okadaic-acid and Skin-Neoplasms

The classic two-stage chemical carcinogenesis in rodents is not directly linked to multistage carcinogenesis in humans. In light of our findings that tumor necrosis factor-α (TNF-α) is an endogenous tumor promoter and that TNF-α-inducing protein (Tipα) of Helicobacter pylori stimulates progression of cancer and epithelial-mesenchymal transition, we think it is necessary to re-examine the concept of tumor promoter, from chemicals to inflammatory proteins.. This paper begins with "inflammation," discovered by Virchow, studies of Yamagiwa and Tsutsui, and briefly reviews numerous topics, such as (1) the classic concept of tumor promoter, (2) tumor promotion on mouse skin induced by protein kinase C activators and okadaic acid class compounds, (3) organ specificity of tumor promoters, presenting numerous tumor promoters in various organs, (4) unique tumor promotion induced by inhibitors of protein phosphatases 1 and 2A in mouse skin, rat glandular stomach, and rat liver, (5) the significant role of TNF-α in tumor-promoting inflammation, (6) progression induced by Tipα of H. pylori, and (7) enhancement of cancer treatment efficacy with the combination of anticancer drugs and green tea catechins, to inhibit tumor-promoting inflammation.. Human cancer development involves both durable genetic changes caused by carcinogens and proinflammatory cytokines, and simultaneous inflammation in progression induced by proinflammatory cytokines and chemokines.

Topics: Animals; Anticarcinogenic Agents; Carcinogens; Catechin; Gastrointestinal Neoplasms; Helicobacter Infections; Humans; Inflammation; Inflammation Mediators; Neoplasms, Experimental; Okadaic Acid; Organ Specificity; Skin 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; Carcinogens; Ethers, Cyclic; Gene Expression; Humans; Ionophores; Liver Neoplasms, Experimental; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Rats, Inbred F344; Skin Neoplasms; Transcription, Genetic

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

Elevated levels of the oncoprotein, osteopontin (OPN), are associated with poor outcome of several types of cancers including melanoma. We have previously reported an important involvement of DNAJB6, a member of heat-shock protein 40 (HSP40) family, in negatively impacting tumor growth. The current study was prompted by our observations reported here which revealed a reciprocal relationship between DNAJB6 and OPN in melanoma specimens. The 'J domain' is the most conserved domain of HSP40 family of proteins. Hence, we assessed the functional role of the J domain in activities of DNAJB6. We report that the J domain of DNAJB6 is involved in mediating OPN suppression. Deletion of the J domain renders DNAJB6 incapable of impeding malignancy and suppressing OPN. Our mechanistic investigations reveal that DNAJB6 binds HSPA8 (heat-shock cognate protein, HSC70) and causes dephosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser 9 by recruiting protein phosphatase, PP2A. This dephosphorylation activates GSK3β, leading to degradation of β-catenin and subsequent loss of TCF/LEF (T cell factor1/lymphoid enhancer factor1) activity. Deletion of the J domain abrogates assembly of this multiprotein complex and renders GSK3β inactive, thus, stabilizing β-catenin, a transcription co-activator for OPN expression. Our in-vitro and in-vivo functional analyses show that silencing OPN expression in the background of deletion of the J domain renders the resultant tumor cells less malignant despite the presence of stabilized β-catenin. Thus, we have uncovered a new mechanism for regulation of GSK3β activity leading to inhibition of Wnt/β-catenin signaling.

Topics: Animals; beta Catenin; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HSC70 Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Humans; Lymphoid Enhancer-Binding Factor 1; Melanoma; Mice; Mice, Nude; Molecular Chaperones; Neoplasm Transplantation; Nerve Tissue Proteins; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Osteopontin; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Structure, Tertiary; Skin Neoplasms; T Cell Transcription Factor 1; Transcription, Genetic; Transcriptome

Akt is a serine/threonine kinase involved in a variety of cellular responses, including cell proliferation and cell survival. Recent studies from our laboratory suggest that Akt signaling may play an important role in skin tumor promotion. To explore this premise, we examined epidermal Akt activation and signaling in response to chemically diverse skin tumor promoters. Mice received single or multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, or chrysarobin. All three tumor promoters were able to activate epidermal Akt as early as 1 h after treatment. Activation of Akt following tumor promoter treatment led to enhanced downstream signaling, including hyperphosphorylation of glycogen synthase kinase-3beta and Bad. Structure activity studies with phorbol ester analogues revealed that the magnitude of activation paralleled tumor-promoting activity. In cultured primary keratinocytes, TPA treatment also led to activation of Akt. Activation of the epidermal growth factor receptor (EGFR) seemed to underlie the ability of TPA to activate Akt as both PD153035, an inhibitor of EGFR, and GW2974, a dual-specific inhibitor of both EGFR and erbB2, were able to effectively reduce TPA-induced Akt phosphorylation as well as TPA-stimulated EGFR and erbB2 tyrosine phosphorylation in a dose-dependent manner. Furthermore, inhibition of protein kinase C (PKC) activity blocked TPA-stimulated heparin-binding EGF production and EGFR transactivation. Inhibition of PKC also led to a decreased association of Akt with the PP2A catalytic subunit, leading to increased Akt phosphorylation. However, combination of EGFR inhibitor and PKC inhibitor completely abrogated TPA-induced activation of Akt. Collectively, the current results support the hypothesis that elevated Akt activity and subsequent activation of downstream signaling pathways contribute significantly to skin tumor promotion. In addition, signaling through the EGFR via EGFR homodimers or EGFR/erbB2 heterodimers may be the primary event leading to Akt activation during tumor promotion in mouse skin.

Topics: Animals; Anthracenes; Anticoagulants; Carcinogens; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermis; ErbB Receptors; Female; Heparin; Indoles; Keratinocytes; Maleimides; Mice; Mice, Inbred ICR; Okadaic Acid; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-akt; Skin Neoplasms; Tetradecanoylphorbol Acetate

Based on our previous results, which pointed to tumor necrosis factor-alpha (TNF-alpha) as the essential cytokine in tumor promotion in mouse skin, we present here three principal findings related to the specific roles of TNF-alpha, interleukin-1 (IL-1) and IL-6 in tumor promotion (using TNF-alpha- and IL-6-deficient mice) and in BALB/3T3 cell transformation: i) The previously reported residual tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in TNF-/- mice was confirmed by experiments with TNF+/+ and TNF-/- 129/Svj mice of the same strain, using two-stage carcinogenesis experiments. TPA produced tumors in 100% of TNF+/+ and 78% of TNF-/- mice at 20 weeks, and the average number of tumors per mouse was 11.1 in the former group and 2.1 in the latter. Judging from the expression of various inflammatory cytokine genes in TNF+/+ and TNF-/- mice, the residual tumor promoting activity of TPA in TNF-/- mice may be dependent on expression of IL-1alpha and IL-1beta genes. ii) Tumor promotion by TPA and okadaic acid in IL-6+/+ and IL-6-/- C57/BL6 mice was studied, with TPA producing tumors in 57.1% of IL-6+/+ and 40.0% of IL-6-/- mice at 20 weeks, and okadaic acid in 40.0% of IL-6+/+ and 53.3% of IL-6-/- mice. Thus, there was no significant difference between TPA or okadaic acid tumor promotion in either group. In addition, expression of IL-6 gene in skin of both types of mice suggested that IL-6 is not the essential cytokine in tumor promotion, since it can be replaced by other cytokines. iii) In transformed clones of BALB/3T3 cells induced by TNF-alpha alone, IL-1alpha gene expression was induced after transformation by TNF-alpha had occurred, which did not occur in parental cells. Expression patterns of TNF-alpha, IL-1beta, IL-6 and IL-10, along with TGF-beta, were similar in both parental and transformed cells. Considering all these results, we conclude that various cytokines have discrete roles in tumor promotion and cell transformation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Division; Cell Transformation, Neoplastic; DNA Primers; Female; Humans; Interleukin-1; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Okadaic Acid; Polymerase Chain Reaction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection; Tumor Necrosis Factor-alpha

Okadaic acid (OA) is a prototypical non-phorbol ester skin tumor-promoting agent that works by inhibiting protein phosphatases, leading to an increase in protein phosphorylation. Increased protein phosphorylation can lead to stimulated signaling through various signal transduction pathways. One or more of the pathways affected by OA leads to increased signaling via the activator protein 1 (AP-1) transcription factor. Because AP-1 signaling has been shown to be required for skin tumor promotion by phorbol ester, studies were undertaken to determine whether AP-1 signaling is also required for 7,12-dimethylbenz(a)anthracene (DMBA)-initiated/OA-promoted skin tumorigenesis. Transgenic mice expressing a dominant negative c-jun (TAM-67) controlled by the keratin 14 promoter in ICR mice were used to determine the effects of OA on AP-1 signaling. By crossing the TAM-67 mice with mice expressing an AP-1-responsive luciferase, it was shown that TAM-67 decreases AP-1 activation in response to OA treatment by 95%. After 7,12-dimethylbenz(a)anthracene initiation, the TAM-67 mice and nontransgenic littermates were promoted with twice weekly applications of OA. These experiments showed that TAM-67 expression decreased tumor multiplicity by 90%. Additional experiments with TAM-67 mice showed that the hyperplastic response to OA is not impaired in these mice, nor were there differences in OA-induced transcription of various genes known to be AP-1 responsive under other conditions. This result suggests that only a subset of AP-1-regulated genes is targeted by TAM-67 when it prevents tumor promotion by OA. A determination of the mechanism by which TAM-67 can block tumor promotion without affecting hyperplasia will be important.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Enzyme Induction; Matrix Metalloproteinase 3; Mice; Mice, Inbred DBA; Mice, Inbred ICR; Mice, Transgenic; Okadaic Acid; Peptide Fragments; Proto-Oncogene Proteins c-jun; Skin; Skin Neoplasms; Transcription Factor AP-1

Okadaic acid (OA), a tumor promoter in the mouse skin carcinogenesis model, has been shown to induce apoptosis in tumor cell lines that harbor H-ras mutations. We examined the effects of OA on mouse keratinocytes with (308) and without (C50) H-ras mutation in vitro and in an in vivo system. Following exposure to varying concentrations of OA over time, the effects of OA in vitro were assessed using microscopic, biochemical and flow cytometric techniques. OA effects on the cells included incorporation of propidium iodide, externalization of phosphatidylserine, and development of hypodiploidy. 308 cells demonstrated typical DNA ladder formation, rapid chromatin and nuclear condensation, while C50 cells demonstrated delayed chromatin condensation and nuclear fragmentation, but no DNA ladder formation. In vivo, OA elicited delayed papilloma formation and reduced tumor multiplicity. Though its mechanism of action is not fully known, we found that OA-induced inhibition of the clonal expansion of initiated cells may be related to the presence or absence of H-ras mutation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Cell Adhesion; Cell Cycle; Cells, Cultured; Colony-Forming Units Assay; Diploidy; Disease Models, Animal; DNA Fragmentation; Exocytosis; Female; Flow Cytometry; Keratinocytes; Mice; Microscopy, Electron; Okadaic Acid; Papilloma; Phosphatidylserines; Skin Neoplasms

Insulin-like growth factor-1 (IGF-1) and its receptor are believed to play an important role in mitogenesis and neoplastic transformation. The purpose of this study was to further examine the role of IGF-1 during tumor promotion in mouse skin. HK1.IGF1 transgenic mice, which overexpress IGF-1 in epidermis via the human keratin 1 promoter, were previously shown to be hypersensitive to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). We examined these mice for their sensitivity to diverse classes of tumor-promoting agents. HK1.IGF-1 transgenic mice initiated with 7,12-dimethylbenz[a]anthracene were more sensitive to treatment with a wide variety of tumor promoters, including chrysarobin, okadaic acid, and benzoyl peroxide, which resulted in more rapid development of tumors and a dramatic increase in the number of tumors per mouse compared with corresponding non-transgenic mice treated with the same compounds. Histological analyses of skin from HK1.IGF-1 mice treated with various tumor promoters revealed that these mice were also more sensitive to the induction of epidermal hyperplasia and cell proliferation. Analysis of the IGF-1 receptor (IGF-1r) and epidermal growth factor (EGFr) in the epidermis of TPA-treated HK1.IGF-1 transgenic and non-transgenic mice revealed that both receptors were activated (hyperphosphorylated on tyrosine residues), and the level of activation was higher in transgenic mice. The mechanism for the increased sensitivity of HK1.IGF-1 mice to tumor promoters may involve cooperation between the IGF-1r and EGFr signaling pathways. Our data suggest that IGF-1r signaling may play an important role in the process of tumor promotion by diverse classes of tumor promoters.

Topics: Animals; Anthracenes; Benzoyl Peroxide; Carcinogens; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Transgenic; Okadaic Acid; Receptor, IGF Type 1; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transgenes

To examine the hypothesis that tumor necrosis factor (TNF) alpha is an essential cytokine in carcinogenesis, we conducted two-stage carcinogenesis experiments with an initiator, 7,12-dimethylbenz(a)anthracene (DMBA), plus either of two tumor promoters, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the skin of TNF-alpha-deficient (TNF-/-) mice. TNF-/- mice treated with DMBA plus okadaic acid developed no tumors for up to 19 weeks, and at 20 weeks, the percentage of tumor-bearing TNF-/- mice was 10%, whereas the percentage of tumor-bearing TNF+/+ mice was 100%. In TNF-/- mice treated with DMBA plus TPA, tumor onset was delayed 4 weeks, and the time to development of small tumors in 100% of mice was 9 weeks later than that seen in TNF+/+ CD-1 mice. The average number of tumors in TPA-treated TNF-/- mice was 2.8, compared with 11.8 for TNF+/+ CD-1 mice. To understand the residual tumor-promoting activity in TNF-/- mice, we also investigated the possible significance of interleukin (IL) 1 as an additional cytokine in tumor promotion. A single application of TPA and okadaic acid increased IL-1alpha and IL-1beta gene expression in TNF-/- mice. All of our results demonstrate that TNF-alpha is the key cytokine for tumor promotion in mouse skin and, very possibly, for carcinogenesis in humans as well.

Topics: 3T3 Cells; 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Division; Cell Line, Transformed; Female; Genes, ras; Interleukin-1; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Okadaic Acid; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection; Tumor Necrosis Factor-alpha

Recent evidence suggests that cholesterol sulfate may be an important second messenger involved in signaling epidermal differentiation in skin. The activity of cholesterol sulfotransferase (Ch-ST) is increased during squamous differentiation of keratinocytes and is believed to be a marker enzyme for terminal differentiation. The primary objective of this study was to examine changes in levels of cholesterol sulfate (CS) and activity of its biosynthetic enzyme, Ch-ST, during multistage carcinogenesis in mouse skin. Using SENCAR mice, we determined the activity of Ch-ST in normal epidermis, in tumor promoter-treated epidermis, in epidermis during wound healing, and in mouse skin tumors generated by initiation-promotion regimens. A single topical application of tumor promoters led to significantly elevated levels of Ch-ST activity and of CS. Epidermal Ch-ST activity was also elevated during wound healing. Dramatic increases in CS levels and in the activity of Ch-ST were found in nearly all of the papillomas and squamous cell carcinomas examined. The increased levels of CS and activity of Ch-ST in tumor promoter-treated epidermis were accompanied by increased transglutaminase-I activity. In contrast, transglutaminase I activity was not elevated in primary papillomas or squamous cell carcinomas. Finally, Ch-ST activity was significantly elevated in the epidermis of newborn HK1.ras transgenic mice, whereas transglutaminase I activity did not correlate with Ch-ST activity in these mice. These results demonstrate that diverse tumor-promoting stimuli all produce elevated CS levels and Ch-ST activity and that CS levels and Ch-ST activity were constitutively elevated in both papillomas and squamous cell carcinomas. The data also suggest a mechanism for upregulation of Ch-ST in skin tumors involving activation/upregulation of Ha-ras.

Topics: Administration, Topical; Animals; Anthracenes; Anticarcinogenic Agents; Carcinogens; Cell Differentiation; Cell Division; Cholesterol Esters; Dehydroepiandrosterone Sulfate; Epidermis; Female; Keratinocytes; Mice; Mice, Inbred SENCAR; Mice, Transgenic; Okadaic Acid; Skin Neoplasms; Sulfotransferases; Tetradecanoylphorbol Acetate; Transglutaminases; Wound Healing

Canventol, a synthetic compound, is a new inhibitor of tumor promotion on mouse skin by okadaic acid. We previously reported that canventol acts by inhibiting both protein isoprenylation and tumor necrosis factor-alpha (TNF-alpha) release. In this study we examined the potencies of 10 newly synthesized canventol analogs through their effect on mevalonate metabolism, and then examined 3 representative analogs for inhibition of protein isoprenylation. Since canventol in vitro did not directly inhibit farnesyl protein transferase or geranylgeranyl protein transferase-I, the effects of canventol and its synthetic analogs on the fate of [3H]mevalonate in cells were studied. Canventol at 500 microM changed the ratio of newly synthesized sterols (cholesterol and lathosterol) to ubiquinones from 0.7 to 8.2 in NIH/3T3 cells which had previously been labeled with [3H]mevalonate, suggesting that the altered pattern of mevalonate metabolism is associated with inhibition of protein isoprenylation in the cells. We named this ratio the inhibition of protein isoprenylation index (IPI index). The 10 analogs showed a wide range of IPI indices. Two analogs, S3 and S9 had effects similar to, or stronger than, canventol. Three analogs, C44, C46 and C47, with lower IPI indices, inhibited tumor promotion on mouse skin slightly less than canventol itself did. This study shows that inhibition of protein isoprenylation in the cells, indicated by an increase in the IPI index, is a new biomarker for estimating inhibition of tumor promotion.

Topics: 3T3 Cells; Alkyl and Aryl Transferases; Animals; Anticarcinogenic Agents; Carcinogens; Cyclohexanols; Enzyme Inhibitors; Female; Lipid Metabolism; Mevalonic Acid; Mice; Mice, Inbred Strains; Okadaic Acid; Protein Prenylation; Skin Neoplasms; Sterols; Transferases; Ubiquinone

(-)-Epigallocatechin gallate (EGCG), the main constituent of green tea, inhibited a tumor promoting activity of okadaic acid in a two-stage carcinogenesis experiment on mouse skin. The group treated with a single application of 100 micrograms 7, 12-dimethylbenz (a) anthracene followed by repeated applications of 1 microgram okadaic acid resulted in 80% of tumor-bearing mice and 4.7 of average numbers of tumors per mouse in week 20. Repeated applications of 5 mg EGCG, prior to okadaic acid, completely inhibited the tumor formation in mice up to week 20. The inhibitory effects of EGCG with two different doses of each application, 1 mg and 5 mg, were dose-dependent. A topical application of 5 mg EGCG immediately reduced the specific binding of [3H]okadaic acid to a particulate fraction of mouse skin to as low as 30% of control. According to the Scatchard analysis, the reduction of specific [3H]okadaic acid binding was mainly due to the reduction of the binding sites, not due to the change of the affinity. The reduction of the specific binding was closely related to the inhibitory effct of EGCG on tumor promotion of okadaic acid. Since EGCG is a non-toxic compound, ingested in green tea in daily life in Japan, EGCG is one of the candidates for practical cancer chemopreventive agents.

Topics: Animals; Antineoplastic Agents; Binding Sites; Carcinogens; Catechin; Mice; Okadaic Acid; Skin Neoplasms; Tea

A synthetic compound named canventol, 2-isopropyl-4-isopropylidencyclohex-2-ene-1-ol, inhibited tumor promotion of okadaic acid on mouse skin initiated with 7,12-dimethylbenz(a)anthracene in two-stage carcinogenesis experiments more strongly than sarcophytol A, isolated from a soft coral, although canventol has a simpler structure than sarcophytol A. Their mechanisms of action were studied based on our recent evidence that tumor necrosis factor alpha release induced by okadaic acid is an essential mechanism of tumor promotion. Canventol inhibited mouse tumor necrosis factor alpha release from BALB/3T3 cells less strongly than sarcophytol A, indicating that canventol has additional activity. Canventol inhibited isoprenylation of proteins with various molecular weights, such as M(r) 22,000, 17,000, and 13,000, whereas sarcophytol A did not show significant inhibition. Thus, a potent anticarcinogenic activity of canventol is mediated through the inhibitory bifunctions of tumor necrosis factor alpha release and of protein isoprenylation. Since canventol is less toxic to cells than sarcophytol A, these bifunctions are useful markers for screening for new cancer chemopreventive agents.

Topics: 3T3 Cells; 9,10-Dimethyl-1,2-benzanthracene; Animals; Anticarcinogenic Agents; Cyclohexanols; Diterpenes; Ethers, Cyclic; Female; Mevalonic Acid; Mice; Okadaic Acid; Protein Prenylation; Skin Neoplasms; Tumor Necrosis Factor-alpha

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, and teleocidin, an activator of protein kinase C, are both potent tumor promoters on mouse skin. The effects of simultaneous treatment of the two different types of tumor promoters on tumor promotion as well as on their biochemical activities were studied. Three independent experiments with different doses of tumor promoters revealed that simultaneous repeated applications of okadaic acid and teleocidin did not induce any synergistic or additive effects on tumor promotion in mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). In Experiment 1, the group treated with a single application of DMBA, followed by repeated applications of 1.0 micrograms (1.2 nmol) okadaic acid and 2.5 micrograms (5.7 nmol) teleocidin, resulted in 64.3% tumor-bearing mice at week 20. But the groups treated with DMBA plus okadaic acid or DMBA plus teleocidin gave 73.3% and 71.4%, respectively. The biochemical activities were studied by means of induction of ornithine decarboxylase in mouse skin and protein phosphorylation in the cells. Simultaneous application of okadaic acid at three different doses with teleocidin did not induce ornithine decarboxylase activity synergistically or additively. Phosphorylation of proteins, cytokeratins, or heat shock protein 27 was not synergistically increased in human keratinocytes treated with okadaic acid and teleocidin, although the cotreatment in a cell-free system synergistically increased protein phosphorylation. Thus, the absence of synergistic effects on tumor promotion in mouse skin was also confirmed in two systems, induction of ornithine decarboxylase in mouse skin and protein phosphorylation in human keratinocytes. The effect of cotreatment of okadaic acid and teleocidin is discussed at the molecular level.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Drug Synergism; Enzyme Induction; Ethers, Cyclic; Female; Lyngbya Toxins; Mice; Okadaic Acid; Ornithine Decarboxylase; Phosphorylation; Proteins; Skin Neoplasms; Tetradecanoylphorbol Acetate

Levamisole is an effective antihelminthic drug with immunomodulatory and anticancer activities in model systems. Combined with fluorouracil (5-FU) as adjuvant treatment following resection of Dukes' stage C colon carcinomas, levamisole significantly reduces mortality. However, neither 5-FU nor levamisole alone has a significant effect on survival in this patient group. Previously, we noted that in vitro levamisole potentiated the antiproliferative activity of 5-FU.. Because levamisole is known to inhibit alkaline phosphatases and has been reported to inhibit dephosphorylation of some membrane phosphoproteins, we studied the effects of levamisole analogues and of chemically unrelated inhibitors of phosphatases for their ability to potentiate 5-FU inhibition of tumor cell line proliferation in vitro.. Human cancer cell lines were exposed to drugs alone or in combination with 5-FU. Antiproliferative activity was measured by determining the extent of reduction of colony formation by the cell lines in test plates compared with control plates.. We found that potentiation of 5-FU cytotoxicity by levamisole and by p-hydroxytetramisole, a metabolite of levamisole, is mimicked by orthovanadate, an inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine and threonine phosphatases, Furthermore, l-p-bromotetramisole, a synthetic analogue of levamisole that is 10-fold more potent in inhibition of alkaline phosphatase than levamisole, potentiates the antiproliferative activity of 5-FU to a greater extent than d-p-bromotetramisole, a stereoisomer of l-p-bromotetramisole with little antiphosphatase activity.. Inhibition of tyrosine phosphatases may be responsible for the potentiation by levamisole of the inhibitory activity of 5-FU in vitro.. Inhibition of dephosphorylation of regulatory phosphoproteins may be related to the therapeutic efficacy of the combination of levamisole and 5-FU in the adjuvant treatment of colon carcinoma and may underlie at least some of the multiple effects of levamisole on immune parameters.

Topics: Breast Neoplasms; Colonic Neoplasms; Drug Synergism; Ethers, Cyclic; Fluorouracil; Humans; Levamisole; Melanoma; Okadaic Acid; Phosphoric Monoester Hydrolases; Skin Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay; Vanadates

Cryptoporic acids D and E, isolated from the fungus Cryptoporus volvatus, are inhibitors of superoxide anion radical release. Cryptoporic acid E inhibited tumor promotion of okadaic acid in two-stage carcinogenesis experiments on mouse skin, initiated with 7,12-dimethylbenz[a]anthracene. Treatment with cryptoporic acid E using two different doses per application, 1 (1.2 mumol) and 5 mg (5.9 mumol), reduced the percentage of tumor-bearing mice from 73.3 to 53.3% and 20.0%, and the average number of tumors per mouse from 4.2 to 2.3 and 0.5 respectively in week 20 of tumor promotion. However, cryptoporic acid D slightly enhanced tumor promotion rather than inhibition of okadaic acid. Cryptoporic acid D was expected to have additional biochemical activities, such as activation of protein kinases. Cryptoporic acid D at concentrations of up to 100 microM activated protein kinase C and stimulated other protein kinase activity in vitro, whereas cryptoporic acid E did not. These two compounds provided differential effects on tumor promotion of okadaic acid on mouse skin.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Enzyme Activation; Ethers, Cyclic; Female; Free Radicals; Mice; Okadaic Acid; Protein Kinase C; Protein Kinases; Sesquiterpenes; Skin Neoplasms; Superoxides

Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin A are the okadaic acid class of non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, which do not bind to the phorbol ester receptors in cell membranes or activate protein kinase C in vitro. They have potent tumor-promoting activities on mouse skin, as strong as TPA-type tumor promoters, such as TPA, teleocidin, and aplysiatoxin. DNA samples isolated from tumors induced by dimethylbenz[alpha]anthracene and each of the okadaic acid class tumor promoters had the same mutation at the second nucleotide of codon 61 (CAA to CTA) in the c-H-ras gene. Okadaic acid receptors, protein phosphatases 1 and 2A, are present in the particulate as well as cytosolic fractions of various mouse tissues. The apparent "activation" of protein kinases by the okadaic acid class tumor promoters, after their incubation with 32P-ATP, protein kinases, and protein phosphatases, was observed. This activation was caused by inhibition of protein phosphatases 1 and 2A by the okadaic acid class tumor promoters. Treatment of primary human fibroblasts and human keratinocytes with the okadaic acid class tumor promoters induced the hyperphosphorylation of a 60-kDa protein in nuclear and cytosolic fractions, due to the inhibition of protein phosphatases. The 60-kDa protein is a proteolytic fragment of nucleolin, a major nonhistone protein and is designated as "N-60." The mechanisms of action of the okadaic acid class tumor promoters are discussed with emphasis on the inhibition of protein phosphatase activity.

Topics: Animals; Carcinogens; DNA; DNA Damage; DNA, Neoplasm; Enzyme Activation; Ethers, Cyclic; Genes, ras; Humans; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Proteins; Pyrans; Receptors, Drug; Skin; Skin Neoplasms; Structure-Activity Relationship

Calyculin A, isolated from a marine sponge, has a novel spiro ketal skeleton. Structurally unrelated to okadaic acid, calyculin A bound to the okadaic acid receptors in particulate and soluble fractions of mouse skin. The biochemical and tumor-promoting activities of calyculin A were studied with those of okadaic acid. Calyculin A inhibited the activity of protein phosphatases, which serve as the okadaic acid receptors. The effective dose of calyculin A for 50% inhibition was 0.3 nM, similar to that of okadaic acid. Like okadaic acid, calyculin A induced ornithine decarboxylase in mouse skin and hyperphosphorylation of a Mr 60,000 protein in human papilloma virus type 16-transformed human keratinocytes. A two-stage carcinogenesis experiment on mouse skin, initiated by 100 micrograms (390 nmol) of 7,12-dimethylbenz(a)anthracene and followed by 1 microgram (1.0 nmol) of calyculin A, revealed that calyculin A is an additional member of the okadaic acid class of tumor promoters. The percentages of tumor-bearing mice in the groups treated with DMBA plus calyculin A, and with DMBA followed by 1 microgram (1.2 nmol) of okadaic acid were 86.7 and 80.0%, respectively, in week 30. The mechanisms of action of calyculin A and okadaic acid, in addition to dinophysistoxin-1 (35-methylokadaic acid), are discussed. Calyculin A is the first tumor promoter to be screened by the okadaic acid receptor binding test.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Binding Sites; Ethers, Cyclic; Female; Marine Toxins; Mice; Mice, Inbred Strains; Molecular Weight; Nuclear Proteins; Nucleolin; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; RNA-Binding Proteins; Skin; Skin Neoplasms

Topics: Animals; Binding, Competitive; Brain; Carcinogens; Cytosol; DNA, Neoplasm; Ethers, Cyclic; Humans; Marine Toxins; Mice; Molecular Structure; Mutation; Nuclear Proteins; Nucleolin; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Protein Kinases; Proteins; Proto-Oncogene Proteins p21(ras); Pyrans; Receptors, Drug; RNA-Binding Proteins; Skin; Skin Neoplasms; Spiro Compounds; Structure-Activity Relationship

Three okadaic acid class tumor promoters, okadaic acid, dinophysistoxin-1, and calyculin A, have potent tumor-promoting activity in two-stage carcinogenesis experiments on mouse skin. DNA isolated from tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and each of these tumor promoters revealed the same mutation at the second nucleotide of codon 61 (CAA----CTA) in the c-Ha-ras gene, determined by the polymerase chain reaction procedure and DNA sequencing. Three potent 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, TPA, teleocidin, and aplysiatoxin, showed the same effects. These results provide strong evidence that this mutation in the c-Ha-ras gene is due to a direct effect of DMBA rather than a selective effect of specific tumor promoters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Base Sequence; Blotting, Western; Carcinogens; Codon; DNA Mutational Analysis; DNA-Directed DNA Polymerase; DNA, Neoplasm; Ethers, Cyclic; Genes, ras; Male; Mice; Okadaic Acid; Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Taq Polymerase

Okadaic acid is a polyether compound of a C38 fatty acid, isolated from a black sponge, Halichondria okadai. Previous studies showed that okadaic acid is a skin irritant and induces ornithine decarboxylase (OrnDCase; 3-hydroxyl-L-glutamate 1-carboxy-lyase, EC 4.1.1.17) in mouse skin 4 hr after its application to the skin. This induction was strongly inhibited by pretreatment of the skin with 13-cis-retinoic acid. A two-stage carcinogenesis experiment in mouse skin initiated by a single application of 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) and followed by application of 10 micrograms of okadaic acid twice a week revealed that okadaic acid is a potent additional tumor promoter: tumors developed in 93% of the mice treated with DMBA and okadaic acid by week 16. In contrast, tumors were found in only one mouse each in the groups treated with DMBA alone or okadaic acid alone. An average of 2.6 tumors per mouse was found in week 30 in the group treated with DMBA and okadaic acid. Unlike phorbol 12-tetradecanoate 13-acetate (TPA), teleocidin, and aplysiatoxin, okadaic acid did not inhibit the specific binding of [3H]TPA to a mouse skin particulate fraction when added up to 100 microM or activate calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in vitro when added up to 1.2 microM. Therefore, the actions of okadaic acid and phorbol ester may be mediated in different ways. These results show that okadaic acid is a non-TPA-type tumor promoter in mouse skin carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Enzyme Induction; Ethers, Cyclic; Humans; Leukemia, Myeloid, Acute; Lyngbya Toxins; Mice; Okadaic Acid; Ornithine Decarboxylase; Porifera; Protein Kinase C; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Dinophysistoxin-1, 35-methylokadaic acid, is a causative agent of diarrhetic shellfish poisoning. The biological activities and tumor-promoting activity of dinophysistoxin-1 were studied together with those of okadaic acid and 7-O-palmitoyl okadaic acid. Dinophysistoxin-1 is a skin irritant and induces ornithine decarboxylase in mouse skin with the same potency as okadaic acid. 7-O-Palmitoyl okadaic acid induced a lower activity than the other compounds. Dinophysistoxin-1 inhibited the specific [3H]okadaic acid binding to a particulate fraction of mouse epidermis. The binding affinities of dinophysistoxin-1 and okadaic acid to a particulate fraction were almost the same. Dinophysistoxin-1 showed a tumor-promoting activity as strong as that of okadaic acid in a two-stage carcinogenesis experiment on mouse skin. The percentages of tumor-bearing mice in the groups treated with 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed by 5 micrograms of dinophysistoxin-1, twice a week, and with DMBA followed by 5 micrograms of okadaic acid twice a week were 86.7% and 80.0% in week 30, respectively. The average number of tumors per mouse was 4.6 in the former group and 3.9 in the latter. Dinophysistoxin-1 and okadaic acid act on cells through different pathways from the 12-O-tetradecanoylphorbol-13-acetate-type tumor promoters.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cocarcinogenesis; Enzyme Induction; Ethers, Cyclic; Female; Irritants; Marine Toxins; Mice; Okadaic Acid; Ornithine Decarboxylase; Pyrans; Skin Neoplasms; Structure-Activity Relationship