okadaic-acid and Carcinoma--Squamous-Cell

The study of green tea polyphenols as a cancer preventative is approaching a new era, with significant results accumulating rapidly. This paper briefly reviews four topics related to mechanisms of action of tea polyphenols: (I) identification of the genes commonly affected by EGCG, as demonstrated by Clontech's Atlas cDNA Expression Array; (II) the significance of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1) as a new biomarker for early detection of lung cancer, and inhibition of its expression by EGCG; (III) the synergistic or additive effects of EGCG with the cancer preventive agents, sulindac and tamoxifen, on induction of apoptosis in PC-9 cells and on inhibition of intestinal tumor development in multiple intestinal neoplasia (Min) mice; (IV) the results of a 10 year prospective cohort study demonstrating the effectiveness of daily consumption of green tea in preventing cancer, and a prototype study for developing green tea beverage as cancer preventive.

Topics: Animals; Anticarcinogenic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Catechin; Chemoprevention; Cohort Studies; Drug Synergism; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Intestinal Neoplasms; Japan; Lung Neoplasms; Male; Mice; Mice, Mutant Strains; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Prospective Studies; Ribonucleoproteins; Sulindac; Tea; Tumor Cells, Cultured

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.

Topics: Carcinoma, Squamous Cell; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Ligands; Membrane Glycoproteins; Neoplasm Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Tongue Neoplasms; Transfection; Tumor Cells, Cultured

Studies assessed if the serine/threonine protein phosphatase-2A (PP-2A) maintains cytoskeletal integrity of normal keratinocytes and if this differs in malignant cells. Murine and human keratinocyte cell lines contained more PP-2A activity than did the murine SCC VII/SF squamous cell carcinoma cells or primary cultures of human head and neck squamous cell carcinoma (HNSCC) cells. Since tyrosine phosphorylation of the focal adhesion proteins paxillin and FAK is indicative of more stable focal adhesions, cells were immunostained for phosphotyrosine plus either paxillin or FAK, and then examined by confocal microscopy. In non-malignant keratinocytes, phosphotyrosine staining co-localized with paxillin and FAK. This co-localization occurred at the cell periphery in a pattern resembling focal adhesions. In contrast, the co-localization of phosphotyrosine with either paxillin or FAK along the cell periphery was almost absent in the SCC cells or in keratinocytes that were treated with okadaic acid to inhibit PP-2A activity. Consistent with this was a rounded cellular morphology with less extended processes as compared to control keratinocytes. These studies indicate PP-2A maintains the organization and tyrosine-phosphorylated state of the focal adhesion proteins FAK and paxillin, and that the loss of PP-2A activity results in a loss of cytoskeletal organization, as is seen in SCC.

Topics: Animals; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Keratinocytes; Mice; Okadaic Acid; Paxillin; Phosphoprotein Phosphatases; Phosphoproteins; Protein Phosphatase 2; Protein-Tyrosine Kinases

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level.

Topics: Carcinogens; Carcinoma, Squamous Cell; DNA-Binding Proteins; Down-Regulation; Early Growth Response Protein 1; Electrophoresis, Agar Gel; Gene Expression Regulation; Humans; Immediate-Early Proteins; Immunohistochemistry; Mouth Neoplasms; Neoplasm Proteins; Okadaic Acid; Reverse Transcriptase Polymerase Chain Reaction; Suppression, Genetic; Transcription Factors; Tumor Cells, Cultured

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to Fas receptor leads to activation of the latter and the induction of intracellular signals that result in apoptotic cell death. In the present study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis to examine the expression of mRNAs and proteins of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. The PCR product of Fas receptor mRNA was detected in the cells and a protein with an estimated molecular weight of 35,000 was also expressed in them. Expression of Fas receptor mRNA stimulated by okadaic acid was elevated in dose- and time-dependent manners as judged by semiquantitative RT-PCR analysis, with the maximum expression level at 50 nM and 8 h treatment. Fas ligand mRNA expression was also stimulated by okadaic acid in SCC-25 cells in dose- and time-dependent manners. Okadaic acid also stimulated the expression of Fas ligand protein in the cells. Okadaic acid in serum-free medium induced apoptosis in SCC-25 cells in a time-dependent manner up to 24 h as determined by nuclear condensation and fragmentation of chromatin and DNA ladder formation. The present results indicate that the expression of Fas receptor and Fas ligand is negatively regulated by a protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in SCC-25 cells. Our results also suggest that Fas receptor and Fas ligand system might regulate apoptosis in SCC-25 cells in an autocrine fashion.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Humans; Membrane Glycoproteins; Neoplasm Proteins; Okadaic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tongue Neoplasms; Tumor Cells, Cultured

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluo

Topics: Antigens; Antigens, Nuclear; Apoptosis; Benzimidazoles; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Cell Nucleus; Chromatin; Coloring Agents; Cytosol; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fluorescent Dyes; Humans; Molecular Weight; Mouth Neoplasms; Nuclear Proteins; Nucleolin; Nucleolus Organizer Region; Okadaic Acid; Phosphoproteins; RNA-Binding Proteins; Salivary Glands; Silver; Silver Nitrate; Tumor Cells, Cultured

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in oral epithelial cells we examined the effects of protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CA), on cultured human oral squamous carcinoma (SCC) cell line, SCC-25 cells. After reaching subconfluence these cells were exposed to varying concentrations of the protein phosphatase inhibitors, OA and CA. Both OA and CA induced cell death in SCC-25 cells in a dose-dependent fashion as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin was observed. DNA ladder formation also was detected in SCC-25 cells by treatment with OA and CA. The induced nuclear fragmentation and DNA ladder formation were dose-dependent with maximal effect at concentrations of 20 nM OA and 2 nM CA, respectively. OA also induced DNA ladder formation in other human oral SCC cell lines, SCCKN and SCCTF. To further determine if new gene transcription and protein synthesis are required for OA-induced apoptosis in SCC-25 cells, the cells were treated for 48 h with varying concentrations of cycloheximide in the presence of 20 nM OA. Cycloheximide did not protect the cells against OA-induced cytotoxicity and DNA ladder formation. Based on the known selectivity of OA and CA, the present results indicate that the pathway of the apoptosis in the cultured oral SCC cells is in part regulated by protein phosphatase type 1 and type 2A. Our results also indicate that new protein synthesis is not involved in OA-induced apoptosis in SCC-25 cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Enzyme Inhibitors; Humans; Marine Toxins; Mouth Neoplasms; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Tumor Cells, Cultured

The role of protein phosphatase-2A (PP-2A) in regulating the motility and adhesion of human head and neck squamous cell carcinomas (HNSCC) was investigated. Immunofluorescent staining of these HNSCC cells showed PP-2A can co-localize with microtubules. That the PP-2A influences motility was shown by the increase in HNSCC cell migration through laminin and vitronectin when PP-2A was selectively inhibited with low dose okadaic acid, and by the reduction in invasion through these same matrix components by elevators of PP-2A activity. Motility of HNSCC cells through collagen I or fibronectin was not modulated by PP-2A. The reduction in HNSCC migration through vitronectin or laminin that resulted from treatment with PP-2A elevators was associated with an increase in cellular adhesiveness to these same ECM components. These studies show the association of PP-2A with the cellular cytoskeleton and its role in restricting the invasiveness of tumor cells through select extracellular matrix components.

Topics: Calcitriol; Carcinoma, Squamous Cell; Ceramides; Head and Neck Neoplasms; Humans; Microtubules; Neoplasm Invasiveness; Neoplasm Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Tumor Cells, Cultured

Human head and neck squamous cell carcinoma (HNSCC) cultures were established from cancers of two patients. These cells were used to study if phosphorylation reactions by protein kinase A (PKA) and dephosphorylation reactions by protein phosphatases-1 and -2A (PP-1/2A) regulate tumor motility and adhesion to extracellular matrix components, and if this might be associated with cytoskeletal reorganization. Both cultures were motile and adherent to collagen I, fibronectin, vitronectin and laminin. Motility and adhesiveness was dependent on production of prostaglandin E2 PGE2 and on PKA activation. Blocking PP-1/2A activity with okadaic acid resulted in a PKA-dependent increase in m otility and, in some instances, adhesiveness by the HNSCC cells. The okadaic acid-induced increase in motility and adhesiveness coincided with a reduction in filamentous actin. These data suggest PKA and PP-1/2A have opposing effects in regulating the motility, adherence, and actin polymerization.

Topics: Actins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Movement; Collagen; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Dinoprostone; Down-Regulation; Fibronectins; Humans; Indomethacin; Laminin; Microscopy, Fluorescence; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Tumor Cells, Cultured; Vitronectin

Diarrheic toxins, especially okadaic acid, are detected nearly every year in mussels on French coasts. The monitoring network determines the toxicity of these shellfish by using a mouse test now considered unsatisfactory from an ethical point of view. Two alternative methods have been investigated: the daphnia test, for which there is a standardized method used routinely in ecotoxicology, and a cytotoxicity test on the KB cell line developed for this study. Using the same samples, the results of these two tests were compared with those obtained by chemical analysis (HPLC okadaic acid assay) or the mouse test. Linear regression studies showed that results for the two bioassays were well correlated with those for HPLC or the mouse test.

Topics: Animals; Anthracenes; Biological Assay; Bivalvia; Carcinoma, Squamous Cell; Cell Survival; Chromatography, High Pressure Liquid; Daphnia; Ethers, Cyclic; Fluorescent Dyes; Foodborne Diseases; France; Humans; Linear Models; Male; Mice; Okadaic Acid; Reference Standards; Tumor Cells, Cultured