A Kunitz-type protease inhibitor,bikunin, inhibits ovarian cancer cell invasion by blocking the calcium-dependent transforming growth factor-beta 1 signaling cascade

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.     

A resorcylic acid lactone, 5Z-7-oxozeaenol,prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase

TAK1, a member of the mitogen-activated kinase kinase kinase (MAPKKK) family, participates in proinflammatory cellular signaling pathways by activating JNK/p38 MAPKs and NF-kappaB. To identify drugs that prevent inflammation, we screened inhibitors of TAK1 catalytic activity. We identified a natural resorcylic lactone of fungal origin, 5Z-7-oxozeaenol, as a highly potent inhibitor of TAK1. This compound did not effectively inhibit the catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that 5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell culture, 5Z-7-oxozeaenol blocked interleukin-1-induced activation of TAK1, JNK/p38 MAPK, IkappaB kinases, and NF-kappaB, resulting in inhibition of cyclooxgenase-2 production. Furthermore, in vivo 5Z-7-oxozeaenol was able to inhibit picryl chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.     

A dominant negative TAK1 inhibits cellular fibrotic responses induced by TGF-beta

Transforming growth factor-beta (TGF-beta) is crucially virulent in the progression of fibrotic disorders. TAK1 (TGF-beta activated kinase 1) is one of the mitogen-activated kinase kinase kinase (MAPKKK) that is involved in TGF-beta signal transduction. To elucidate the importance of TAK1 in TGF-beta-induced fibrotic marker expression, we investigated whether dominant negative TAK1 could suppress TGF-beta signaling. Based on the finding that TAB1 (TAK1 binding protein 1) binding to TAK1 is required for TAK1 activation, a minimal portion of TAK1 lacking kinase activity that binds to TAB1 was designed as a TAK1 dominant negative inhibitor (TAK1-DN). The effect of TAK1-DN was assessed in the cells that respond to TGF-beta stimulation and that lead to the increase in production of extracellular matrix (ECM) proteins. TAK1-DN, indeed, decreased the ECM protein production, indicating that TAK1-DN retains the ability to intercept the TGF-beta signaling effectively.     

CrkL directs ASAP1 to peripheral focal adhesions

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.     

Sildenafil-nitric oxide donor combination promotes ventricular tachyarrhythmias in the swine right ventricle

We tested the hypothesis that sildenafil, singly or in combination with nitric oxide (NO) donors, promotes ventricular tachycardia (VT) and ventricular fibrillation (VF). Vulnerability to VT/VF was tested by rapid pacing in eight isolated normal swine right ventricles (RV). The endocardial activation was optically mapped, and the dynamic action potential duration (APD) restitution curves were constructed with metal microelectrodes. At baseline, no VT/VF could be induced. Sildenafil (0.2 microg/ml) or NO donor singly or in combination did not alter VT/VF vulnerability. However, when 2 microg/ml sildenafil was combined with NO donors, the incidence of VT and VF rose significantly (P < 0.01). VT with a single periodic wavefront was induced in five of eight RVs, and VF with multiple wavefronts was induced in all eight RVs. The sildenafil-NO donor pro-VT/VF combination significantly increased the maximum slope of the APD restitution curve and the amplitude of the APD alternans. The pro-VT/VF effects of sildenafil were reversible after drug-free Tyrode solution perfusion. We conclude that a sildenafil (2 microg/ml) and NO donor combination increases VT/VF vulnerability in the normal RV by a mechanism compatible with the restitution hypothesis.     

Mitochondrial swelling and cytochrome c release: sensitivity to cyclosporin A and calcium

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca(2+)-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca(2+)-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca(2+)-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T3. Fe2+/ADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca(2+)-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.     

Mevastatin,an inhibitor of HMG-CoA reductase,induces apoptosis,differentiation and Rap1 expression in HL-60 cells

It has been reported that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppress cell proliferation and induce apoptosis. One inhibitor which induces apoptosis is mevastatin. However, the molecular mechanism of apoptosis induction is not well understood so the effects of mevastatin on various functions of HL-60 cells were investigated. We confirmed that mevastatin activated caspase-3 by release of cytochrome c (Cyt. c) from mitochondria through a membrane permeability transition mechanism and also induced typical fragmentation and ladder formation of DNA in HL-60 cells. These effects were inhibited by mevalonate, a metabolic intermediate of cholesterol biosynthesis. Mevalonate and geranylgeraniol (GGOH) inhibited DNA fragmentation whereas farnesol (FOH) did not. Mevastatin also induced cell differentiation to monocytic cells via a mevalonate inhibitable mechanism. Furthermore, mevastatin decreased the amount of an isoprenylated membrane bound Rap1 small GTPase concomitant with an increase in cytosolic Rap1 which occurred before apoptosis and differentiation. On the contrary, both mevastatin and geranylgeranylacetone (GGA), which competes with geranylgeranyl pyrophosphate, induced membrane depolarization of isolated mitochondria without swelling and Cyt. c release. These results suggest that mevastatin-induced apoptosis of HL-60 cells might be caused indirectly by activation of the caspase cascade through the modulation of mitochondrial functions and that some relationship between a certain small GTPase molecule, such as Rap1, and mevastatin-induced apoptosis may exist.     

Identification of the capsular polysaccharides of Type D and F Pasteurella multocida as unmodified heparin and chondroitin,respectively

Pasteurella multocida is a pathogenic Gram-negative bacterial species that infects a wide variety of animals and humans. A notable morphological feature of many isolates is the extracellular capsule. The ability to remove the capsule by treatment with certain glycosidases has been utilized to discern various capsular types called A, D and F. Based on this preliminary evidence, these microbes have capsules made of glycosaminoglycans, linear polysaccharides composed of repeating disaccharide units containing an amino sugar. Glycosaminoglycans are also abundant components of the vertebrate extracellular matrix. It has been shown previously that the major Type A capsular material was hyaluronan (hyaluronic acid). We report that the Type D polymer is an unmodified heparin (N-acetylheparosan) with a -->4)-beta-D-Glcp-UA-(1-->4)-alpha-D-Glcp-NAc-(1--> repeating unit and the Type F polymer is an unmodified chondroitin with a -->4)-beta-D-Glcp-UA-(1-->3)-beta-D-Galp-NAc-(1--> repeating unit. The monosaccharide compositions, disaccharide profiles, and 1H NMR analyses are consistent with these identifications. The molecular size of the Pasteurella polymers is approximately 100-300 kDa as determined by gel electrophoresis and multi-angle laser light scattering; this size is much greater than the 10-30 kDa size of the analogous polymers isolated from animal tissues. The glycosaminoglycan capsular polymers are relatively non-immunogenic virulence factors that enhance microbial pathogenicity.     

Structural studies on kappa-carrageenan derived oligosaccharides

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum carrageenan. Three oligosaccharides were purified by strong-anion exchange high-performance chromatography. Their structure was elucidated using mass spectral and NMR data. Negative-ion electrospray ionization (ESI) mass spectra at different fragmentor voltages provided the molecular weight of the compounds and unraveled the fragmentation pattern of the kappa-carrageenan oligosaccharides. 2D NMR techniques, including 1H-(1)H COSY, 1H-(1)H TOCSY and 13C-(1)H HMQC, were performed to determine the structure of a trisulfated pentasaccharide. 1D NMR and ESIMS were used to determine the structures of a kappa-carrageenan-derived pentasaccharide, heptasaccharide, and an undecasaccharide. All the oligosaccharides characterized have a 4-O-sulfo-D-galactopyranose residue at both the reducing and nonreducing ends.