formazans and Carcinoma--Squamous-Cell

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Topics: Animals; Betaine; Carcinoma, Squamous Cell; Cariostatic Agents; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Culture Media, Serum-Free; Detergents; Diamines; Epithelial Cells; Fibroblasts; Fluorescent Dyes; Fluorides; Formazans; Gingiva; Humans; Indicators and Reagents; Materials Testing; Mice; Mouth Neoplasms; Polyethylene Glycols; Sodium Dodecyl Sulfate; Tetrazolium Salts; Toothpastes

To investigate the effect of metformin on the proliferation and cell apoptosis of oral squamous cell carcinoma (OSCC) (HSC-3, HSC-4) in vitro and in vivo.. HSC-3, HSC-4 cells were treated with metformin at different concentration (2-50 mmol/L) for 24, 48 or 72 hours. In vitro cell proliferation ability was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation assay. Cell cycle progression was assessed by flow cytometry. Cell apoptosis was tested by both TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry. The activation of related cell markers was examined by immunohistochemistry. Xenograft mouse model was used to demonstrate the in vivo anti-tumor effect of metformin. A total of 30 BALB/c mice were randomly divided into control groups (water + phosphate buffer saline, PBS) and treatment groups (pre-oral, oral or intraperitoneal injection). Each group had 6 mice. The tumor size was measured once every three days until endpoint (35 days). After sacrificing the mice, tumor tissue was removed, sectioning and then analyzed by TUNEL or immunohistochemistry (IHC) assays.. Metformin inhibited proliferation and colony formation of HSC-3, HSC-4 in a time- and dose-dependent manner. The cell proliferation was significantly reduced when treated with 5, 10, 20 and 40 mmol/L metformin for 48 and 72 hours (P < 0.05).The colony formation of OSCC cells treated with metformin for 72 hours in vitro had the same result. Treated with 2, 20 and 50 mmol/L metformin for 24 hours increased the ratio of G0/G1 1.2-1.8 fold compared with the control group on HSC-4 cell. The percentage of apoptosis cell rose from 10% (control) to around 30% (treatment) in vitro. Metformin also decreased the size of xenografts by 82.5% (pre-oral), 63.9% (oral), and 62.8% (oral or intraperitoneal injection). The percentage of apoptosis cell rose from lower than 10% (control) to 70% (pre-oral), 50% (oral), and 25% (oral or intraperitoneal injection). The percentage of PCNA positive cell was lower than 60% (control group was normalized to 100%).. Metformin could inhibit the growth of OSCC cell line (HSC-3, HSC-4) by reducing cell proliferation and increasing cell apoptosis in vitro and in vivo. Therefore metformin could be a potential new treatment candidate for human OSCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Formazans; Humans; In Vitro Techniques; Metformin; Mice; Mice, Inbred BALB C; Mouth Neoplasms; Random Allocation; Tetrazolium Salts; Time Factors; Tumor Stem Cell Assay

There is a correlation between the multidrug-resistance (MDR) of cancer cells and their enhanced invasive or metastatic potential. We studied the expression of CD147, a plasma membrane glycoprotein that plays a key role in tumor metastasis by stimulating the production of matrix metalloproteinases (MMPs), in sensitive human oral squamous KB and MDR derivative KB/V cells. Reverse transcription-PCR and flow cytometric analysis revealed that KB/V cells expressed CD147 at significantly higher levels than their parental KB cells. Using stable RNA interference, we succeeded in establishing a CD147 knock-down KB/V cell line (KB/VsiCD147). MTT colorimetric assay showed an increase in the chemosensitivity to vincristine (VCR), all transretinoic acid (ATRA), taxol, and 5-fluorouracil (5-Fu) of KB/VsiCD147 cells. Proteome analysis of KB, KB/V, and KB/VsiCD147 cell lines identified 21 differently expressed proteins. The enhanced expression of representative active proteins, GRP75 and CyPA, was confirmed by Western blotting and RT-PCR. In addition, pretreatment of KB/V cells with a CyPA-binding immunosuppressive drug, cyclosporine A (CsA), enhanced their chemosensitivity to VCR and 5-Fu. We document an abundance of molecules that interact with CD147 in the MDR of human oral squamous carcinoma cells. Additional studies are needed to investigate these novel target proteins of CD147.

Topics: Basigin; Carcinoma, Squamous Cell; Cell Line, Tumor; Coloring Agents; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Formazans; Humans; Proteome; Proteomics; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Transfection

Identification of reliable markers of chemo- and radiosensitivity and the key molecules that enhance the susceptibility of squamous esophageal cancer cells to anticancer treatments would be highly desirable. To test whether regenerating gene (REG) I expression enhances chemo- and radiosensitivity in esophageal squamous cell carcinoma cells, we used MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays to compare the chemo- and radiosensitivities of untransfected TE-5 and TE-9 cells with those of cells stably transfected with REG Ialpha and Ibeta. We then used flow cytometry to determine whether REG I expression alters cell cycle progression. No REG I mRNA or protein were detected in untransfected TE-5 and TE-9 cells. Transfection with REG Ialpha and Ibeta led to strong expression of both REG I mRNA and protein in TE-5 and TE-9 cells, which in turn led to significant increases in both chemo- and radiosensitivity. Cell cycle progression was unaffected by REG I expression. REG I thus appears to enhance the chemo- and radiosensitivity of squamous esophageal cancer cells, which suggests that it may be a useful target for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.

Topics: Antimetabolites, Antineoplastic; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Radiation; Esophageal Neoplasms; Fluorouracil; Formazans; Humans; Lithostathine; Proteins; Radiation Tolerance; RNA, Messenger; Tetrazolium Salts; Transfection

The chemokine receptor 7 (CCR7) mediates survival and invasiveness of metastatic squamous cell carcinoma of the head and neck (SCCHN) to regional lymph nodes. Constitutive prosurvival signaling by the phosphoinositide-3 kinase/Akt pathway has been observed in SCCHN cells independent of epidermal growth factor receptor (EGFR) signaling.. Human SCCHN cell lines were treated with agents that block or activate CCR7-mediated signaling, and Akt activation, cell viability in the presence or absence of EGFR inhibition, and cisplatin-induced apoptosis (in the presence or absence of Akt inhibition) were assessed by immunoblotting, the MTT assay, and the detection of annexin V, respectively. Expression and secretion of chemokines by primary tumors, metastatic nodes, and benign nodes of patients with SCCHN were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The role of paracrine activation of CCR7 on tumor growth was analyzed by comparing the growth of orthotopic tumors derived from B7E3 murine oral carcinoma cells in wild-type BALB/c mice, in paucity of lymphoid T cell (plt, deficient in CCL19 and CCL21 expression) mice, and in plt mice in which the implanted B7E3 cells overexpressed CCR7 (n = 14 mice per group).. In the absence of exogenous ligand treatment, blockade of CCR7 signaling reduced levels of phosphorylated (activated) Akt and decreased SCCHN cell viability by up to 59% (95% confidence interval [CI] = 58.2% to 59.8%), enhancing the effect of EGFR inhibition. CCR7 stimulation protected metastatic SCCHN cells from cisplatin-induced apoptosis in an Akt-dependent manner. Metastatic nodes expressed and secreted higher levels of CCL19 than benign nodes or primary tumors. CCR7-positive murine SCCHN tumors grew more slowly in plt mice than in control BALB/c mice (mean average tumor volume on day 20 = 12.2 and 26.5 mm(3), respectively; difference = 14.3 mm(3), 95% CI = 12.3 to 17.1 mm(3)).. Secretion of CCL19 and CCL21 by SCCHN cells and by paracrine sources combine to promote activation of CCR7 prosurvival signaling associated with tumor progression and disease relapse. CCR7 and its cognate chemokines may be useful biomarkers of SCCHN progression, and blockade of CCR7-mediated signaling may enhance the efficacy of platinum- and EGFR-based therapies.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Apoptosis; Autocrine Communication; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chemokine CCL19; Chemokine CCL21; Cisplatin; Disease Models, Animal; Disease Progression; ErbB Receptors; Female; Formazans; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Mice; Middle Aged; Paracrine Communication; Receptors, CCR7; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tetrazolium Salts; Thiazoles; Up-Regulation

Coumarin and its derivative 7-hydroxycoumarin (7-OHC) have antitumor and antimetastatic properties. The purpose of this study was to investigate the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in two human lung cancer cell lines, A427 and Calu-1. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 1.0 mM ethanolic coumarin or 1.0 mM ethanolic 7-OHC. Viability was determined in each cell line by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cells cultured with coumarin or 7-OHC. Neither drug changed bcl-2 expression in Calu-1 cells compared to solvent controls, and Bax expression was only slightly increased by coumarin. We conclude that 7-OHC is a more potent inhibitor of cell proliferation than coumarin and has more marked effects on oncoprotein expression. Also, the A427 cell line was more sensitive to the drugs than Calu-1.

Topics: Adenocarcinoma; Antineoplastic Agents; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Squamous Cell; Cell Proliferation; Coumarins; Formazans; Humans; In Vitro Techniques; Lung Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Tumor Cells, Cultured; Umbelliferones

Flavopiridol is a synthetic flavone that has shown an antitumor effect against several cancers. Here, we investigated the in vitro effect of flavopiridol alone and the combined effect of low-dose flavopiridol plus radiation on esophageal squamous cell carcinoma cell lines. Esophageal squamous cell carcinoma cell lines (TE8, TE9 and KE4) were exposed to flavopiridol (0.05-400 nmol/L) for 48 h. Growth inhibition was evaluated by MTT assay, cell cycle distribution was determined by flow cytometry, and cyclin D1, Bcl-2 and Rb protein expression was detected by Western blotting. The effect of 0.05 nmol/L flavopiridol as a radio-sensitizer was determined by clonogenic assay. The IC50 was approximately 110-250 nmol/L. Exposure to 0.05 nmol/L flavopiridol for 48 h increased the G2/M population, while 300 nmol/L increased the G1 population. At a concentration of 300 nmol/L, nuclear fragmentation and chromatin condensation were observed in all three cell lines. Exposure to 300 nmol/L flavopiridol decreased the levels of cyclin D1 and Rb protein in all three cell lines and Bcl-2 protein was also decreased in TE8 and KE4 cells. Moreover, exposure to 0.05 nmol/L flavopiridol slightly decreased the levels of cyclin D1, Rb and Bcl-2 protein in KE4 cells. Flavopiridol treatment (0.05 nmol/L) enhanced the radio-sensitivity in all three cell lines. Low-dose flavopiridol augmented the response of esophageal squamous cell carcinoma cell lines to radiation. Administration of a low dose of flavopiridol could be a potent new therapeutic approach for improving the efficacy of radiotherapy against esophageal cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Esophageal Neoplasms; Flavonoids; Flow Cytometry; Formazans; Genes, bcl-2; Growth Inhibitors; Humans; Piperidines; Radiation Tolerance; Radiation-Sensitizing Agents; Retinoblastoma Protein; Tetrazolium Salts; Treatment Outcome

To investigating the relation between the expression of P-glycoprotein and Glutathione transferase-pi and the chemoresistance.. The expressions of these two proteins in patients with oral and maxillofacial squamous carcinoma and normal oral tissues were detected by immunohistochemistry.. The positive expression rate of P-gp and GST-pi in oral and maxillofacial malignant tumor was 57.1% and 53.6% respectively, and no expression in normal oral tissues; the expression of GST-pi was relevant to the resistance to cisplatin, while the expression of P-gp was relevant to the resistance to chemotherapeutic drug in general.. The method of immunohistochemistry combining MTT assay in vitro may become an efficient way to predict the sensitivity to chemotherapeutic drug.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Squamous Cell; Drug Resistance, Neoplasm; Facial Neoplasms; Formazans; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Immunohistochemistry; Isoenzymes; Maxillary Neoplasms; Mouth Neoplasms; Tetrazolium Salts

Human non-small cell lung cancer (N-SCLC), a common malignancy generally unmanageable by conventional cytotoxic chemotherapy, represents a major world health burden. Suramin, a polyanionic drug which appears to interfere with growth-factor/receptor interaction, has recently been shown to be cytostatic for small cell lung cancer cells; it may also be effective for N-SCLC. As insulin-like growth factor I (IGF-I) is a known progression agent for N-SCLC, we have examined the effects of suramin on the 'IGF-I system' in a panel of human N-SCLC cell lines. Colorimetric and thymidine incorporation assays were used to assess cell chemosensitivity whereas a radio-receptor assay was employed to evaluate IGF-I/receptor binding. Suramin reversibly reduced, in a concentration- and time-dependent manner, the growth of each N-SCLC cell line examined either cultured in serum-containing or serum-free medium. Furthermore, suramin caused a concentration-related inhibition of labeled IGF-I peptide specific binding on all cell lines studied. Suramin caused a significant reduction in the Bmax values with only weak variations in the affinity constants (Kd). We hypothesize that suramin interference with IGF-I mitogenic activity is a pathway by which this drug produces its effect in vitro. These data indicate further studies on the mechanism of action and pharmacology of suramin in vivo are warranted.

Topics: Adenocarcinoma; Binding, Competitive; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Division; DNA; Dose-Response Relationship, Drug; Formazans; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Mitogens; Radioligand Assay; Suramin; Tetrazolium Salts; Thymidine; Tumor Cells, Cultured