okadaic-acid and Neoplasm-Metastasis

This study shows that the ECM degradation-associated pathway, including uPA and tPA and the downstream MMP-2/-9 protein, was significantly suppressed in HA22T cells treated with a Zanthoxylum avicennae extract (YBBE). The endogenous inhibitors, including TIMP-1/-2 and PAI-1, were enhanced in HA22T cells by the YBBE treatment. The expression of MMP-2/-9 and TIMP-1/-2 was respectively assessed by using RT-PCR and a zymography assay. The mRNA levels and enzymatic activity of MMP-2/-9 were down-regulated by the YBBE treatment in a dose-dependent manner, while the TIMP-1/-2 levels were conversely markedly increased. The PP2A siRNA or PP2A inhibitor totally reversed the YBBE effects, confirming the essential role of PP2A in YBBE inhibiting the HA22T cell migration and invasion effects. Xenografted animal experiments on nude mice demonstrated similiar results to the in vitro system. Both the in vitro and in vivo models clearly demonstrate that YBBE inhibited the highly metastatic HA22T liver cancer cell migration and invasion effects through PP2A activation.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Down-Regulation; Enzyme Activation; Humans; Liver Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Okadaic Acid; Plant Extracts; Plasminogen Activators; Protein Phosphatase 2; Tissue Inhibitor of Metalloproteinases; Xenograft Model Antitumor Assays; Zanthoxylum

Phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by protein kinase C alpha (PKC alpha) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that once released into the cytoplasm, phosphoMARCKS directly promotes motility of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas in weakly motile B16 F1 cells it is undetectable. Following treatment with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibited a dramatic increase in phosphoMARCKS that was co-incident with a 5-fold increase in motility. Both MARCKS phosphorylation and motility were substantially decreased when prior to OA addition, MARCKS expression was knocked out by a MARCKS-specific shRNA, thereby implicating MARCKS as a major component of the motility pathway. Decreased motility and phosphoMARCKS levels in OA-treated cells were observed with a PKC inhibitor (calphostin C), thus indicating that PKC actively phosphorylates MARCKS in F1 cells but that this reaction is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility was further established with a pseudo-phosphorylated mutant of MARCKS-GFP in which Asp residues replaced Ser residues known to be phosphorylated by PKC alpha. This mutant localized to the cytoplasm and engendered three-fold higher motility in F1 cells. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localized to the cytoplasm, blocked motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These results demonstrate that phosphoMARCKS contributes to the metastatic potential of melanoma cells, and reveal a previously undocumented signaling role for this cytoplasmic phospho-protein.

Topics: Animals; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Melanoma, Experimental; Membrane Proteins; Mice; Mutation; Myristoylated Alanine-Rich C Kinase Substrate; Neoplasm Metastasis; Okadaic Acid; Phosphorylation

Guggulsterone, derived from Commiphora mukul and used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, has been recently shown to antagonize the farnesoid X receptor and decrease the expression of bile acid-activated genes. Because activation of NF-kappaB has been closely linked with inflammatory diseases affected by guggulsterone, we postulated that it must modulate NF-kappaB activation. In the present study, we tested this hypothesis by investigating the effect of this steroid on the activation of NF-kappaB induced by inflammatory agents and carcinogens. Guggulsterone suppressed DNA binding of NF-kappaB induced by tumor necrosis factor (TNF), phorbol ester, okadaic acid, cigarette smoke condensate, hydrogen peroxide, and interleukin-1. NF-kappaB activation was not cell type-specific, because both epithelial and leukemia cells were inhibited. Guggulsterone also suppressed constitutive NF-kappaB activation expressed in most tumor cells. Through inhibition of IkappaB kinase activation, this steroid blocked IkappaBalpha phosphorylation and degradation, thus suppressing p65 phosphorylation and nuclear translocation. NF-kappaB-dependent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK was also blocked by guggulsterone but without affecting p65-mediated gene transcription. In addition, guggulsterone decreased the expression of gene products involved in anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, and survivin), proliferation (cyclin D1 and c-Myc), and metastasis (MMP-9, COX-2, and VEGF); this correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents. Overall, our results indicate that guggulsterone suppresses NF-kappaB and NF-kappaB-regulated gene products, which may explain its anti-inflammatory activities.

Topics: Active Transport, Cell Nucleus; Apoptosis; Blotting, Western; Cell Line; Cell Line, Tumor; Cyclooxygenase 2; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation; Genes, Reporter; Humans; I-kappa B Proteins; Inflammation; Interleukin-1; Isoenzymes; Jurkat Cells; Luciferases; Membrane Proteins; Models, Chemical; Neoplasm Metastasis; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Phosphorylation; Poly(ADP-ribose) Polymerases; Pregnenediones; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Protein Structure, Tertiary; Temperature; Tetradecanoylphorbol Acetate; Transcription, Genetic

Increased urokinase plasminogen activator (u-PA) production is associated with tumor invasion and metastasis in several malignancies, including breast cancer. The mechanisms underlying constitutive u-PA expression are not well understood. We examined the relationship between the signal strength of the ERK pathway and the level of u-PA expression in the metastatic human breast cancer cell line MDA-MB-231. Treatment with the MEK1 inhibitor PD98059 resulted in decreased ERK1/2 phosphorylation and decreased u-PA mRNA and protein expression. Inhibition of ERK1/2 activity also led to decreased cell proliferation and to decreased cyclin D1 expression. Less than 5% of total ERK1/2 was phosphorylated in exponentially growing MDA-MB-231 cells, and ERK1/2 activity could be stimulated by okadaic acid. Okadaic acid did not stimulate u-PA expression, but induced strong expression of the cdk-inhibitor p21Cip1. These findings suggest that ERK1/2 signaling is tuned to a level which results in high u-PA expression and rapid cell proliferation.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Okadaic Acid; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-raf; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.

Topics: Base Sequence; Benzoquinones; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Dactinomycin; DNA, Complementary; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Genistein; Humans; Hydroquinones; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; NF-kappa B; Okadaic Acid; Phenols; Proto-Oncogene Proteins c-raf; Quinones; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboplastin; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The role of protein kinase A (PKA) and protein phosphatases (PP) -1 and -2A in regulating the metastatic phenotype of Lewis lung carcinoma (LLC) cells was evaluated. The metastatic LLC-LN7 cells were more motile and invasive than were nonmetastatic LLC-C8 cells. Compared to the nonmetastatic cells, the LLC-LN7 cells had increased PKA activity and a deficiency in PP-2A. Nonmetastatic LLC-C8 cells became migratory and invasive when PP-1 and P-2A activities were inhibited with okadaic acid. This stimulation of LLC-C8 motility was tempered by PKA inhibition. Also examined was if the okadaic acid-stimulated LLC-C8 motility was associated with a change in the cytoskeletal organization to that typical of metastatic cells. Treatment of nonmetastatic LLC-C8 cells with okadaic acid caused a redistribution of F-actin toward the periphery of the cells, and eventually to a loss of the filamentous actin network. All of these effects were reversed upon removal of okadaic acid. Our results show that PP-1/2A maintain reduced motility and increased cytoskeletal organization within nonmetastatic LLC cells.

Topics: Actins; Animals; Carcinoma, Lewis Lung; Cell Movement; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Enzyme Inhibitors; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Tumor Cells, Cultured

Topics: Animals; Cell Movement; Dinoprostone; Indomethacin; Lung Neoplasms; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Okadaic Acid; Phosphoprotein Phosphatases; Tumor Cells, Cultured

Increasing phosphorylation reactions by protein kinase A (PKA) or reducing dephosphorylation reactions of protein phosphatase-2A (PP-2A) increases the invasiveness of Lewis lung carcinoma (LLC) cells, as measured by their capacity to traverse extracellular matrix (ECM)-coated filters. Metastatic LLC-LN7 variants have reduced PP-2A activity when compared to nonmetastatic LLC-C8 variants. Immunoblotting showed that this reduced level of PP-2A activity was not due to reduced levels of the PP-2A catalytic (C) subunit. The cellular PP-2A activity could be stimulated by addition of C2-ceramide to LLC-LN7 lysates, or by incubating cells with either C2-ceramide or with a noncalcemic analog of vitamin D3, which has previously been shown to stimulate the release of ceramide. These treatments to elevate PP-2A activity in metastatic LLC-LN7 cells resulted in a decline in their capacity to invade through select (ECM) components, particularly through vitronectin and laminin. Underscoring the importance of PP-2A in limiting the invasiveness of tumor cells was the demonstration that LLC-LN7 cell transfectants overexpressing the PP-2A C alpha subunit were less invasive through ECM components than the wild-type cells. Invasion by these cells was further reduced by additionally increasing PP-2A activity by incubation with C2-ceramide or the vitamin D3 analog. These results suggest a role of a vitamin D3/ceramide/PP-2A pathway in limiting the invasiveness of tumor cells through select ECM components.

Topics: Animals; Blotting, Western; Cholecalciferol; Enzyme Activation; Extracellular Matrix; Humans; Laminin; Lung Neoplasms; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Sphingosine; Transfection; Vitronectin

Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.

Topics: Cell Movement; Dose-Response Relationship, Drug; Ethers, Cyclic; Neoplasm Metastasis; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; Protein Kinases; Tumor Cells, Cultured