okadaic-acid and Stomach-Neoplasms

Okadaic acid is functionally a potent tumor promoter working through inhibition of protein phosphatases 1 and 2A (PP1 and PP2A), resulting in sustained phosphorylation of proteins in cells. The mechanism of tumor promotion with okadaic acid is thus completely different from that of the classic tumor promoter phorbol ester. Other potent inhibitors of PP1 and PP2A - such as dinophysistoxin-1, calyculins A-H, microcystin-LR and its derivatives, and nodularin - were isolated from marine organisms, and their structural features including the crystal structure of the PP1-inhibitor complex, tumor promoting activities, and biochemical and biological effects, are here reviewed. The compounds induced tumor promoting activity in three different organs, including mouse skin, rat glandular stomach and rat liver, initiated with three different carcinogens. The results indicate that inhibition of PP1 and PP2A is a general mechanism of tumor promotion applicable to various organs. This study supports the concept of endogenous tumor promoters in human cancer development.

Topics: Animals; Carcinogens; Disease Progression; Enzyme Inhibitors; Humans; Marine Toxins; Mice; Models, Molecular; Neoplasms; Neoplasms, Experimental; Okadaic Acid; Oxazoles; Protein Phosphatase 1; Protein Phosphatase 2; Rats; Skin Neoplasms; Stomach Neoplasms; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Topics: Animals; Antifungal Agents; Carcinogens; Ethers, Cyclic; Liver Neoplasms; Marine Toxins; Mice; Molecular Structure; Okadaic Acid; Oxazoles; Peptides, Cyclic; Pyrans; Rats; Skin Neoplasms; Spiro Compounds; Stomach Neoplasms; Structure-Activity Relationship

Several previous studies have demonstrated that cyclin‑dependent kinase (CDK)‑5 expression serves an important role in promoting the development of malignant tumours. We have previously reported that CDK5 suppresses gastric tumourigenesis. The aim of the present study was to investigate the mechanistic basis of CDK5. The results of immunoprecipitation and western blot analysis demonstrated that CDK5 could interact with serine/threonine‑protein phosphatase 2A (PP2A). The use of an inhibitor of PP2A in CDK5‑overexpressing gastric cancer (GC) cell lines antagonized CDK5‑mediated suppression in GC cells. Further analysis revealed that PP2A expression was downregulated in GC and patients with low levels of PP2A had worse survival outcomes than those with high levels of PP2A (P=0.035). Therefore, the present study provided a novel mechanism for CDK5‑mediated tumour suppression, suggesting that CDK5 may be an attractive target for future therapeutic strategies for treating GC. In addition, low levels of PP2A may indicate a tendency for poor prognosis in patients with GC.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 5; Down-Regulation; Female; Follow-Up Studies; Humans; Male; Middle Aged; Neoplasm Invasiveness; Okadaic Acid; Prognosis; Protein Binding; Protein Phosphatase 2; Stomach; Stomach Neoplasms

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Brain; Brain Chemistry; Cell Line; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Epitopes; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Kidney; Liver; Liver Diseases; Mice; Mutation; Okadaic Acid; Phosphorylation; Rabbits; Serine; Stomach Neoplasms; Transfection; Vaccination; Vanadates

It is now well accepted that (-)-epigallocatechin gallate (EGCG) inhibits carcinogenesis in the digestive tract in rodents. To understand the mechanisms of anticarcinogenesis, we first studied growth inhibition by EGCG in human stomach cancer cell lines established at Seoul National University (SNU cell lines). Inhibition by EGCG of [3H]thymidine incorporation into eight SNU cell lines was examined, in relation to transforming growth factor-beta (TGF-beta) responsiveness. Various tea polyphenols derived from green tea and black tea induced growth inhibition and apoptosis of human stomach cancer cell line KATO III, and inhibition of tumor necrosis factor-alpha (TNF-alpha) release from the cells, in the order of (-)-epicatechin gallate (ECG), EGCG, (-)-epigallocatechin (EGC), teaflavins (TF) and (-)-epicatechin (EC). In addition, we demonstrated that EGCG inhibited TNF-alpha gene expression in KATO III cells, as well as okadaic acid-induced AP-1 and NF-kappa B activation. The inhibitory potencies of EGCG for AP-1 and NF-kappa B binding to DNA were different between KATO III cells and mouse fibroblast cell line BALB/3T3. Thus, EGCG and other tea polyphenols may interact with various transcription factors, in addition to AP-1 and NF-kappa B, in nuclei of various cells, resulting in inhibition of TNF-alpha gene expression and TNF-alpha release.

Topics: 3T3 Cells; Animals; Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Catechin; Cell Division; Flavonoids; Gene Expression; Humans; Mice; Mice, Inbred BALB C; NF-kappa B; Okadaic Acid; Phenols; Phytotherapy; Polymers; Stomach Neoplasms; Tea; Transcription Factor AP-1; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Tautomycin isolated from Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Tautomycin induced hyperphosphorylation of cytokeratin peptides in human keratinocytes (PHK 16-I cells) 30 times less strongly than did okadaic acid. Repeated applications of tautomycin (30 micrograms, 40 nmol/application) did not induce tumor promotion in a two-stage carcinogenesis experiment on mouse skin initiated with 7,12-dimethylbenz[a]anthracene, whereas okadaic acid (1 microgram, 1.2 nmol/application) as a control induced tumor promotion strongly. As for mucosa of rat glandular stomach, tautomycin induced ornithine decarboxylase 4 h after intubation into the stomach. The tumor-promoting activity of tautomycin was next studied in the glandular stomach initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Administration of tautomycin in the diet (1 mg rat-1 day-1), from week 9 to week 52 of the experiment, inhibited rather than enhanced tumor development in the glandular stomach initiated with MNNG. The percentages of tumor-bearing rats of the groups treated with MNNG plus tautomycin, MNNG alone, and tautomycin alone were 20.0%, 40.6%, and 0% respectively in week 52. The reason for the absence of tumor-promoting activity of tautomycin was studied in relation to tumor necrosis factor alpha (TNF alpha), an endogenous tumor promoter. We found that tautomycin neither enhanced TNF alpha mRNA expression in mouse skin nor induced TNF alpha release in a human stomach cancer cell line (KATO III cells), whereas okadaic acid did both. These results indicate that not all inhibitors of protein phosphatases are tumor promoters, and suggest that tumor promotion of the okadaic acid class of compounds is mediated by TNF alpha.

Topics: Animals; Antifungal Agents; Carcinogens; Enzyme Induction; Enzyme Inhibitors; Ethers, Cyclic; Female; Gene Expression; Humans; Keratins; Male; Mice; Okadaic Acid; Ornithine Decarboxylase; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Spiro Compounds; Stomach Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

To challenge the theory of tissue specificity of tumor promoters, the biochemical and tumor promoting effects of okadaic acid (OA), a potent tumor promoter on mouse skin, were studied in the mucosa of rat glandular stomach. OA strongly inhibited protein phosphatases 1 and 2A, and increased 4-fold the phosphorylation of elongation factor 2 in vitro in the mucosa. Intubation of 10 micrograms (12.4 nmol) OA induced ornithine decarboxylase in the mucosa. Tumor promotion of OA was studied in the glandular stomach initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a two-stage carcinogenesis experiment. OA in drinking water, 10 micrograms (12.4 nmol) per rat per day from weeks 9-55 of the experiment, and 20 micrograms (24.8 nmol) from weeks 56-72, significantly enhanced development of the neoplastic changes in the glandular stomach (P < 0.05). The neoplastic changes included adenomatous hyperplasias and adenocarcinomas, both of which correspond to papillomas and carcinomas in a two-stage mouse skin carcinogenesis experiment. The percentages of neoplastic change-bearing rats of the groups treated with MNNG plus OA, MNNG alone or OA alone were 75.0, 46.4 and 0% respectively. OA enhanced tumorigenesis in the MNNG-initiated glandular stomach of rats through the same mechanisms of action as in mouse skin. The OA pathway mediated through inhibition of protein phosphatases 1 and 2A is applicable to various organs as a general mechanism of tumor promotion.

Topics: Animals; Body Weight; Carcinogens; Enzyme Induction; Ethers, Cyclic; Female; Gastric Mucosa; Male; Methylnitronitrosoguanidine; Okadaic Acid; Organ Specificity; Ornithine Decarboxylase; Phosphoprotein Phosphatases; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Stomach; Stomach Neoplasms