Lysophosphatidic acid induces a migratory phenotype through a crosstalk between RhoA-Rock and Src-FAK signalling in colon cancer cells

Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.     

Rho-dependent inhibition of the induction of connective tissue growth factor (CTGF) by HMG CoA reductase inhibitors (statins)

It was supposed that inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase (statins) might inhibit the expression of the fibrosis-related factor CTGF (connective tissue growth factor) by interfering with the isoprenylation of Rho proteins. The human renal fibroblast cell line TK173 was used as an in vitro model system to study the statin-mediated modulation of the structure of the actin cytoskeleton and of the expression of CTGF mRNA. Incubation of the cells with simvastatin or lovastatin time-dependently and reversibly changed cell morphology and the actin cytoskeleton with maximal effects observed after about 18 h. Within the same time period, statins reduced the basal expression of CTGF and interfered with CTGF induction by lysophosphatidic acid (LPA) or transforming growth factor beta. Simvastatin and lovastatin proved to be much more potent than pravastatin (IC(50) 1 - 3 microM compared to 500 microM). The inhibition of CTGF expression was prevented when the cells were incubated with mevalonate or geranylgeranylpyrophosphate (GGPP) but not by farnesylpyrophosphate (FPP). Specific inhibition of geranylgeranyltransferase-I by GTI-286 inhibited LPA-mediated CTGF expression whereas an inhibitor of farnesyltransferases FTI-276 was ineffective. Simvastatin reduced the binding of the small GTPase RhoA to cellular membranes. The effect was prevented by mevalonate and GGPP, but not FPP. These data are in agreement with the hypothesis that interference of statins with the expression of CTGF mRNA is primarily due to interference with the isoprenylation of RhoA, in line with previous studies, which have shown that RhoA is an essential mediator of CTGF induction. The direct interference of statins with the synthesis of CTGF, a protein functionally related to the development of fibrosis, may thus be a novel mechanism underlying the beneficial effects of statins observed in renal diseases.     

Crotoxin induces actin reorganization and inhibits tyrosine phosphorylation and activity of small GTPases in rat macrophages

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom. Previous work of our group demonstrated that this toxin or its phospholipase A₂ subunit inhibits macrophage spreading and phagocytosis. The phagocytic activity of macrophages is controlled by the rearrangement of actin cytoskeleton and activity of the small Rho GTPases. The effect of crotoxin and its subunit on actin reorganization and tyrosine phosphorylation in rat peritoneal macrophages, during phagocytosis of opsonized zymosan, was presently investigated. The crude venom was used as positive control. In addition, the effect of crotoxin on the activity of Rho and Rac1 small GTPases was examined. Transmission electron studies showed that the venom or crotoxin decreased the extent of spread cells and increased microprojections often extended from macrophage surface. Immunocytochemical assays demosntrated that the venom or toxins increased F-actin content in the cytoplasm of these cells, but induced a marked decrease of phosphotyrosine. These effects were abolished by treatment with zileuton, a 5-lipoxygenase inhibitor. Furthermore, crotoxin decreased membrane-associated RhoA and Rac1 in translocation assays. The present results indicate that the crotalid venom and crotoxin are able to induce cytoskeleton rearrangement in macrophages. This effect is associated with inhibition of tyrosine phosphorylation and of the activity of proteins involved in intracellular signalling pathways important for the complete phagocytic activity of these cells.     

Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER(+) MCF-7 and ER(-) MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G(2)/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G(2) exit and initiation of mitosis, namely the p34(CDC2)/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21(WAF), proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (DeltaPsi) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells.     

Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling

Cucurbitacin B, a member of the cucurbitaceae family, can act as a STAT3 signaling inhibitor to regulate the growth of hepatocellular carcinoma. STAT3 signaling has been shown to inhibit adipocyte differentiation through C/EBPα and PPARγ. Based on these studies, we hypothesized that cucurbitacin B would prevent PPARγ mediated adipocyte differentiation through STAT3 signaling. To test this hypothesis, mesenchymal C3H10T1/2 and 3T3-L1 preadipocyte cells were treated with a sub-cytotoxic concentration of cucurbitacin B. Cucurbitacin B treatment inhibits lipid accumulation and expression of adipocyte markers including PPARγ and its target genes in a dose-dependent manner. Cucurbitacin B treatment impairs STAT3 signaling as manifested by reduced phosphorylation of STAT3 and suppression of STAT3 target gene expression in preadipocytes. The anti-adipogenic effects of cucurbitacin B are significantly blunted in cells with STAT3 silenced by introducing small interfering RNA. Finally, our data show that cucurbitacin I, another cucurbitacin family member, also inhibits adipocyte differentiation by suppressing STAT3 signaling. Together, our data suggest the possibility of utilizing cucurbitacins as a new strategy to treat metabolic diseases and implicate STAT3 as a new target for the development of functional foods and drugs.     

Involvement of Rho signaling in PAR2-mediated regulation of neutrophil adhesion to lung epithelial cells

Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH2, 1-100 microM) for 0.5-3 h dose- and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.     

Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21

Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.     

Cleavage of tensin during cytoskeleton disruption in YTX-induced apoptosis.

Yessotoxin (YTX) is a marine algal toxin previously shown to induce apoptosis in L6 and BC3H1 myoblast cell lines. Disassembly of the F-actin cytoskeleton and cleavage of tensin, a cytoskeletal protein localised at the focal adhesion contacts, appear during this apoptotic process. Tensin binds to actin filaments at the focal adhesion contacts and it links the actin cytoskeleton to the extracellular matrix (ECM). This binding occurs via integrin receptors and it makes tensin a potential link between the actin cytoskeleton and signal transduction. This study evaluates disruption in the F-actin cytoskeleton and change of tensin in myoblast cell lines exposed to 100 nM YTX up to 72 h. YTX treatment cleaves tensin and makes it translocate to the cell centre. Tensin has normally a role in the maintenance of cell shape and YTX-treatment may therefore alter the shape of the cells. YTX exposure also induces formation of lamellas associated with pseudopodia. Alternative linkages and cytoskeletal proteins anchoring the actin filaments to focal contacts remain to be identified.     

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation

Human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. HEF1 is present at focal adhesions and is involved in integrin signalling mediating cytoskeleton reorganization associated with cell migration, adhesion or apoptosis. HEF1 functions are regulated in part by phosphorylation on tyrosine residues. HEF1 is also phosphorylated on serines/threonines leading to two isoforms refered to as p105 and p115. In most cases, the serine/threonine kinase(s) responsible for HEF1 phosphorylation have not been identified. In the present study, we have investigated HEF1 ser/thr phosphorylation. In the HCT-116 cell line transiently overexpressing Flag-HEF1 we showed that Hesperadin, a synthetic indolinone displaying antiproliferative effect and described as an inhibitor of various kinases including Aurora-B, prevented HEF1 phosphorylation induced by the ser/thr phosphatase PP2A inhibitor: okadaic acid (OA). In addition we showed that conversion of endogenous HEF1 p105 to p115 in HaCaT cells was prevented upon treatment with Hesperadin, resulting in accumulation of p105HEF1. We also identified serine 369 as the target site of phosphorylation by this Hesperadin-inhibited kinase in HCT-116. Finally, we provide evidence that phosphorylation on serine 369 but not phosphorylation on serine 296, triggers HEF1 degradation by the proteasomal machinery. These data suggest that conversion of p105 to p115 results from a ser-369-dependent phosphorylation mediated by an Hesperadin-sensitive kinase and regulates the half-life of HEF1.     

Influence of Clostridium botulinum C2 toxin on FcɛRI-mediated secretion and tyrosine phosphorylation in RBL cells

We studied the effects of the binary Clostridium botulinum C2 toxin on stimulated [³H]serotonin release and protein tyrosine phosphorylation in RBL 2H3 hm1 cells. Actin was specifically ADP-ribosylated by C2 toxin in intact cells resulting in a 2–3 fold increase in antigen- or calcium ionophore (A23187)-induced degranulation. The effects of C2 toxin were time- and concentration-dependent. Toxin treatment, which dramatically changes the morphology of RBL cells, was not sufficient to induce mediator release in the absence of activators of secretion. Antigen- and A23187-stimulated tyrosine phosphorylation of 60–80kDa and 110–120kDa proteins was reduced or blocked after C2 toxin incubation. Treatment of RBL cells with the tyrosine phosphatase inhibitor pervanadate reversed the inhibitory effect of C2 toxin on stimulated protein tyrosine phosphorylation indicating activation of phosphatases by C2 toxin. The data indicate that disassembly of the actin cytoskeleton by C2 toxin facilitates Fc?RI-mediated signal-secretion coupling and suggest a role of the actin cytoskeleton in phosphatase regulation in RBL cells.     

Cucurbitacin B induces inhibitory effects via the CIP2A/PP2A/C-KIT signaling axis in t(8;21) acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common subtype of hematological malignancy in humans, and its incidence increases with age. The treatment of AML still faces challenges. Therefore, there is an urgent need to develop more effective targeted therapies. The receptor tyrosine kinase C-KIT confers critical proliferative signals to AML. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an endogenous inhibitor of protein phosphatase 2A (PP2A), which promotes the growth and transformation of various solid tumors. These actions make CIP2A a promising target for tumor treatment. Here, we reported the effects and underlying mechanisms of a natural compound, cucurbitacin B (CuB), on AML. We reported that CuB suppressed growth and induced apoptosis in AML cells. The inhibition of growth and activation of apoptosis were mediated through CuB-induced downregulation of the CIP2A/PP2A/C-KIT signal pathway. Furthermore, CuB inactivated the JAK2 and STAT3 molecules downstream of C-KIT via the downregulation of CIP2A. These results advance our understanding of CuB-induced growth inhibition and apoptosis and support further investigation of CuB as a CIP2A inhibitor for AML therapies.     

Low-dose exposure of intestinal epithelial cells to formaldehyde results in MAP kinase activation and molecular alteration of the focal adhesion protein paxillin

We investigated the potential pathophysiological role of non-lethal formaldehyde concentrations on human intestinal epithelial HT-29 cells. Expression levels of actin, tubulin and detectable cytokeratin isoforms 5, 13, 18, 19 and 20 were not affected after 24h of exposure to 1mM formaldehyde. By contrast, cellular organization of cytoskeletal constituents was already changed after 60 min. Within 15 min, formaldehyde induced profound tyrosine phosphorylation of the focal adhesion protein paxillin and of proteins at about 120-130 kDa. Concomitantly, phosphorylation of ERK-1/2 and p38 MAP kinase occurred. Paxillin was not only tyrosine phosphorylated but underwent a sustained molecular weight shift representing serine/threonine phosphorylation that was independent of MAP kinase activity and EGF-R-mediated signalling. Our data show that exposure of intestinal epithelial cells to low-dose formaldehyde is followed by rapid and profound signalling events. The data suggest a modifier role of environmental or endogenous formaldehyde for epithelial cell functions.     

Inhibition of cytokine-induced JAK-STAT signalling pathways by an endonuclease inhibitor aurintricarboxylic acid

1. Inducible nitric oxide (iNOS) is thought to involve in host defence and tissue damage in inflammatory loci. In previous study, we have found that the endonuclease inhibitor aurintricarboxylic acid (ATA) can protect macrophages from cell death induced by bacterial lipopolysaccharide. This action is through the interruption with signalling pathways for NF-kappa B and AP-1 activation, and thus iNOS expression. In this study we have addressed the effects of ATA on JAK-STAT signalling pathways. 2. In murine RAW 264.7 macrophages, IFN-gamma-mediated NO production and iNOS expression were concentration-dependently reduced by the presence of 3-100 micro M ATA. 3. IFN-gamma-induced STAT1 activation, as assessed from its tyrosine phosphorylation, nuclear translocation, binding to specific DNA response element and evoked IRF-1 reporter gene assay, were concomitantly inhibited by ATA. However, ATA did not alter IFN-gamma binding to RAW 264.7 cells. 4. The activities of JAK1 and JAK2, the upstream kinases essential for STAT1 signalling in response to IFN-gamma, were also reduced by ATA. 5. Moreover, IL-4, IL-10, GM-CSF and M-CSF elicited tyrosine phosphorylation of STAT3, STAT5 and/or STAT6 in macrophages were diminished by the presence of ATA. 6. Taken together, we conclude that ATA can interfere JAK-STAT signalling pathways in response to cytokines. This action contributes to the inhibition of IFN-gamma-induced iNOS expression.     

Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide

1.Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-gamma (IFN-gamma). 2. Inhibition of NOS activity by concomitant N(G)-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. 3. Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. 4. Rapid IFN-gamma-induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and confirmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-gamma for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. 5. We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-gamma pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. 6. We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-gamma, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.