Treatment with anti-NAP monoclonal antibody reduces disease severity in murine model of novel angiogenic protein-induced or ovalbumin-induced arthritis.

Rheumatoid arthritis (RA) is a polyarticular inflammatory, angiogenic disease. Synovial angiogenesis contributes to inflammation in RA. In this study we have developed an arthritic model in rats using a novel angiogenic protein (NAP), isolated from human synovial fluid of RA patients. We produced anti-NAP monoclonal antibodies (mAbs) and investigated the therapeutic efficacy of the same in adjuvant-induced or NAP-induced arthritis as a model of human RA. The treatment of arthritic rats with anti-NAP mAbs resulted in effective amelioration of paw oedema, radiological arthritic characteristics, serum levels of vascular endothelial growth factor (VEGF) and NAP, compared to that of untreated arthritic animals. Further, profiling of angiogenic markers such as synovial microvessel density, angiogenesis, CD31, VEGF and fms-like tyrosine kinase (Flt1) by immunohistochemistry both in arthritic and anti-NAP mAb-treated animals revealed the efficacy of mAb as an anti-angiogenic functional antibody. Therefore, NAP may be an attractive target to design anti-angiogenic and anti-arthritic therapies to control the pathogenesis of arthritis.

Inhibition of metastasis-associated gene 1 expression affects proliferation and osteogenic differentiation of immortalized human mesenchymal stem cells.

OBJECTIVES: MTA1 is known to be responsible for independent nucleosome remodelling and deacetylase complexes with ability to regulate divergent cellular pathways. However, additional biological functions have, up to now, remained largely unexplored. The present study was initiated to investigate involvement of MTA1 in osteogenic differentiation of immortalized human mesenchymal stem cells (MSCs). MATERIALS AND METHODS: MSCs were examined for expression of MTA1 and stably transfected clones expressing shRNA to MTA1 were generated. Cells were grown under osteogenic and non-osteogenic conditions. Effects of silencing on cell proliferation, calcium deposition and alkaline phosphatase (ALP) activity were studied. mRNA expression of bone sialoprotein (BSP), osteopontin (OSP), runt-related transcription factor 2 (Runx2), osteocalcin (OC), collagen type I (Col1A) and ALP were analysed. RESULTS: Transfected cells showed reduction in proliferation and significant increase in calcium deposition and expression of osteogenic marker genes, BSP, OSP, Runx2, OC and Col1A, when they were grown under osteogenic conditions. Under non-osteogenic conditions, expression of BSP and OSP were also markedly upregulated, whereas expression of osteogenic marker genes, Runx2, OC and Col1A, was almost unaffected. Expression of ALP was slightly suppressed under non-osteogenic conditions but significantly increased under osteogenic differentiation conditions, as assessed by enzyme activity and mRNA expression assays. CONCLUSIONS: Our data collectively suggest that endogenously produced MTA1 constrains osteogenic differentiation of MSCs and that targeting of this molecule may provide a novel strategy for enhancing bone regeneration.

Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent.

We previously reported a vascular endothelial growth factor (VEGF) autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach, we screened the HNSCC cell line, squamous cell carcinoma-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation 1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion and matrix metalloproteinase (MMP) expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix-coated coverslips showed enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared with a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also show for the first time that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1's recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.

Butyrate-induced phosphatase regulates VEGF and angiogenesis via Sp1.

Sp1 is a ubiquitous transcription factor and master regulator of various eukaryotic gene expression. Better understanding of the role of increased Sp1 levels on angiogenic regulation and the regulatory regions of that transcription factor may act as a useful target in 'transcriptional therapy'. At the molecular level, butyrate inhibits Sp1-DNA binding activity by promoting Sp1 protein dephosphorylation in EAT cells. It also inhibits Sp1 binding activity and reduces expression of VEGF gene, thereby inhibiting angiogenesis. It was confirmed that butyrate induces expression of a tyrosine phosphatase by RT-PCR, cDNA sequence analysis, protein ESI-MS analysis and protein sequence homology comparison. Thus our result strongly suggests that inhibition of angiogenesis by butyrate involves Sp1 dephosphorylation and down-regulation of VEGF gene expression. Further, butyrate inhibits neoangiogenesis induced by tumor cells and VEGF in peritoneum of EAT bearing mice and rat cornea.

Inhibition of in vivo angiogenesis by Anacardium occidentale L. involves repression of the cytokine VEGF gene expression.

Lethal tumor growth and progression cannot occur without angiogenesis, which facilitates cancer cell proliferation, survival, and dissemination. Among the many growth factors and cytokines engaged in angiogenesis, the cytokine vascular endothelial growth factor (VEGF) is regarded as the most potent and specific. Angiogenesis inhibitors are recognized as potentially useful agents for treating angiogenesis-associated diseases and VEGF represents a promising and well-studied target for antiangiogenic agents. In this study, we have tested the crude ethanolic extract of the leaves of Anacardium occidentale Linn, on Ehrlich ascites tumor cells (EAT) in vivo and in vitro. Anacardium occidentale extract (AOE) was able to suppress VEGF-induced angiogenesis in vivo in the chorioallantoic membrane, rat cornea, and tumorinduced angiogenesis in the peritoneum of EAT bearing mice. The extract inhibited cell proliferation of different tumor cells such as EAT, BeWo, and MCF-7 in vitro in a dose-dependent manner and it reduced the VEGF level in the ascites of treated mice. A decrease in the microvessel density count and CD31 antigen staining of treated mice peritoneum provide further evidence of its antiangiogenic activity. Our results from Northern blot analysis and ELISA demonstrate that AOE can downregulate endogenous VEGF gene expression at the mRNA and protein level. Furthermore, results of our gene analysis of VEGF-promoter luciferase reporter indicated that this effect is mediated by transcriptional repression of VEGF promoter activity in EAT cells treated with AOE. Taken together, the data suggest that the VEGF system of angiogenesis is the molecular target for the antiangiogenic action of AOE.

Induction of caspase-3 activated DNase mediated apoptosis by hexane fraction of Tinospora cordifolia in EAT cells.

Tinospora cordifolia (Guduchi) has been used for centuries for treating various ailments including cancer in Ayurvedic system of medicine. In this study, we report the mechanism of cell death exhibited by the hexane extract fraction of T. cordifolia (TcHf) against Ehrlich ascites tumor (EAT) in mice. Treatment of EAT bearing animals with TcHf resulted in growth inhibition and induction of apoptosis in a dose-dependent manner. TcHf induced the formation of apoptotic bodies, nuclear condensation, typical DNA ladder, activation of caspase-3, decreased cell number and ascites volume. We examined TcHf for its effect on proliferation and cell cycle progression in EAT cells. The results showed that TcHf inhibited the proliferation of EAT cells by blocking cell cycle progression in the G1 phase. In Western blot analysis, apoptosis in the EAT cells was associated with the constitutive expression of caspase activated DNase (CAD) in both nucleus and cytoplasm after TcHf treatment. Further more the expression of pro-apoptotic gene, Bax, was increased and the expression of anti-apoptotic gene, Bcl-2, was decreased in a time dependent manner by TcHf treatment. All results indicate that the hexane fraction of T. cordifolia is capable of inducing apoptosis in EAT cells in vivo.

Targeted brain tumor treatment-current perspectives.

Brain tumor is associated with poor prognosis. The treatment option is severely limited for a patient with brain tumor, despite great advances in understanding the etiology and molecular biology of brain tumors that have lead to breakthroughs in developing pharmaceutical strategies, and ongoing NCI/Pharma-sponsored clinical trials. We reviewed the literature on molecular targeted agents in preclinical and clinical studies in brain tumor for the past decade, and observed that the molecular targeting in brain tumors is complex. This is because no single gene or protein can be affected by single molecular agent, requiring the use of combination molecular therapy with cytotoxic agents. In this review, we briefly discuss the potential molecular targets, and the challenges of targeted brain tumor treatment. For example, glial tumors are associated with over-expression of calcium-dependent potassium (K(Ca)) channels, and high grade glioma express specific K(Ca) channel gene (gBK) splice variants, and mutant epidermal growth factor receptors (EGFRvIII). These specific genes are promising targets for molecular targeted treatment in brain tumors. In addition, drugs like Avastin and Gleevec target the molecular targets such as vascular endothelial cell growth factor receptor, platelet-derived growth factor receptors, and BRC-ABL/Akt. Recent discovery of non-coding RNA, specifically microRNAs could be used as potential targeted drugs. Finally, we discuss the role of anti-cancer drug delivery to brain tumors by breaching the blood-brain tumor barrier. This non-invasive strategy is particularly useful as novel molecules and humanized monoclonal antibodies that target receptor tyrosine kinase receptors are rapidly being developed.

Dexamethasone induced expression of phosphatase inhibits generation of reactive oxygen species in Ehrlich ascites tumor cells.

Both pre-activated and phorbol ester tetradecanoyl phorbol myristate acetate (TPA) activated reactive oxygen species (ROS) generation were inhibited by dexamethasone in vivo. Time kinetics on influence of dexamethasone on cytosolic phosphoprotein phosphatase activity revealed that, when compared to phosphatase activity in cytosol of control Ehrlich ascites tumor (EAT) cells, a 5-fold increase in specific activity is seen in the cytosol of EAT cells treated (in vivo, 0-90 min. 1 mg/kg body weight) with dexamethasone. Dexamethasone induced phosphatase was partially purified by conventional ion-exchange and gel filtration column chromatographic techniques. Purified phosphatase had a molecular weight of 70 KDa by SDS-PAGE. A dose-dependent inhibition of TPA activated ROS generation by partially purified phosphatase in permeabilized EAT cells suggested that dephosphorylation is a major regulatory mechanism in "switching off" of the respiratory burst. Anti-phosphatase antibodies were raised, purified and were used to quantitate cytosolic phosphatase by ELISA, which revealed that dexamethasone induces 6-fold increase in expression of phosphatase in EAT cells by 120 min. The expression of phosphatase in EAT cell cytosol was further confirmed by immunostaining using anti-phosphatase antibodies, the results of which showed intense blue staining on development with BCIP/NBT.

Cross-talk between protein kinase C and protein kinase A down-regulates the respiratory burst in polymorphonuclear leukocytes.

In this paper it has been shown that increase in intracellular cAMP by epinephrine or its analogue dibutyryl cAMP (Bt2cAMP) abolishes in a dose-dependent manner the protein kinase C (PKC)-mediated respiratory burst in polymorphonuclear leukocytes. The mechanism of inhibition has been shown to involve induction of cytosolic phosphoprotein phosphatase activity specific to cells receiving dual signals (PKC, PKA), as minimum respiratory burst was associated with cells with maximum phosphatase activity. Inclusion of specific PKA inhibitor completely restricted the development of dual signal-induced phosphatase activity in vitro, demonstrating the requirement of multisite phosphorylation of the phosphatase for the development of its activity. Purified phosphatase had a molecular weight of 78,000 and could exert its inhibitory effect on PKC-triggered respiratory burst in permeabilized cells, clearly showing that down-regulation of oxidase activity involved dephosphorylation by the phosphatase. Interaction of the purified phosphatase with eight-fold purified NADPH oxidase as revealed by fluorescence studies further confirmed the role of the phosphatase in the respiratory burst event. Taken together, we have been able to establish that cross-talk between protein kinase C and protein kinase A is essential to 'turn off' generation of reactive oxygen species.

A novel role for calcium and membrane-binding proteins in regulation of respiratory burst in Ehrlich ascites tumour cells.

The effect of the signals, dexamethasone and phorbol-ester (TPA) and their interplay on the respiratory burst in Ehrlich ascites tumour (EAT) cells and the effect of this interplay has on the phosphorylation state and thus function of EAT cell cytosolic calcium and membrane-binding proteins (CaMBPs) was studied. Dexamethasone (1 mg/kg body weight) inhibited basal and TPA-activated EAT cell respiratory burst. CaMBPs isolated from EAT cells under the influence of either or both signals by a membrane-affinity binding technique showed similar profiles on SDS-PAGE. Autoradiography showed that only an 11,000 M(r) protein is subject to phosphorylation and that dexamethasone brings down the extent of phosphorylation of CaMBPs isolated from control and TPA-treated EAT cells. Phosphorylated CaMBPs from control and TPA-treated cells could activate the respiratory burst in Triton-permeabilized EAT cells in a dose-dependent manner (four- and six-fold activations, respectively, at 16 micrograms protein concentration) when included in the assay, whereas dephosphorylated CaMBPs from EAT cells from mice treated with dexamethasone alone, or later activated with TPA, could not. A phosphatase activity was detected in EAT cell cytosol receiving the dual-signal, dexamethasone and TPA.

Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells.

The superoxide anion generation in Ehrlich ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt2 cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt2 cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt2 cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells.

Capsaicin inhibits calmodulin-mediated oxidative burst in rat macrophages.

In this study we seek to elucidate the interaction of capsaicin with the calmodulin mediated signal pathways in macrophages, by comparing its action on macrophage functions with a known calmodulin antagonist, fluphenazine. Kinetics of capsaicin uptake by macrophages (10(3) cells) revealed that a maximum of 200 microM capsaicin was taken up within 10 min. Ca2+ ionophore triggered generation of superoxide anion and hydrogen peroxide by macrophages was inhibited in a dose-dependent manner by fluphenazine (IC50, 20 microM and 12 microM, respectively) and also by capsaicin (IC50, 30 microM and 9 microM, respectively), suggesting an involvement of calmodulin in the regulation of NADPH oxidase. In vitro both fluphenazine and capsaicin inhibited Ca2(+)-Mg2+ ATPase and cAMP-phosphodiesterase from macrophages and this inhibition was reversed by exogenous addition of calmodulin. Fluorescence studies revealed a direct Ca2+ dependent interaction of capsaicin with calmodulin. From these results we suggest that capsaicin acts via calmodulin to inhibit stimulus-induced macrophage oxidative burst and also that calmodulin regulates the oxidative burst in macrophages.

Inhibition of calcium and calmodulin-dependent phosphodiesterase activity in rats by capsaicin.

Capsaicin, reported to elevate hormone sensitive lipase (HSL), is also found to inhibit the Ca++ and calmodulin-dependent cAMP phosphodiesterase (PDE) activity in adipose tissue of rats, fed high fat diet. The dependence of the enzyme activity on Ca++ and calmodulin in vitro, in control rats, is shown by its substantial lowering in the presence of EGTA and inhibition by trifluoperazine (TFP) (IC50 between 10-20 microM). This enzyme activity is also inhibited by both red pepper extract (80% inhibition with 50 microliter) and capsaicin (IC50 between 0.3-1 microM) in a dose dependent manner. Capsaicin has been found to inhibit Ca++-dependent PDE activity by 60% in the test rats. Enzyme inhibition in vivo, due to capsaicin, was overcome by addition of calmodulin to the assay system. Inclusion of fluphenazine or capsaicin in assay inhibited not only the calmodulin-restored enzyme activity from test rats but also that of control rats. These results suggest a possible mechanism for the stimulation of lipolytic activity by capsaicin in vivo.

Microsomal CA2+ uptake in human platelets is stimulated by calmodulin.

Calcium accumulating vesicles were prepared from washed human platelets. Ca2+ uptake could be stimulated with calmodulin. The stimulation of Ca2+ uptake was not abolished by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, thus excluding enhanced cAMP formation as a possible cause for the calmodulin effect. Our findings suggest that, in addition to cAMP, calmodulin is also involved in the regulation of platelet free Ca2+ concentrations, i.e. Ca2+ homeostasis in platelets is under dual control.

Isolation and characterization of CAB-63, a novel calcium-binding protein.

A novel calcium-binding protein named CAB-63 (formerly called calregulin) has been purified from bovine liver 100,000 X g supernatant. The purified protein has been characterized with respect to its physical, chemical, and calcium-binding properties. It has an apparent molecular weight of 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 55,000 by sedimentation equilibrium centrifugation under nondenaturing conditions. It is an asymmetric molecule with a frictional coefficient of 1.69 and a Stokes radium of 44.2 A. Amino acid analysis has revealed 34.0% acidic residues, 14.0% basic residues, and 4.0% tryptophan. The acidic nature of the molecule is further confirmed by its isoelectric point of 4.65. In the presence of 3 mM MgCl2 and 150 mM KCl, CAB-63 binds 3.0 mol of calcium/mol of protein with an apparent Kd = 0.1 microM. Immunoblotting and Ouchterlony double-diffusion procedures have identified CAB-63 in a variety of bovine tissues. Immunocytochemical staining of both fibroblasts and cryotome-sectioned bovine liver further indicates that CAB-63 immunoreactivity is restricted to an elaborate system of perinuclear membranous vacuoles and cisternae indistinguishable from immunocytochemical staining of the endoplasmic reticulum. It is concluded that CAB-63 represents a major calcium-binding protein whose subcellular organization suggests a possible role in the function of the endoplasmic reticulum.

Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls.

Protein-kinase activity has been found to be associated with a membrane fraction obtained from dark-grown zucchini (Cucurbita pepo L., cv. Senator) hypocotyl hooks. Proteins of this membrane fraction were used as protein substrates. The effects of Mg(2+), Na(+) and K(+) on phosphorylation, measured as (32)P incorporation, was investigated. The kinetics of phosphorylation of the individual protein peptides indicate the presence of specific phosphatase activity. Phosphorylation activity is strongly influenced by Ca(2+). One peptide (relative molecular weight: 180,000) exhibits strong inhibition of (32)P incorporation at physiological Ca(2+) concentrations between 0.1 and 1 µM. Phosphorylation of about 10 other proteins was enhanced by Ca(2+), being maximal in most cases at a concentration of about 3 µM free Ca(2+). Five out of these 10 peptides show increased phosphorylation in the presence of 1 µM calmodulin. This calmodulin-dependent enhancement of phosphorylation could be completely inhibited by the calmodulin antagonist fluphenazine. Cyclic AMP was found to have no stimulating effect on protein phosphorylation.