Sheng-n Li

Epiregulin promotes migration and invasion of salivary adenoid cystic carcinoma cell line SACC-83 through activation of ERK and Akt.

Hematogenous metastasis is one of the most important factors determining the outcome of the patients with salivary adenoid cystic carcinoma (SACC). In the present study, we examined expression profile of genes in SACC cell lines to look for molecules responsible for its unique metastatic trait. A transcriptomic microarray analysis between the lower lung-metastatic rate cell line SACC-83 and the higher lung-metastatic rate cell line SACC-LM were performed, and eight genes, showed by microarray to be highly expressed in SACC-LM, were picked for validation by quantitative real-time PCR. Among the genes, the expression of epiregulin, a novel member of epidermal growth factor family, was 350-folds higher in SACC-LM than in SACC-83. Accordingly, we examined the effects of epiregulin on migration and invasion in SACC-83 as well as its targeted downstream molecules, and found that epiregulin could promote in vitro migration and invasion in SACC-83. Furthermore, epiregulin not only induced activation of both ERK1/2 and Akt, but also expression of COX-2. In addition, all these effects could be partially blocked by U0126, a specific inhibitor of mitogen-activated protein kinase kinase (MEK or MAPKK), or LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Conclusively, the results suggest that epiregulin may play an important role in lung metastasis of SACC.

Reactive Oxygen Species and Autophagy Associated Apoptosis and Limitation of Clonogenic Survival Induced by Zoledronic Acid in Salivary Adenoid Cystic Carcinoma Cell Line SACC-83

Salivary adenoid cystic carcinoma is an epithelial tumor in the head and neck region. Despite its slow growth, patients with salivary adenoid cystic carcinoma exhibit poor long term survival because of a high rate of distant metastasis. Lung and bone are common distant metastasis sites. Zoledronic acid, a third generation bisphosphonate, has been used for tumor-induced osteolysis due to bone metastasis and has direct antitumor activity in several human neoplasms. Here, we observed that zoledronic acid inhibited salivary adenoid cystic carcinoma cell line SACC-83 xenograft tumor growth in nude mice. In vitro, zoledronic acid induced apoptosis and reduced clonogenic survival in SACC-83. Flow cytometry and western blotting indicated that the cell cycle was arrested at G0/G1. Zoledronic acid treatment upregulated reactive oxygen species as well as the autophagy marker protein LC-3B. Reactive oxygen species scavenger N-acetylcysteine and autophagy antagonist 3-methyladenine decreased zoledronic acid-induced apoptosis and increased clonogenic survival. Silencing of the autophagy related gene Beclin-1 also decreased zoledronic acid-induced apoptosis and inhibition of clonogenic formation. In addition, isobolographic analysis revealed synergistic effects on apoptosis when zoledronic acid and paclitaxel/cisplatin were combined. Taken together, our results suggest that zoledronic acid induced apoptosis and reduced clonogenic survival via upregulation of reactive oxygen species and autophagy in the SACC-83 cell line. Thus, zoledronic acid should be considered a promising drug for the treatment of salivary adenoid cystic carcinoma.

Transforming growth factor-β and epithelial-mesenchymal transition are associated with pulmonary metastasis in adenoid cystic carcinoma.

OBJECTIVES Adenoid cystic carcinoma (ACC) is one of the most common malignancies of salivary glands, characterized by poor prognosis, particularly due to pulmonary metastasis. We previously reported that transforming growth factor (TGF)-β1 promoted ACC cell migration and invasion via the Smad pathway in vitro. The aim of this study was to establish the underlying mechanisms. MATERIALS AND METHODS TGF-β1, phospho-Smad2 and β-catenin expression in ACC tissues derived from patients was evaluated by immunohistochemistry. The role of TGF- β1 on the invasive capacity of ACC cells was determined by transwell assays in SACC-83 cells transfected with TGF-β1 and TGF-β type II dominant-negative receptor (TβRIIDN) plasmids or silenced by TGF-β1 siRNA. Expression of the epithelial-mesenchymal transition (EMT) markers, β-catenin, E-cadherin and Nectin-1, was determined by real-time PCR and immunochemistry. In vivo investigations were performed by inoculating nude mice with the transfected ACC cells and examining metastasis in bilateral lung tissues by immunohistochemistry. RESULTS Overexpression of TGF-β1 and phospho-Smad2, and reduced expression of membrane β-catenin, were closely associated with lung metastasis in ACC. Furthermore, the EMT markers were downregulated. In vitro, cells transfected with TGF-β1 exhibited altered morphology and increased invasive capacity compared to TβRIIDN-transfected cells or TGF-β1 siRNA silenced cells. In vivo, mice inoculated with TGF-β1 transfected ACC cells exhibited more metastases than other cells. CONCLUSION TGF-β1, phospho-Smad2 and β-catenin were significantly correlated with ACC metastasis. Blockade of TGF-β signaling by TβRIIDN or siRNA may offer potential gene therapies against pulmonary metastasis in patients with ACC.

Survival analysis of oral squamous cell carcinoma in a subgroup of young patients.

Oral squamous cell carcinoma (OSCC) is predominantly a disease of middle-aged men with long-term exposure to tobacco and alcohol. An increasing trend has been reported at a younger age worldwide. Clinical records of 100 patients under the age of 45 years treated specifically for oral cavity SCC in our hospital during a 10-year period were retrospectively analyzed to calculate the survival rates. An obvious male predominance coincided with smoking trend among Chinese young individuals and female patients were more likely to have no traditional risk factors such as smoking or drinking. The 5-year overall survival rate and disease-free survival rate were 61.0% and 75.5%, respectively, consistent with other published series over the decade showing a relatively better survival among the young. No significant differences clearly correlated with outcome when comparing non-smokers non-drinkers to ever-smokers and ever drinkers (P>0.05). Overall survival rate and disease free survival rate was found to be significantly higher in patients with early-stage disease than with advanced stage disease (P=0.001, P=0.009 respectively). The strong influence of clinical stage on prognosis emphasizes the importance of early diagnosis and treatment of oral malignancies for this unique clinical subgroup.

Decreased adiponectin level is associated with aggressive phenotype of tongue squamous cell carcinoma

Circulating adiponectin levels are inversely associated with risk of various obesity‐related cancers. However, the effect of adiponectin on carcinogenesis and progression of tongue squamous cell carcinoma (TSCC) remains unknown. We measured serum adiponectin levels in 59 patients with TSCC and 50 healthy controls. Expression of adiponectin and its receptors in paired tumor and paracancerous specimens were determined by immunohistochemical staining (n = 37) and western blot (n = 30), respectively. Serum adiponectin level was lower in patients than in controls (5.0 ± 2.4 vs 8.4 ± 3.5 μg/mL, P < 0.01), and was inversely associated with histological grade and lymph node metastasis but not tumor size. Local adiponectin levels in tumor tissue gradually decreased as tumor‐node‐metastasis stage increased, while the expression of adiponectin receptors was unchanged. In addition, serum adiponectin levels in the TSCC patients without metabolic and cardiovascular diseases, or without smoking and drinking habits, were still lower than in controls. Furthermore, adiponectin inhibited the migration, but not proliferation, of SCC15 cells in vitro. These results indicate that a decreased adiponectin level is associated with risk of TSCC. Hypoadiponectinemia might be used as a biomarker to predict an aggressive phenotype of TSCC.

Thioredoxin 1 mediates TGF-β-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-β 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-β 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-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.

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/m2) induced apoptosis and inhibited colony formation significantly. However, low-dose UV-C (10 J/m2), 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/m2) 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.

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.

Ca 2+ -CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca2+ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca2+ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca2+ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca2+-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca2+, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca2+ modulation in rat submandibular gland acinar cells. Ca2+-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.

β-adrenoceptor activation increased VAMP-2 and syntaxin-4 in secretory granules are involved in protein secretion of submandibular gland through the PKA/F-actin pathway

Autologous submandibular gland transplantation is an effective treatment for severe dry eye syndrome. However, the protein secretion in transplanted gland is altered by a mechanism that remains to be elucidated. In the present study, we found that β1-adrenoceptor (β1-AR) and β2-AR expression and the phosphorylation of the downstream molecule protein kinase A (PKA) were elevated in transplanted submandibular glands obtained from epiphora patients. Synaptobrevin/vesicle-associated membrane protein 2 (VAMP-2) interacted with syntaxin-4 and actin in human submandibular gland. The contents of syntaxin-4 and actin interacting with VAMP-2 were increased in transplanted gland. Moreover, VAMP-2 and syntaxin-4 expression in the secretory granule fraction, and VAMP-2 expression in the membrane protein fraction were increased in isoproterenol-treated and transplanted glands. Isoproterenol increased F-actin polymerization in the apical and lateral regions of the cytoplasm in both control and transplanted glands. Inhibiting PKA activity and/or F-actin formation abolished the isoproterenol-enhanced expression of VAMP-2 and syntaxin-4 in the secretory granule fraction and the isoproterenol-enhanced expression of VAMP-2 in the membrane protein fraction. Taken together, these results indicate that the activation of β-ARs induces secretory granules and cell membrane fusion via the interaction of VAMP-2 and syntaxin-4 in a PKA- and F-actin-dependent manner in human submandibular gland. Up-regulated β-ARs might participate in altering protein secretion in transplanted submandibular gland by promoting the interaction of VAMP-2 with syntaxin-4.