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.

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.

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 transcription factor nuclear factor-kB (NF-kB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-KB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Numerous evidences showed that over expression of TGase 2 might be linked with constitutive activation of NF-KB. To understand the pathways responsible for constitutive activation of NF-kB is important for rational design of NF-kB inhibitors for cancer therapy. Human salivary gland adenocarcinoma has been most aggressive solid tumors. Current therapy does not significantly improve survival rates. Thus, to investigate new therapeutic modalities against this adenocarcinoma is necessary. The purpose of this study was to study a constitutive activation of NF-kB with the expression of TG2 in SGT cell line origianted from human salivary gland adenocarcinoma by TGase 2 activity, RT-PCR and immunoslot blotting method. SGT cell line showed the highest TGase 2 activity and NF-kB activation than any other cell line. All the cell lines showed increased NF-kB mRNA activation after A231027 treatment than that of control. In immunoslot blotting, SGT cell line showed NF-kB activation correlated with TGase 2 expression after A231027 and BSA. It suggested that there might be a direct correlation between TGase 2 expression and NF-kB activation in SGT cell line.

Adenocarcinoma NOS of salivary glands is characterized by a high rate of local recurrences and metastasis. Long-term survival rate of Adenocarcinoma NOS lis not promising. Thus, different chemotherapeutical approaches had been proposed for this neoplasm, including apoptosis induction by drugs. The current treatment of choice of adenocarcinoma NOS is controversible, and an effective treatment for them is not yet available. Chemotherpeutic agents that can be inhibit or reverse the tumor growth by targeting apoptotic pathways will be new candidates for cancer prevention and therapy. The purpose of this study were to study the effect of Brefeldin A(BFA) as apoptotic inducing agent in SGT cell line from human submandibular adenocarcinoma NOS and apply these results to make a plan of treatment and prognosis of salivary gland tumors involving adenocarcinoma NOS. SGT cells were treated with a 300μM BFA solution in serum-free medium during 18 hours. SGT cells were grown in DMEM with 10% fetal bovine serum served as controls. The growth curve and MTT assay for succinyl dehydrogenase activity were performed. For apoptotic analysis, fragmentation of genomic DNA was confirmed with gel electrophoresis. Transmission electron microscopy was assessed for the effect of BFA on SGT cells phenotype. Apoptotic cell recognition and counting were carried out with Annexin-V, caspase 3 and APo2.7 antibody through flow cytometry. Growth of SGT cell line was abrutply decreased after 1 day of BFA treatment. MTT assay for succinyl dehydrogenase activity of the cells showed about 55% after 300μM BFA treatment. Destruction of cellular organells, numerous vacuolation in the cytoplasm & nucleus, chromatin margination, & fragments of nucleus were seen with TEM after 300μM BFA treatment. DNA fragmentation of SGT cell line was induced by 300μM BFA treatment and confirmed by gel electrophoresis from genomic DNA extraction. Late apoptosis of the cells through flow cytometric analysis of Annexin-V staining as induced by 300μM BFA treatment. Early apoptosis of the cells through flow cytometric analysis of caspase 3 and Apo 2.7 staining was induced by 300μM BFA treatment. It suggested that early and late apoptosis of SGT cell line would be induced by Brefeldin A treatment in vitro study. This work evaluated the efficacy of BFA, a potent apoptosis inducer, on SGT cultured cell line. And BFA as chemotherapeutic agent will be used as the treatment choice for adenocarcinoam NOS, and be need to apply BFA to in vivo study & clinical approach in future.

Established SGT cell line from human submandibular gland adenocarcinoma was used to study the TGase expression on a cellular level in vitro. Transglutaminase 2(TGase 2) is assoacitated with apoptosis, GTP binding protein, and cell marix interaction. The role of TGase 2 in salivary gland tumors is not clear yet. The pupose of this study were to examine the TGase expression of SGT cell line compared to other tumor cell lines, and to apply these results to the pathogenesis of salivary gland tumor. TGase enzyme assay of SGT, SCC-15, HN 4 and HeLa tumor cell line was 3 times repeated, and calculated. Immunoslot blot for semiquantitative protein analysis was done. The obtained results were as follows. 1. SGT cell line showed the highest TGase 2 enzyme activity(about 6-16 folds) irrespective of pre or postconfluency. 2. HN 4 cell line showed the highest TGase 1 enzyme activity(about 2-3 folds) irrespective of pre or postconfluency. 3. Under postconfluency TGase 1 induction was not induced, but slightly increased in all tumor cell lines. 4. TGase enzyme activity in all tumor cell lines was accompanied with TGase protein formation. From the aboving results, the higher TGase 2 expression of SGT cell line suggested that they would come from submandibular ductal cells and have a important role in the pathogensis of salivary gland tumors.