agar and Mouth-Neoplasms

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.

Topics: Agar; Animals; Biomarkers, Tumor; Blotting, Western; Carcinoma; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Female; Gene Silencing; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; Nicotinamide N-Methyltransferase; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Thiazoles

A high-dose-rate (HDR) 192-Ir brachytherapy using a customized intraoral mold is effective for superficial oral cavity cancer, and the surrounding normal tissue is kept away from the radioactive source with gauze pads and/or mouth piece for reducing the dose on the normal tissues. In the Tokushima university hospital, the mold has a lead shield which utilizes the space prepared with sufficient border-molding by a specific dental technique using modeling compound. In HDR 192-Ir brachytherapy using a lead shielded customized intraoral mold, there are no reports measuring the absorbed dose. The purpose of the present study is to measure the absorbed dose and discuss the optimum thickness of lead in HDR 192-Ir brachytherapy using a customized intraoral mold with lead shield using a 1 cm thickness mimic mold. The thickness of lead in the mold could be changed by varying the arrangement of 0.1 cm thickness sheet of the acrylic resin plate and lead. The measured doses at the lateral surface of the mold with thermo-luminescence dosimeter were reduced to 1.12, 0.79, 0.57, 0.41, 0.31, 0.24 and 0.19 Gy and the ratios to the prescription dose were reduced to 56, 40, 29, 21, 16, 12 and 10 percent as lead thickness increased from 0 to 0.6 cm in 0.1 cm increments, respectively. A 0.3 cm thickness lead was considered to be required for a 1 cm thickness mold, and it was necessary to thicken the lead as much as possible with the constraint of limited space in the oral cavity, especially at the fornix vestibule.

Topics: Absorption; Acrylic Resins; Agar; Brachytherapy; Dental Impression Materials; Dose Fractionation, Radiation; Dose-Response Relationship, Radiation; Equipment Design; Humans; Iridium Radioisotopes; Lead; Materials Testing; Mouth Neoplasms; Mouth Protectors; Organs at Risk; Radiation Injuries; Radiation Protection; Radiometry; Radiotherapy Dosage

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Topics: Agar; Animals; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Epithelial Cells; Epithelium; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Microtubule-Associated Proteins; Mouth Neoplasms; Neoplasm Invasiveness; Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; S-Phase Kinase-Associated Proteins; Transfection; Tumor Suppressor Proteins

Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3.1.4.5). In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells. (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein. (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000. (c) It has an amino-acid composition closely similar to that of actin. (d) The peptide maps of the two proteins are nearly identical. (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I. (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts. (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody.The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions.

Topics: Actins; Agar; Amino Acid Sequence; Animals; Carcinoma; Cattle; Cell Line; Chickens; Chromatography, Affinity; Deoxyribonucleases; Electrophoresis, Polyacrylamide Gel; Epitopes; Fibroblasts; Fluorescent Antibody Technique; Humans; Hydrolysis; Immunoassay; Molecular Weight; Mouth Neoplasms; Muscles; Peptide Fragments; Polysaccharides; Spleen; Thymus Gland; Trypsin

Areas of cytopathic effect can be circumscribed in cell monolayers by adding antiserum to the liquid nutrient medium after adsorption of virus. This procedure represents a simple and reliable tool for the titration of virus infectivity and provides an experimental model for studying some aspects of virus infection.

Topics: Adsorption; Agar; Animals; Carcinoma; Cell Line; Chick Embryo; Culture Techniques; Cytopathogenic Effect, Viral; Evaluation Studies as Topic; Haplorhini; Humans; Immune Sera; Kidney; Methods; Mouth Neoplasms; Orthomyxoviridae; Poliovirus; Rabbits; Viral Plaque Assay

Topics: Agar; Animals; Antineoplastic Agents; Carcinoma; Cell Line; Cells, Cultured; Disease Models, Animal; Humans; Leukemia L1210; Melanoma; Mice; Mice, Inbred Strains; Mouth Neoplasms; Neoplasm Transplantation; Neoplasms, Experimental; Oncogenic Viruses; Plant Extracts