Salivary gland dysfunction is a common complication of diabetes. Decreased saliva production and changes in saliva composition may cause oral diseases. Reactive oxygen species (ROS) generation in the salivary glands results in the loss of acinar cells and decreased saliva secretion. Glucagon-like peptide 1 (GLP-1) is the incretin hormone that regulates blood glucose level and can suppress ROS production and inflammation through its antioxidant effects. Dipeptidyl peptidase-4 (DPP-4) is an enzyme that breaks down GLP-1. In this study, we evaluated the pathological role of DPP-4 and GLP-1 on salivary gland dysfunction in type 2 diabetic db/db mice. We observed reduced salivary secretion and histopathological alteration of salivary glands in the db/db mice. The increased DPP-4 and decreased GLP-1 levels in the salivary glands were also detected in the db/db mice. Furthermore, the db/db mice had increased apoptosis and oxidative injury in salivary glands. There was an accumulation of advanced glycation end products and mucus in the salivary glands of the db/db mice. In conclusion, these results showed the possible involvement of DPP-4 and GLP-1, leading to increased ROS-induced apoptosis in diabetes-related salivary gland dysfunction. DPP-4 and GLP-1 may be a pharmacological target for patients with diabetes-related salivary gland dysfunction.

Declined salivary gland function is commonly observed in patients with diabetes. Advanced glycation end products (AGEs) are believed to contribute to the pathogenesis of diabetes-induces hypofunction of the salivary glands. Polydatin (resveratrol-3-O-β-mono-D-glucoside) is a polyphenol that can be easily accessed from peanut, grape, and red wines. Although polydatin is known to have anti-glycation, anti-oxidation, and anti-inflammation effects, its effect in the salivary gland is not known. In the present study, we evaluated the AGEs burden in the salivary gland in streptozotocin (STZ)-induced diabetic rats and the protective effect of polydatin on diabetes-related salivary hypofunction. Polydatin (50 and 100 mg/kg/day) was orally administered in the STZ-induced diabetic rats for 4 weeks. The results showed that the salivary flow rate of the STZ-induced diabetic rats was reduced compared with that in the normoglycemic control rats. The circulating AGEs in serum and secreted AGEs in saliva increased in the STZ-induced diabetic rats. The reactive oxygen specises (ROS) were highly generated in the salivary gland in these diabetic rats. The salivary gland from the diabetic rats showed increased acinar cell apoptosis compared to normoglycemic control mice. However, polydatin suppressed all of these diabetes-related salivary changes. Overall, polydatin could provide a beneficial option for diabetes-related salivary hypofunction.

Despite evidence that bacteria-sensing Toll-like receptors (TLRs) are activated in salivary gland tissues of Sjogren syndrome (SS) patients, the role of oral bacteria in SS etiopathogenesis is unclear. We previously reported that two SS-associated oral bacteria, Prevotella melaninogenica (Pm) and Rothia mucilagenosa (Rm), oppositely regulate the expression of major histocompatibility complex class I (MHC I) in human salivary gland (HSG) cells. Here, we elucidated the mechanisms underlying the differential regulation of MHC I expression by these bacteria. The ability of Pm and Rm to activate TLR2, TLR4, and TLR9 was examined using TLR reporter cells. HSG cells were stimulated by the TLR ligands, Pm, and Rm. The levels of MHC I expression, bacterial invasion, and viability of HSG cells were examined by flow cytometry. The hypoxic status of HSG cells was examined using Hypoxia Green. HSG cells upregulated MHC I expression in response to TLR2, TLR4, and TLR9 activation. Both Pm and Rm activated TLR2 and TLR9 but not TLR4. Rm-induced downregulation of MHC I strongly correlated with bacterial invasion and cell death. Rm-induced cell death was not rescued by inhibitors of the diverse cell death pathways but was associated with hypoxia. In conclusion, Pm upregulated MHC I likely through TLR2 and TLR9 activation, while Rm-induced hypoxia-associated cell death and the downregulation of MHC I, despite its ability to activate TLR2 and TLR9. These findings may provide new insight into how oral dysbiosis can contribute to salivary gland tissue damage in SS.

Salivary duct carcinoma is a highly malignant tumor that arises from salivary gland. Pathologically, Salivary duct carcinoma is similar to high-grade breast ductal carcinoma. The tumor is commonly associated with pain and facial paralysis and is known to have high rates of recurrence and metastasis. Surgical resection and postoperative radiation is mainstream of the treatment. In this study, we report a case of this tumor which was seen predominantly benign-looking in fine needle aspiration biopsy. The features of this tumor are presented with a review of literature.

Non-keratinizing squamous cell carcinoma (NKSCC) is a rare malignancy of the nose and paranasal sinuses which is characterized by a unique anastomosing ribbon-like growth pattern with absent of limited maturation and keratinization. NKSCC accounts for 10-27% of sinonasal squamous cell carcinomas and some of the NKSCCs are reported to be associated with high risk-HPV infection. Advanced lesion can involve the oral cavity with oral symptoms of palatal bulging, surface ulceration mimicking salivary gland tumors. Herein, we report a case of NKSCC of a 46-year old male, which clinically presented as a bulging mass on the mid palate and mimicked a palatal salivary gland tumor. We reviewed the clinical and histopathological considerations required for differential diagnosis of sinonasal carcinoma involving the oral cavity.

Salivary gland adenocarcinoma(AdCa NOS) is one of the major causes of mortality among malignant salivary gland tumors. New therapeutic measure are needed to improve the outcome for patients with AdCa NOS because current therapy does not significantly improve survival rates. Transglutaminase 2(TGase 2) was implicated in forming cross-linked protein polymer, apoptosis and matrix interaction. And also TGase 2 expression is up-regulated in proliferation, migration, invasion, and metastasis of cancer cells. shRNA which has emerged as an effective method to target specific genes for silencing has provided new opportunities for cancer therapy. But there has been rarely reported using shRNA-TGase 2 transfection in AdCa NOS. The purpose of this study were to examine the specific inhibition of TGase 2 mRNA and protein expression by siRNA transfection of TGase 2 through RT-PCR and immunoslot blotting, and to study proliferation, migration and invasion assay of SGT cell line from AdCa NOS. Cell cycle analysis showed that the downregulation of shRNA-TGase 2 caused the accumulation of cells in the sub-G0/G1 phase. In migration assay, suppressing shRNA-TGase 2 inhibited the capacity of the cells to migrate compared to parental cells. In invasion assay, cells transfected with shRNA-TGase 2 decreased in invasion when compared to SGT and vector transfected cells. shRNA-TGase 2 expressing plasmids efficiently downregulated TGase 2 mRNA and TGase 2 protein expression. It suggested that the shRNA-TGase 2 targeting system against TGase 2 could have a therapeutic potentiality for malignant salivary gland tumors, especially in inhibiting and/or preventing cancer cell proliferation, migration and invasion.

Despite existing chemotherapy and surgical resection strategies, salivary gland adenocarcinoma(AdCa NOS) is one of the major causes of mortality among malignant salivary gland tumors. New therapeutic measure are needed to improve the outcome for patients with AdCa. Overexpression of urokinase-type plasminogen activator receptor/urokinase-type plasminogen activator(uPAR-uPA) has been implicated in progression and metastasis of oral cancer. RNA interference(RNAi) which has emerged as an effective method to target specific genes for silencing has provided new opportunities for cancer therapy. But there has been rarely reported using RNAi-uPAR/uPA transfection in salivary gland AdCa. The purpose of this study were to examine the specific inhibition of uPAR/uPA mRNA and protein expression by RNAi transfection of uPAR/uPA through RT-PCR and Immunoslot blot, and to study tumor cell proliferation activity, adhesion, invasion and migration of SGT cell line in vitro compared to the controls. In adhesion assay, cells transfected with RNAi-uPAR/uPA inhibited markedly adhesion to vitronectin compared to parental cells. Angiogenic assays revealed a significant decrease in the angiogenic potential of SGT cells downregulated by both uPAR and uPA. In migration assay, suppressing uPAR and uPA inhibited the capacity of the cells to migrate compared to parental cells. In invasion assay, cells transfected with RNAi-uPAR/uPA showed the maximum decrease in invasion when compared to all other treatment conditions. RNAi expressing plasmids efficiently downregulated mRNA and protein expression of uPAR and uPA. Cell cycle analysis showed that the simultaneous downregulation of uPAR and uPA caused the accumulation of cells in the sub-G0/G1 phase in SGT cells. Immunoslot blot analysis revealed that downregulation of uPAR and uPA caused the prominent activation of caspase 8. It suggested that the RNAi targeting of the uPAR/uPA system could have a therapeutic potentiality for malignant salivary gland tumors.

Mucinous adenocarcinoma (MAC) refers to a malignant neoplasm that belongs to a subtype of adenocarcinoma which produces mucin. The World Health Organization has classified MAC as a tumor of salivary gland origin and it is extremely rare. Therefore, its characteristics are not well-known. This study reports a case of MAC of minor salivary gland origin, which occurred in the oral commissure of an 82-year-old female patient, and a systematic review on existing cases of MAC of salivary origin. Based on the study’s findings, it is safe to assume that the occurrence of lymph node metastasis is an important factor in determining the prognosis of patients with MAC.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this paper, we investigated the molecular structure, tissue distribution patterns, and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was detected by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of sequestration and hydrolysis of acetylcholine (ACh)/choline (Ch) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. Kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions as a sequestering enzyme specific to ACh (not to Ch) by having a very strong affinity to ACh but an extremely long turnover time.

Type1 diabetes mellitus (DM) is generally known to be caused by destruction of insulin-producing pancreatic β cells or an immune-related problem. Polydipsia is a representative symptom of DM, and it has been reported that this condition is closely related to xerostomia and is considered that hyposalivation from the salivary gland results in this phenomenon. Although various studies have reported that induction of diabetes reduces endogenous stem cells in other organs (heart, brain etc.), diabetes-related changes in endogenous stem cells in the salivary gland have not yet been well established. Therefore, in this study, to verify the change in salivary gland stem cells after diabetes, salivary gland tissues in the control and diabetes-induced groups were processed by histochemistry (Masson’s trichrome staining) for morphological analysis, TUNEL assay for cell death, and immunohistochemistry (Ki-67 and c-Kit) for cell proliferation and maturation. Diabetes induced by STZ leads to vacuolization, apoptosis, and reduction in proliferating cells/salivary gland stem cells in salivary glands of rats. This result suggests that diabetes may be associated with reduction in salivary gland function such as degeneration and inhibition of regeneration in the salivary gland.

Although salivary gland adenocarcinoma NOS accounts for third prevalence rate of all salivary gland tumors, it is one of the most aggressive solid tumors. Current therapy does not significantly improve survival rates. Thus, investigating new therapeutic modalities against salivary gland adenocarcinoma NOS is necessary. It is well known that docetaxel(TXT) as an antimicrotubulin agent induces mitotic block in proliferating cells. TXT has significant antitumor effects, and it is currently being tested in patients with malignant tumors, but TXT has not yet been tested in malignant salivary gland tumors. The purpose of this study were to examine the effects of TXT and to evaluate the biological mechanisms of TXT on salivary gland adenocarcinoma NOS. Proliferation, cell cycle regulation, connexin43 expression, apoptosis, and Fas receptor(FasR) expression were measured in cultured SGT cell line. Proliferation was little changed after 10ng/ml TXT exposure, but cellular proliferation was inhibited according to increasing concentration of TXT and time. Especially it was prominently inhibited after 96 hrs at 20ng/ml. G2-M arrest stage showed about up to 5 fold increase after exposure of TXT by flow cytometry. Apoptosis index showed about up to 8 fold increase after exposure of TXT by flow cytometry. Fas expression showed about up to 3 fold increase after exposure of TXT by flow cytometry. Apoptosis showed about up to 3 fold increase at 20ng/ml after exposure of TXT and anti-Fas agonist by flow cytometry. In Immunoslot blotting, Cx 43 protein expression was increased after TXT treatment. It suggested that TXT might induce apoptosis in SGT cells and could be used as a potent and specific chemotherapeutic tool for the treatment of salivary gland adenocarcinoma NOS in future.

RNA interference (RNAi) has been widely adopted as a primary reverse genetic tool to determine the physiological function of genes of interest. Nevertheless, the lack of optimized RNA delivery method has been a major obstacle for non-model organisms, such as Cimex lectularis. In this study, we have established a RNAi protocol for the silencing of C. lectularis salivary gland-specific cholinesterase (SChE) gene based on micro-injection of double stranded RNA (dsRNA). An aliquot (20 nl) of dsRNA solution (4.5 ng/nl) was injected to body cavity through the arthrodial membrane between metathoracic coxa and sternum of adult females. Observed mortality was less than 5% and at 6-day post injection, while the gene silencing efficiency reached 97~99% at 2-6 day post injection. This result demonstrates the efficacy of injection RNAi via the arthrodial membrane in C. lectularius.

In order to establish symbiotic host-bacterial relationships, symbionts in insects evolved a mechanism to overcome host immune responses. Here we provide the resistance of symbiotic bacteria on the insect immune system. As a result, through the transposon mutagenesis, we found a salivary gland (SG) susceptible mutant. The disrupted gene was identified as nlpB involved in lipoprotein synthesis. The nlpB, bla double deletion mutant was sensitive to SG like nlpB-Tn5 inserted mutant. This mutant increases outer membrane permeability. It provides an explanation for SG susceptibility, because the antimicrobial peptide in SG would be able to translocate across the outer membrane more easily than in the wild type. These results indicate that nlpB and bla are likely to be important factors in terms of determining resistance against SG of Riptortus that is connected with the successful colonization of the Riptortus midgut.

Curcumin (diferuloylmethane), a constituent of turmeric powder derived from the rhizome of Curcuma longa, has been shown to inhibit the growth of various types of cancer cells by regulating cell proliferation and apoptosis. However, a need exists to design more effective analogs because of curcumin's poor intestinal absorption. EF-24 (diphenyl difluoroketone), the monoketone analog of curcumin, has shown good efficacy in anticancer screens. However, the effects of curcumin and EF-24 on salivary gland epidermoid carcinoma cells are not clearly established. The main goal of this study was to investigate the effects of curcumin and EF-24 on cell growth and induction of apoptosis in human salivary gland epidermoid carcinoma cells. Our studies showed that curcumin and EF-24 inhibited the growth of HTB-41 cells in a dose- and time-dependent manner, and the potency of EF-24 was > 34-fold that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in HTB-41 cells, whereas the control HTB-41 cell nuclei retained their normal regular and oval shape. Curcumin and EF-24 promoted proteolytic cleavages of procaspase-3/-7/-9, resulting in an increase in the amount of cleaved caspase-3/-7/-9 in the HTB-41 cells. Caspase-3 and -7 activities were detected in viable HTB-41 cells treated with curcumin or EF-24. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptosis in HTB-41 human salivary gland epidermoid carcinoma cells, and that they may have potential properties as an anti-cancer drug therapy.

The carcinogenesis mechanism of human salivary gland adenocarcinoma NOS is poorly understood. MicroRNA155(miRNA155) has been involved in the carcinogenesis of many malignant tumors. The purpose of this study was to examine the role of miRNA155 in tumor growth and invasion of adenocarcinoma NOS. Using SGT cells as a model for adenocarcinoma NOS, cell proliferation was examined by MTT assay after knocking down miRNA155 expression, and cell cycle analysis was performed. Invasive capacity by a Transwell culture assay, and miRNA155 expression in SGT cell line by RT-PCR were examined. In MTT assay, proliferation of SGT-miRNA155 cells was decreased prominently after 96 hrs. Proliferation of SGT cells was markedly inhibited by knocking down miRNA155, resulting from a blockade of cell cycle in the G1 phase, but apoptosis was increased about 4 folds. In adhesion assay, SGT-miRNA155 cells decreased about 60% compared to SGT cells. In invasion assay, inhibition of miRNA155 significantly suppressed the invasive capacity of about 34% SGT cells. mRNA expression of SGT-miRNA155 cells prominently were decreased compared to SGT cells by RT-PCR. It suggested that miRNA155 could play an role in cell cycle progression and invasion in SGT cells, including antitumor effect. These results have provided insights into the carcinogenic mechanisms and new intervention method of salivary gland adenocarcinoma NOS.

The Riptortus pedestris-Burkholderia symbiotic system is a promising model for understanding molecular mechanism of symbiosis. In previous studies, the Burkholderia symbiont has been shown to play important biological roles in the growth and fitness of host R. pedestris. The Burkholderia symbiont, one of bacteria found in the soil, is the only bacterium that can colonize the symbiotic midgut region of R. pedestris. However, the molecular mechanism of host selectivity for the Burkholderia symbiont remains unknown. To determine where the selection occurs, we firstly compared initial infectivity of different mid-gut regions after oral infection of Escherichia coli and Burkholderia. Interestingly, E. coli were not detected in any regions of mid-gut, while Burkholderia could reach to the posterior region of mid-gut. Therefore, we hypothesized that host selectivity for the Burkholderia symbiont is occurred in the salivary gland. To address this hypothesis, we treated E. coli and Burkholderia with lysate of salivary gland and examined their survival by estimation of colony forming unit (CFU) on the plate. We found that E. coli, but not Burkholderia, was susceptible to the lysate of salivary gland. To determine molecular basis of the selective mechanism in the salivary gland, we analyzed antimicrobial proteins (AMPs) from lysate of salivary gland. we identified three AMPs, namely rip-trialysin1, rip-trialysin2 and lysozyme and further purified rip-trialysin1 and rip-trialysin2. When E. coli and Burkholderia were treated with rip-trialysin1 and rip-trialysin2, rip-trialysin1 exhibited little antimicrobial activity, but rip-trialysin2 exhibited antimicrobial activity. Furthermore, we found that E. coli was susceptible, but Burkholderia is resistant to commerciallypurchased egg white lysozyme. Our results suggest that R. pedestris salivary gland provides a chance of selection for the Burkholderia symbiont and lysozyme in salivary gland seems to play an important role for the selection of gut symbiont.

Toxic heavy metals like mercury and cadmium are known to involve in altering the salivary flow so that can be appeared sialorrhea or ptyalism, the condition of increased salivary flow, or xerostomia (“dry mouth”), the condition related to inhibited or decreased salivary flow. Although many people were exposed to these heavy metal in work environment, dental clinics, the mechanism is rarely discussed in the clinical literature. The present study is to carried out analysis of AQP5 expression that play a key role in saliva fluid secretion and cell membrane water permeability on mercury- or cadmium-exposed mice submandibular gland. To investigate AQP 5 expression, immunohistochemical study and western blot assay were carried out on mercury- or cadmium-exposed mice. Additionally, RT-PCR, real- time PCR with specific primers were carried out. Cadmium or mercury exposure led ductal extension, ductal cell increase, and blood vessel increase in mouse submandibular gland. The mRNA and protein expression of AQP5 were increased in time dependent manners on cadmium or mercury exposed mouse. Also, AQP5 were translocated from basolateral membrane to apical membrane of acini cell. In conclusion, toxic heavy metal such as mercury and cadmium appear to alter the AQP5 expression and distribute to apical membrane of ductal cell and lead to alter salivary secretion.

To determine differential gene expression profiles in the salivary gland of a water stick-insect, Ranatra chinensis Mayrt, a subtractive cDNA library was constructed by suppression subtractive hybridization. The salivary gland was determined among three salivary gland-like tissues by investigating transcription levels of five trypsin genes isolated from R. chinensis. The major transcripts encoding trypsins (64.4% of the total ESTs) were eliminated from the library and then remaining salivary gland-specific genes were searched. A total of 643 expressed sequence tags (ESTs) were clustered and assembled into 148 contigs (49 multiple sequences and 99 singletons), among which 35 contigs had matched BLASTx hits (E ≤ 1.00E-4). Salivary apyrase occupied 5.6% (36 ESTs) of the library. Apyrase is known to be released by female mosquitoes or blood-sucking assassin bugs to prevent blood clots during blood sucking. Therefore, apyrase in the salivary of R. chinensis might allow R. chinensis to facilitate feeding. Several contigs encoding acid phosphatase, hyaluronidase, prophenoloxidase, and dipeptidylpeptidase IV, commonly found in venoms of Hymenoptera, were also identified from the salivary gland-specific library. Discovery of salivary glandspecific genes should promote further studies on biologically active components in the saliva of R. chinensis.

To determine differential gene expression profiles in the salivary gland of a predatory flower bug species, Orius laevigatus Fieber, a subtractive cDNA library was constructed by suppression subtractive hybridization. The major transcripts encoding trypsins (28.6% of the total ESTs) were eliminated from the library and then remaining salivary gland-specific genes were searched. A total of 513 expressed sequence tags (ESTs) were clustered and assembled into 129 contigs (64 multiple sequences and 65 singletons). About 58% were matched with insect genes. In total, 38 genes (179 ESTs) were found from the library by BLASTx search. A hemolysin-like protein occupied ca. 8% (41 ESTs) of the library. Hemolysin is known to destruct cells including blood cells by forming pores on the cell membrane. A hemolysin-like salivary protein of O. laevigatus might be hemolytic against the prey cells, thereby allowing O. laevigatus to facilitate feeding. Several contigs encoding lipase was also identified from the salivary gland-specific library. Discovery of salivary gland-specific genes should promote further studies on biologically active components in the saliva of O. laevigatus.