the effect of stem cells  from  exfoliated  deciduous  teeth on the hyposalivation caused by Sjögren syndrome (SS) and investigate the mechanism. METHODS: Stem cells were injected into the tail veins of non-obese diabetic mice, the animal model of SS. The saliva flow was measured after pilocarpine intraperitoneal injection. Apoptosis and autophagy were evaluated by TUNEL and Western blot. Lymphocyte proportions were detected by flow cytometer. RESULTS: Fluid secretion was decreased in 21-week-old mice. Stem cell treatment increased fluid secretion, alleviated inflammation in the submandibular glands and reduced inflammatory cytokine levels in the serum, submandibular glands and saliva. Stem cells decreased the apoptotic cell number and the expressions of ATG5 and Beclin-1 in the submandibular glands. Stem cells have no effect on other organs. Furthermore, the infused stem cells migrated to the spleen and liver, not the submandibular gland. Stem cells directed T cells towards Treg cells and suppressed Th1 and Tfh cells in spleen lymphocytes. CONCLUSION: Stem cells  from  exfoliated  deciduous  teeth alleviate the hyposalivation caused by SS via decreasing the inflammatory cytokines, regulating the inflammatory microenvironment and decreasing the apoptosis and autophagy. The stem cells regulated in T-cell differentiation are involved in the immunomodulatory effects.

Hsa_circRNA_0059655 plays a role in salivary adenoid cystic carcinoma by functioning as a sponge of miR-338-3p.

Circular RNAs(circRNA) are recently demonstrated to have a close relationship with tumors.To investigate the role of circular RNA in the pathogenesis of salivary adenoid cystic carcinoma(SACC), ten SACC tissues and paired normal submandibular gland(SMG) tissues were collected as the tumor group and the control group. Total RNA was extracted and then measured using ceRNA microarray (including mRNA, lncRNA, and circRNA) and miRNA microarray. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis were performed in order to investigate the function of the differential expressing genes. The ceRNA regulatory network was constructed to find the core circRNAs. Then the role of circRNA on proliferation was examined in the SACC cell line SACC-83 using CCK-8,qRT-PCR and western blotting, and its roles on migration and invasion were examined using wound healing assay and transwell assay. The results of the microarrays showed that 3792 mRNAs, 7649 lncRNAs, 11553 circRNAs, and 132 miRNAs expressed differentially. The ceRNA regulatory network analysis showed that hsa_circ_0059655 and other 14circRNAs derived from PYGB target on several similar genes by miR-338-3p.Among the 15 circRNAs derived from PYGB, hsa_circ_0059655has the most relationships in the ceRNA network. Furthermore, after hsa_circ_0059655 was knocked down in SACC-83 cells, the expression of hsa-miR-338-3p was up-regulated while CCND1was down-regulated. The proliferation, migration, and invasion of SACC-83 cells also decreased after hsa_circ_0059655 knock-downed.Taken together, the circRNAs derived from PYGB may regulate the tumorigenesis and development of SACC through competing with miR-338-3p.

Nimotuzumab suppresses epithelial-mesenchymal transition and enhances apoptosis in low-dose UV-C treated salivary adenoid cystic carcinoma cell lines in vitro.

Salivary adenoid cystic carcinoma (SACC), which is one of the most common malignant tumors of the salivary glands, is associated with a poor long-term outcome. There are currently few therapeutic options for patients with SACC. Recent studies have shown the potential of the application of ultraviolet-C (UV-C) irradiation for the treatment of human cancer. In the present study, we investigated the effects of UV-C in the SACC cell lines SACC-83 and SACC-LM. High-dose UV-C (200 J/m) induced apoptosis and inhibited colony formation significantly. However, low-dose UV-C (10 J/m), which had little effect on apoptosis and colony formation, increased the ability of migration in SACC cells accompanied by a decrease in E-cadherin and an increase in vimentin, suggesting the occurrence of epithelial-mesenchymal transition (EMT). Low-dose UV-C (10 J/m) also resulted in upregulation of the phosphorylated forms of epidermal growth factor receptor (EGFR) and Akt (p-EGFR and p-Akt, respectively). Pretreatment with Nimotuzumab, an anti-EGFR monoclonal antibody, reversed the EMT as well as upregulation of p-EGFR/p-Akt induced by UV-C. Moreover, Nimotuzumab enhanced UV-C induced apoptosis and inhibition of colony formation. Our results indicate that EMT exerts a protective effect against apoptosis induced by low-dose UV-C. Thus, the combined application of Nimotuzumab and low-dose UV-C in vitro has an advantageous antitumor effect in SACC compared with the application of UV-C alone.

Extracellular vesicles containing circMYBL1 induce CD44 in adenoid cystic carcinoma cells and pulmonary endothelial cells to promote lung metastasis.

Adenoid cystic carcinoma (ACC) is a rare malignant epithelial neoplasm that arises in secretory glands and commonly metastasizes to the lungs. MYBL1 is frequently overexpressed in ACC and has been suggested to be a driver of the disease. Here, we identified a circRNA derived from MYBL1 pre-mRNA that accompanied overexpression of MYBL1 in ACC. Overexpression of circMYBL1 was correlated with increased lung metastasis and poor overall survival in ACC patients. Ectopic circMYBL1 overexpression promoted malignant phenotypes and lung metastasis of ACC cells. Mechanistically, circMYBL1 formed a circRNA-protein complex with CCAAT enhancer binding protein beta (CEBPB), which inhibited ubiquitin-mediated degradation and promoted nuclear translocation of CEBPB. In the nucleus, circMYBL1 increased the binding of CEBPB to the CD44 promoter region and enhanced its transcription. In addition, circMYBL1 was enriched in small extracellular vesicles (sEVs) isolated from the plasma of ACC patients. Treatment with sEVs containing circMYBL1 in sEVs enhanced pro-metastatic phenotypes of ACC cells, elevated the expression of CD44 in human pulmonary microvascular endothelial cells (HPMECs), and enhanced the adhesion between HPMECs and ACC cells. Moreover, circMYBL1 encapsulated in sEVs increased the arrest of circulating ACC cells in the lung and enhanced the lung metastatic burden. This data suggests that circMYBL1 is a tumor-promoting circRNA that could serve as a potential biomarker and therapeutic target in ACC.

ALMS1-IT1: A Key Player in the Novel Disulfidptosis-Related LncRNA Prognostic Signature for Head and Neck Squamous Cell Carcinoma.

Disulfidptosis is a newly discovered form of programmed cell death that is induced by disulfide stress. It is closely associated with various cancers, including head and neck squamous cell carcinoma (HNSCC). However, the factors involved in the modulation of disulfidptosis-related genes (DRGs) still remain unknown. In this study, we established and validated a novel risk score model composed of 11 disulfidptosis-related lncRNAs (DRLs) based on 24 DRGs in HNSCC. The results revealed strong correlations between the 11-DRL prognostic signature and clinicopathological features, immune cell infiltration, immune-related functions, and disulfidptosis-associated pathways, including NADPH and disulfide oxidoreductase activities. Furthermore, we studied and verified the involvement of ALMS1-IT1, one of the 11 model DRLs, in the disulfidptosis of HNSCC cell lines. A series of assays demonstrated that ALMS1-IT1 modulated cell death under starvation conditions in a pentose phosphate pathway (PPP)-dependent manner. Knockdown of ALMS1-IT1 inhibited the PPP, contributing to a decline in NADPH levels, which resulted in the formation of multiple intermolecular disulfide bonds between actin cytoskeleton proteins and the collapse of F-actin in the cytoplasm. Therefore, ALMS1-IT1, which is highly expressed in SLC7A11high cells, can be considered a promising therapeutic target for disulfidptosis-focused treatment strategies for cancer and other diseases.

Applications of Polydopamine in Implant Surface Modification.

There is great clinical demand for orthopedic and dental implant surface modification methods to prevent osseointegration failure and improve implant biological functions. Notably, dopamine (DA) can be polymerized to form polydopamine (PDA), which is similar to the adhesive proteins secreted by mussels, to form a stable bond between the bone surface and implants. Therefore, PDA has the potential to be used as an implant surface modification material with good hydrophilicity, roughness, morphology, mechanical strength, biocompatibility, antibacterial activity, cellular adhesion, and osteogenesis. In addition, PDA degradation releases DA into the surrounding microenvironment, which is found to play an important role in regulating DA receptors on both osteoblasts and osteoclasts during the bone remodeling process. Furthermore, the adhesion properties of PDA suggest its use as an intermediate layer in assisting other functional bone remodeling materials, such as nanoparticles, growth factors, peptides, and hydrogels, to form "dual modifications." The purpose of this review is to summarize the recent progress in research on PDA and its derivatives as orthopedic and dental implant surface modification materials and to analyze the multiple functions of PDA.

Facile distribution of an alkaline microenvironment improves human bone marrow mesenchymal stem cell osteogenesis on a titanium surface through the ITG/FAK/ALP pathway.

PURPOSE: Osseointegration at the titanium surface-bone interface is one of the key factors affecting the success rate of dental implants. However, the titanium surface always forms a passive oxide layer and impacts bone marrow-derived mesenchymal stem cell (BMSC) osteogenic differentiation after exposure to the atmosphere, which further leads to poor osseointegration. Given that wet storage helps prevent titanium aging and that weakly alkaline conditions stimulate BMSC osteogenic differentiation, the aim of the present study was to explore whether sodium bicarbonate, a well-known hydrogen ion (pH) buffer, forms an alkaline microenvironment on titanium surfaces to promote BMSC osteogenic differentiation. MATERIAL AND METHODS: In this work, sand-blasted and acid-etched (SLA) titanium discs were soaked in 20 mM, 50 mM, 100 mM, and 200 mM sodium bicarbonate at room temperature for 5 min without rinsing. The influence of this surface modification on BMSC adhesion, proliferation, and osteogenic differentiation was measured. Additionally, cellular osteogenic differentiation-associated signaling pathways were evaluated. RESULTS: We showed that titanium discs treated with sodium bicarbonate created an extracellular environment with a higher pH for BMSCs than the normal physiological value for 5 days, strongly promoting BMSC osteogenic differentiation via the activation of integrin-focal adhesion kinase-alkaline phosphatase (Itg-FAK-ALP). In addition, the proliferation and adhesion of BMSCs were increased after alkaline treatment. These cellular effects were most significant with 100 mM sodium bicarbonate. CONCLUSION: The results indicated that the titanium surface treated with sodium bicarbonate improved BMSC osteogenic differentiation mainly by creating an alkaline microenvironment, which further activated the Itg-FAK-ALP signaling pathway. CLINICAL RELEVANCE: Surfaces modified with 100 mM sodium bicarbonate had the highest initial pH value and thus showed the greatest potential to improve BMSC performance on titanium surfaces, identifying a novel conservation method for dental implants.

Dopamine D1 receptor-mediated activation of the ERK signaling pathway is involved in the osteogenic differentiation of bone mesenchymal stem cells.

BACKGROUND: Osteogenic differentiation of bone mesenchymal stem cells (BMSCs) is regulated by numerous signaling pathways. Dopamine (DA), a neurotransmitter, has previously been demonstrated to induce new bone formation by stimulating the receptors on BMSCs, but the essential mediators of DA-induced osteogenic signaling remain unclear. METHODS: In this work, we evaluated the influence of both dopamine D1 and D2 receptor activation on BMSC osteogenic differentiation. Gene and protein expression of osteogenic-related markers were tested. The direct binding of transcriptional factor, Runx2, to those markers was also investigated. Additionally, cellular differentiation-associated signaling pathways were evaluated. RESULTS: We showed that the expression level of the D1 receptor on BMSCs increased during osteogenic differentiation. A D1 receptor agonist, similar to DA, induced the osteogenic differentiation of BMSCs, and this phenomenon was effectively inhibited by a D1 receptor antagonist or by D1 receptor knockdown. Furthermore, the suppression of protein kinase A (PKA), an important kinase downstream of the D1 receptor, successfully inhibited DA-induced BMSC osteogenic differentiation and decreased the phosphorylation of ERK1/2. Compared with P38, MAPK, and JNK, DA mainly induced the phosphorylation of ERK1/2 and led to the upregulation of Runx2 transcriptional activity, thus facilitating BMSC osteogenic differentiation. On the other hand, an ERK1/2 inhibitor could reverse these effects. CONCLUSIONS: Taken together, these results suggest that ERK signaling may play an essential role in coordinating the DA-induced osteogenic differentiation of BMSCs by D1 receptor activation.

MicroRNA­103a­3p promotes metastasis by targeting TPD52 in salivary adenoid cystic carcinoma.

Salivary adenoid cystic carcinoma (SACC) exhibits slow continuous growth, frequent local recurrences and a high incidence of blood metastasis, with advanced lung metastasis frequently occurring and being among the primary causes of mortality. MicroRNAs (miR) serve a significant role in the initiation and development of cancer and may be tumour­specific molecular targets. However, the role of miR­103a­3p in SACC remains largely unknown. In the present study, the expression levels of miR­103a­3p and tumour protein D52 (TPD52) were detected by reverse transcription­quantitative PCR. In addition, wound­healing assays, Transwell assays and mouse models of lung metastasis were used to investigate the biological functions exerted by miR­103a­3p. The present results suggested that miR­103a­3p expression was significantly upregulated in SACC samples. Gain­of­function and loss­of­function studies in SACC cells demonstrated that miR­103a­3p acted as an oncogene by promoting tumour cell migration in vitro and lung metastasis in vivo. Dual­luciferase reporter gene assays indicated that miR­103a­3p exerted its regulatory functions by binding to the 3' untranslated region of TPD52 mRNA. TPD52 overexpression rescued the effect of miR­103a­3p on promoting SACC cell migration, suggesting that miR­103a­3p acted as an oncogene to promote cancer metastasis by directly targeting TPD52. Thus, the newly identified miR­103a­3p/TPD52 axis contributes to the understanding of SACC pathogenesis, providing insights into the identification of novel biomarkers or potential therapeutic targets in SACC.

Long Noncoding RNA MRPL23-AS1 Promotes Adenoid Cystic Carcinoma Lung Metastasis.

Lung metastasis is a major factor affecting long-term survival in patients with adenoid cystic carcinoma. Here, we showed that the long noncoding RNA (lncRNA) MRPL23 antisense RNA 1 (MRPL23-AS1) was highly expressed and correlated with lung metastasis and overall survival in patients with salivary adenoid cystic carcinoma (SACC). MRPL23-AS1 positively regulated epithelial-mesenchymal transition by forming an RNA-protein complex with enhancer of zeste homolog 2 (EZH2). MRPL23-AS1 increased the binding of EZH2 and H3K27me3 on the E-cadherin promoter region. Moreover, MRPL23-AS1 levels were higher in exosomes isolated from the blood plasma of patients with SACC, and exosomal MRPL23-AS1 affected pulmonary microvascular endothelial cells in an "exosomecrine" manner. MRPL23-AS1-enriched exosomes increased microvascular permeability and facilitated the metastasis of SACC in vivo. Collectively, these findings highlight a molecular mechanism of lung metastasis in SACC. MRPL23-AS1 may represent a biomarker and target for clinical intervention to control this intractable disease. SIGNIFICANCE: This study identifies a novel metastasis-promoting lncRNA MRPL23-AS1, which mediates the transcriptional silencing of E-cadherin through forming an RNA-protein complex with EZH2.

Titanium discs coated with 3,4-dihydroxy-l-phenylalanine promote osteogenic differentiation of human bone mesenchymal stem cells in vitro.

The bioinspired material 3,4-dihydroxy-l-phenylalanine (DOPA) is commonly used as a basic layer in surface modification for osteogenesis; however, its effects on bone remodeling and the underlying mechanisms remain unclear. Here, we investigated the effect of DOPA-coated surfaces on human bone marrow-derived mesenchymal stem cells in vitro. Cells cultured on DOPA-modified titanium discs exhibited enhanced cellular adhesion and spreading compared with cells on non-treated surfaces. Moreover, DOPA-coating promoted greater cell proliferation and osteogenic differentiation, as determined using cell counting kit-8 (CCK-8) assay, alkaline phosphatase activity test and quantitative mineralization measurements. Furthermore, microarray analysis revealed that genes participating in focal adhesion were upregulated on DOPA-coated surfaces. Our results indicate that the application of a simple DOPA coating can promote osteogenic differentiation of osteoprogenitor cells, improving new bone formation and bone remodeling around implantable devices in tissue engineering.

MYB promotes the growth and metastasis of salivary adenoid cystic carcinoma.

The incidence of recurrent t(6;9) translocation of the MYB proto­oncogene to NFIB (the gene that encodes nuclear factor 1 B­type) in adenoid cystic carcinoma (ACC) tumour tissues is high. However, MYB [the gene that encodes transcriptional activator Myb (MYB)] overexpression is more common, indicating that MYB serves a key role in ACC. The current study aimed to investigate the role of MYB in salivary (S)ACC growth and metastasis. A total of 50 fresh­frozen SACC tissues and 41 fresh­frozen normal submandibular gland (SMG) tissues were collected to measure MYB mRNA expression, and to analyse the associations between MYB and epithelial­mesenchymal transition (EMT) markers. Compared with normal SMG tissue, SACC tissues demonstrated significantly increased MYB expression, with a high expression rate of 90%. Interestingly, MYB tended to be negatively correlated with CDH1 [the gene that encodes cadherin­1 (E­cadherin)] and positively correlated with VIM (the gene that encodes vimentin), suggesting that MYB is associated with SACC metastasis. To explore the role of MYB in SACC, the authors stably overexpressed and knocked down MYB in SACC cells. The authors of the current study demonstrated that MYB overexpression promoted SACC cell proliferation, migration and invasion, whereas its knockdown inhibited these activities. Additionally, when MYB was overexpressed, CDH1 expression was downregulated, and CDH2 (the gene that encodes cadherin­2), VIM and ACTA2 (the gene that encodes actin, aortic smooth muscle) expression was upregulated. Then, the effect of MYB on lung tumour metastasis was investigated in vivo in non­obese diabetic/severe combined immunodeficiency mice. MYB overexpressing and control cells were injected into the mice through the tail vein. The results revealed that MYB promoted SACC lung metastasis. Collectively, these results demonstrated that MYB is aberrantly overexpressed in SACC tissues, and promotes SACC cell proliferation and metastasis, indicating that MYB may be a novel therapeutic target for SACC.

Simple 3,4-Dihydroxy-L-Phenylalanine Surface Modification Enhances Titanium Implant Osseointegration in Ovariectomized Rats.

Osteoporosis presents a challenge to the long-term success of osseointegration of endosseous implants. The bio-inspired 3,4-dihydroxy-L-phenylalanine (Dopa) coating is widely used as a basic layer to bind osteogenetic molecules that may improve osseointegration. To date, little attention has focused on application of Dopa alone or binding inhibitors of bone resorption in osteoporosis. Local use of a bisphosphonate such as zoledronic acid (ZA), an inhibitor of osteoclast-mediated bone resorption, has been proven to improve implant osseointegration. In this study, ovariectomized rats were divided into four groups and implanted with implants with different surface modifications: sandblasted and acid-etched (SLA), SLA modified with Dopa (SLA-Dopa), SLA modified with ZA (SLA-ZA), and SLA modified with Dopa and ZA (SLA-Dopa + ZA). Measurement of removal torque, micro-computed tomography and histology revealed a greater extent of bone formation around the three surface-modified implants than SLA-controls. No synergistic effect was observed for combined Dopa + ZA coating. Microarray analysis showed the Dopa coating inhibited expression of genes associated with osteoclast differentiation, similarly to the mechanism of action of ZA. Simple Dopa modification resulted in a similar improvement in osseointegration compared to ZA. Thus, our data suggest simple Dopa coating is promising strategy to promote osseointegration of implants in patients with osteoporosis.

Inhibition of cathepsin K promotes osseointegration of titanium implants in ovariectomised rats.

The bone mineral deficiency in osteoporosis poses a threat to the long-term outcomes of endosseous implants. The inhibitors of cathepsin K (CatK) significantly affect bone turnover, bone mineral density (BMD) and bone strength in the patients with osteoporosis. Therefore, we hypothesised that the application of a CatK inhibitor (CatKI) could increase the osseointegration of endosseous implants under osteoporotic conditions. Odanacatib (ODN), a highly selective CatKI, was chosen as the experimental drug. Sixteen rats were randomised into 4 groups: sham, ovariectomy (OVX) with vehicle, OVX with low-dose ODN (5 mg/kg) and OVX with high-dose ODN (30 mg/kg). Titanium implants were placed into the distal metaphysis of bilateral femurs of each OVX rat. After 8 weeks of gavaging, CatKI treatment increased the removal torque, BMD and bone-to-implant contact (BIC). Moreover, high-dose CatKI exerted a better influence than low-dose CatKI. Furthermore, CatKI treatment not only robustly suppressed CatK gene (CTSK) expression, but also moderately reduced expression of the osteoblast-related genes Runx2, Collagen-1, BSP, Osterix, OPN, SPP1 and ALP. Thus, CatKI could affect the osteoblast-related genes, although the balance of bone turnover was achieved mainly by CatK inhibition. In conclusion, CatKI prevented bone loss and aided endosseous implantation in osteoporotic conditions.

Epiregulin Promotes Lung Metastasis of Salivary Adenoid Cystic Carcinoma.

Salivary adenoid cystic carcinoma (SACC) is a peculiar malignant tumor, characterized by its slow but inexorable growth, with a high incidence of lung metastasis and poor prognosis. Here, we show the upregulated expression of EGFR ligand epiregulin in a subset of SACC cells correlates with lung metastasis and unfavorable outcome in patients with SACC. We found that upregulation of epiregulin in SACC cells induced epithelial-mesenchymal transition by regulating GLI1/E-cadherin. Elevated epiregulin increased the expression of pro-angiogenic factors, such as VEGFA, bFGF, and IL-8. We also show that epiregulin can be delivered via exosomes and was enriched in exosomes derived from epiregulin-overexpressing SACC cells. Furthermore, treating immunodeficient mice with these epiregulin-enriched exosomes greatly enhanced SACC metastasis to lung. These epiregulin-enriched exosomes significantly enhanced angiogenesis in the neighboring tumor microenvironment and increased vascular permeability in the pre-metastatic lung microenvironment in vivo. Therefore, epiregulin, as well as epiregulin-containing exosomes, may be a novel target for controlling SACC lung metastasis.

Establishment of submandibular gland allotransplantation model in miniature swine.

BACKGROUND: Autologous transplantation of the submandibular gland (SMG) into the temporal fossa with microvascular anastomosis has been successfully applied in severe xerophthalmia patients as a permanent tear substitute. However, severe xerophthalmia can be accompanied by salivary gland dysfunction, making such autotransplantation unsuitable. Therefore, SMG allotransplantation might be a solution. The aim of this study was to assess the technical feasibility of submandibular gland allotransplantation. METHODS: Twelve miniature swine were randomized to serve as donors or recipients. One SMG was transplanted between a donor and a recipient. The donor SMG was revascularized by microvascular anastomosis of its vascular pedicle to the recipient lingual artery and external jugular vein. The secretory duct was implanted into the vestibule of the mouth through a subcutaneous tunnel. No immunosuppressive agent was administered. The results were assessed by visual inspection of the secretion, and histopathological examination of the transplanted SMG. RESULTS: Technically, all surgical procedures were successful. Clear secretion flowed out of the duct as soon as blood supply of the transplanted submandibular gland was reestablished. The secretion of the gland lasted for 5 days. As expected, an acute rejection reaction occurred after surgery because no immunosuppressive agents were used. Secretion from the transplanted SMG ceased within 5 days. CONCLUSIONS: A model of SMG allotransplantation can be established in miniature swine. The technique of submandibular gland allotransplantation is feasible.

Long-term survival of an allografted submandibular gland in a miniature swine model given immunosuppressant drugs.

We used a model of allografts of submandibular glands in miniature swine to test the requirements of immunosuppressants for the survival of such grafts. Animals in the first group (n=6) were given no immunosuppressant, and the submandibular glands were rejected within 7 days. Those in the low-dose group (n=4) were treated with a low dose of cyclosporine, methylprednisolone, and azathioprine. The allografts were rejected on days 15, 17, 19, and 20. The animals in the high-dose group (n=6) were treated with a high dose of cyclosporin, methylprednisolone, and azathioprine. Three allografts were rejected, on days 35, 48, and 60. One animal died from anaesthetic problems on day 30 after transplantation, and there were no signs of rejection in the allograft. The other two allografts survived for 100 days with secretory function and no signs of rejection. After we discontinued the immunosuppression on day 100, the two remaining allografts were rejected on days 121 and 128. Blood tests and biopsy specimens of the heart, lung, liver, and kidneys were normal in all animals that were given immunosuppressants. To our knowledge, this study is the first to describe long-term survival of allografted submandibular glands with secretory function in miniature swine.

Thioredoxin 1 mediates TGF-ß-induced epithelial-mesenchymal transition in salivary adenoid cystic carcinoma.

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-ß is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-ß induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-ß-Akt/GSK-3ß on EMT. TXN could be a potential therapeutic target for SACC.