Native and recombinant phospholipases A2 of Scorpio maurus venom glands impair angiogenesis by targeting integrins α5ß1 and αvß3.

We recently purified an heterodimeric phospholipase A2 named Sm-PLGV from the venom glands of scorpion Scorpio maurus containing a Long chain, a penta-peptide insertion, which is cut out during the maturation, followed by a short chain. Three recombinant forms of Sm-PLGV were produced in Escherichia coli: rPLA2(+5) containing the full-length sequence including the penta-peptide insert, rPLA2(-5) a fused continuous chain of the Long and the short chains without the penta-peptide and the Long chain alone without the short one. In this study, we showed that Sm-PLGV, rPLA2(+5) and rPLA2(-5) displayed more potent anti-angiogenic properties than the recombinant Long chain and the short chain obtained by chemical synthesis. These phospholipases A2 inhibited in a dose-dependent manner adhesion, migration and invasion of human microvascular endothelial cells through the alteration of α5ß1 and αvß3 integrins function. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that Sm-PLGV, rPLA2(+5) and rPLA2(-5) significantly inhibited both in vitro and in vivo angiogenesis. We also showed a clear dissociation of the anti-angiogenic effect of Sm-PLGV and its catalytic activity. This is the first study describing an anti-angiogenic effect for recombinant scorpion venom enzymes.

Targetting αvß3 and α5ß1 integrins with Ecballium elaterium (L.) A. Rich. seed oil.

In the present study, the effect of Ecbalium elaterium seed oil on adhesion, migration and proliferation of human brain cancer cell line (U87) was determined. Treatment of U87 cell line with the seed oil resulted in strong inhibition of their adhesion to fibrinogen (Fg), fibronectin (Fn). It also reduced their migration and proliferation in a dose-dependent manner without being cytotoxic. Concomitantly, by using Matrigel™ assays, the oil significantly inhibited angiogenesis. The anti- tumor effect of the oil is specifically mediated by αvß3 and α5ß1 integrins. The presence of integrin antagonists in seed oil from E. elaterium could be used for the development of anticancer drugs with targeted "multi-modal" therapies combining anti-adhesif, antiproliferative, antimetastasic and anti-angiogenic, approaches.

Modified Panax ginseng Extract Inhibits uPAR-Mediated α[Formula: see text]ß1-Integrin Signaling by Modulating Caveolin-1 to Induce Early Apoptosis in Lung Cancer Cells.

Urokinase receptor (uPAR) is enhanced in many human cancer cells and is frequently an indicator of poor prognosis. Activation of [Formula: see text]1-integrin requires caveolin-1 and is regulated by uPAR. However, the underlying molecular mechanism responsible for the interaction between uPAR and [Formula: see text]1-integrin remains obscure. We found that modified regular Panax ginseng extract (MRGX) had a negative modulating effect on the uPAR/[Formula: see text]1-integrin interaction, disrupted the uPAR/integrin interaction by modulating caveoline-1, and caused early apoptosis in cancer cells. Additionally, we found that siRNA-mediated caveoline-1 downregulation inhibited uPAR-mediated [Formula: see text]1-integrin signaling, whereas caveoline-1 up-regulation stimulated the signaling, which suppressed p53 expression, thereby indicating negative crosstalk exists between the integrin [Formula: see text]1 and the p53 pathways. Thus, these findings identify a novel mechanism whereby the inhibition of [Formula: see text]1 integrin and the activation of p53 modulate the expression of the anti-apoptotic proteins that are crucially involved in inducing apoptosis in A549 lung cancer cells. Furthermore, MRGX causes changes in the expressions of members of the Bcl-2 family (Bax and Bcl-2) in a pro-apoptotic manner. In addition, MGRX-mediated inhibition of [Formula: see text]1 integrin attenuates ERK phosphorylation (p-ERK), which up-regulates caspase-8 and Bax. Therefore, ERK may affect mitochondria through a negative regulation of caspase-8 and Bax. Taken together, these findings reveal that MRGX is involved in uPAR-[Formula: see text]1-integrin signaling by modulating caveolin-1 signaling to induce early apoptosis in A549 lung-cancer cells and strongly indicate that MRGX might be useful as a herbal medicine and may lead to the development of new herbal medicine that would suppress the growth of lung-cancer cells.

Pectin of Prunus domestica L. alters sulfated structure of cell-surface heparan sulfate in differentiated Caco-2 cells through stimulation of heparan sulfate 6-O-endosulfatase-2.

Although previous reports have suggested that pectin induces morphological changes of the small intestine in vivo, the molecular mechanisms have not been elucidated. As heparan sulfate plays important roles in development of the small intestine, to verify the involvement of heparan sulfate (HS) in the pectin-induced morphological changes of the small intestine, the effects of pectin from Prunus domestica L. on cell-surface HS were investigated using differentiated Caco-2 cells. Disaccharide compositional analysis revealed that sulfated structures of HS were markedly changed by pectin administration. Real-time RT-PCR showed that pectin upregulated human HS 6-O-endosulfatase-2 (HSulf-2) expression and markedly inhibited HSulf-1 expression. Furthermore, inhibition analysis suggested that pretreatment with fibronectin III1C fragment, RGD peptide, and ERK1/2 inhibitor suppressed pectin-induced HSulf-2 expression. These observations indicate that pectin induced the expression of HSulf-2 through the interaction with fibronectin, α5ß1 integrin, and ERK1/2, thereby regulating the sulfated structure of HS on differentiated Caco-2 cells.

Effect of chondroitinase ABC on adhesion and behavior of synovial membrane-derived mesenchymal stem cells in rabbit partial-thickness chondral defects.

Transplanted cells may have difficulty attaching to the surface of partial-thickness chondral lesions because of the anti-adhesive properties of the proteoglycan rich matrix. Therefore, the current study attempts to evaluate the effect of chondroitinase ABC (chABC) on the adhesion and behavior of transplanted synovial membrane-derived mesenchymal stem cells (SDSCs) in rabbit partial-thickness chondral defects. In ex vivo adhesion experiments, chABC treatment (0.1 U/ml) was increased in SDSC attachment to the cartilage explants, and significantly diminished by pretreatment with neutralizing antibody against fibronectin. In the in vivo experiments, 1 day and 4 weeks after the chABC treatment (0.1 and 1 U/ml), the immunoreactivity (IR) against CS-56 (intact chondroitin sulfate antibody) was markedly decreased; however, the IR of 2B6 (stub of the chondroitin 4-sulfate chain), 3B3 (stub of the chondroitin 6-sulfate chain), and fibronectin was increased. At 12 weeks, this IR returned to normal except in the high-dose chABC-treated group (1 U/ml). Furthermore, the attachment of SDSCs to the chondral defects after chABC treatment was increased at 7 days compared with that in the chondral defects pretreated with saline. However, the tissue repaired by SDSCs was negatively stained for type II collagen at 12 weeks. In conclusion, these results showed that the exposure to fibronectin by chABC treatment enhances the attachment of SDSCs to partial-thickness chondral defects. However, the tissue regenerated by SDSCs showed lack of hyaline cartilage regeneration. Thus, to understand the fate of transplanted MSCs in cartilage defect is very important for successful cell therapies.

Zinc inhibits magnesium-dependent migration of human breast cancer MDA-MB-231 cells on fibronectin.

Metastasis is the major cause of breast cancer mortality. The strength of cell adhesion to extracellular matrix is critical to cancer cell migration. Integrins, the primary mediators of cell to extra-cellular matrix adhesion, contain distinct divalent cation-binding sites. Binding of manganese and magnesium is vital to integrin-mediated cancer cell adhesion and migration. We hypothesized that zinc, a divalent cation, can modulate breast cancer metastasis through interfering with these divalent cation-dependent integrin-mediated cancer cell adhesion and migration. MDA-MB-231 cells were cultured in a zinc-depleted medium supplemented with 0 (control), 2.5, 5, 10, 25 and 50 µM of zinc to mimic severe zinc-deficiency, moderate zinc-deficiency, adequate zinc and three levels of zinc-supplementation: low-, moderate- and high-levels of zinc-supplementation, respectively. Zinc treatments had no effect on cellular zinc concentration, cell number and cell viability. Zinc at 5-50 µM reduced migration distance of MDA-MB-231 cells on fibronectin by 43-86% and migration rate on fibronectin by 72-90%. Zinc induced a dose-dependent inhibition of cell adhesion to fibronectin (R(2)=-0.98). Zinc at 10-50 µM reduced magnesium-facilitated cell adhesion to fibronectin in a dose-dependent manner (R(2)=-0.90). However, zinc had no effect on manganese-facilitated cell adhesion to fibronectin. Zinc at 5-50 µM caused rounding of the normally elongated, irregular-shaped MDA-MB-231 cells and disappearance of F-actin. Anti-integrin α5- and ß1-subunit blocking antibodies inhibited magnesium-facilitated cell adhesion to fibronectin by 95 and 99%, respectively. In summary, zinc inhibited MDA-MB-231 cell migration on fibronectin by interfering with magnesium-dependent integrin-, likely integrin α5/ß1-, mediated adhesion.

Mediating effects of aryl-hydrocarbon receptor and RhoA in altering brain vascular integrity: the therapeutic potential of statins.

We have demonstrated previously that focal adhesion kinase (FAK)/RhoA alteration by the aryl-hydrocarbon receptor (AhR) agonist 3-methylcholanthrene (3MC) is involved in the antimigratory effects of 3MC in human umbilical vascular endothelial cells. Here, we identified that signaling properties and molecular mechanisms of RhoA/ß-catenin were both implicated in alterations to blood-brain barrier integrity. The mechanisms of action were the down-regulation of integrin, the extracellular matrix, and adherens junction stability. PTEN phosphorylation by 3MC-mediated AhR/RhoA activation increased the proteasomal degradation of ß-catenin through PKCδ/pGSK3ß-mediated ß-catenin phosphorylation; the crucial roles of AhR/RhoA in this process were verified by using gain- or loss-of-function experiments. The decrease in ß-catenin led to decreased expression of fibronectin and α5ß1 integrin. Additionally, protein interactions among FAK, VE-cadherin, vinculin, and ß-actin were simultaneously decreased, resulting in adherens junction instability. Novel functional TCF/LEF1 binding sites in the promoter regions of fibronectin and α5/ß1 integrin were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results indicate that the binding activities of ß-catenin decreased in mouse cerebrovascular endothelial cells treated with 3MC. In addition, simvastatin and pravastatin treatment reversed 3MC-mediated alterations in mouse cerebrovascular endothelial cells by RhoA inactivation, and the in vitro findings were substantiated by an in vivo blood-brain barrier assay. Thus, endothelial barrier dysfunction due to 3MC occurs through AhR/RhoA-mediated ß-catenin down-regulation, which is reversed by simvastatin treatment in vivo.

Caffeoylserotonin suppresses THP-1 monocyte adhesion and migration via inhibition of the integrin ß1/FAK/Akt signalling pathway.

We investigated the effect of caffeoylserotonin (CaS) on THP-1 monocyte migration and adhesion to fibronectin in response to MCP-1. CaS decreased monocyte adhesion and migration induced by MCP-1, together with CCR2 expression and α5ß1 integrin, and activated ß1 integrin expression on the cell surface. CaS also inhibited FAK and Akt phosphorylation. We found that CaS had anti-inflammatory activity based on inhibition of adhesion and migration via inhibition of the integrin ß1/FAK/Akt signalling pathway. Thus, the inhibitory effects of CaS on monocyte function may support the future development of this compound as a potential treatment for inflammation-dependent diseases.

Ginsenoside Rh1 inhibits the invasion and migration of THP-1 acute monocytic leukemia cells via inactivation of the MAPK signaling pathway.

Ginsenoside Rh1 has been reported to possess antiallergic and anti-inflammatory activities, but its effects on monocytes remain to be determined. Herein, we investigated the effects of Rh1 on the expression of MCP-1 and CCR2, activation of MAPK signaling, and chemotaxis of monocytes. Treatment of Rh1 decreased the levels of MCP-1 and CCR2 and the expression of VLA5 and activated ß1 integrin on the cell surface, and attenuated the phosphorylation of MAPKs. Based on these results, the inhibitory effects of Rh1 on monocyte function should be regarded as a promising new anti-inflammatory response with a potential therapeutic role against inflammation-dependent diseases.

Effects of transcutaneous electrical stimulation (1 pulse/sec) through custom-made disposable surface electrodes covering Omura's ST36 area of both legs on normal cell telomeres, oncogen C-fosAb2, integrin alpha5beta1, chlamydia trachomatis, etc. in breast cancer & alzheimer patients.

Our previous study indicated that when extremely reduced normal cell (NC) telomeres in various cancer patients are increased over 500 ng BDORT units, abnormally high cancer cell telomeres and cancer-related markers such as Oncogen C-fosAb2 (Onco.)& Integrin alpha5beta1 (Integ.), & 8-OH-dG as well as bacterial & viral infections, mercury, asbestos, chromium, & beta-amyloid (1-42) markedly reduced due to improved circulation & excretion of these substances in urine. Since 1995, we have been using press-needle stimulation of Omura's ST36 with 200x press-release procedure 4x a day, with significant improvements in various cancer patients. In this study, Transcutaneous Electrical Stimulation (TES) at 60 pulses/min, which is close to patient's heart rate, was given between Omura's ST36 of both legs of the breast cancer & Alzheimer's patients. After about 10 minutes of TES, NC telomeres increased from 1 yg (= 10-24 g) to 500-525 ng; Integ. reduced from 85-75 ng to 0.5 ng & Chlamydia trachomatis (CT) reduced from 4500-3500 ng to 0.5 ng. An additional 10 minutes TES increased NC telomeres to 800-875 ng, while Integ. reduced to 0.5 yg & CT became less than 0.1 yg. After a total 30 minutes of TES, NC telomeres increased to 1000-1200ng BDORT units, with decreases in Integ. and Onco. to less than 0.1 yg. CT reduced to << 0.1 yg. About 24 hours later, NC telomeres were still 300 ng & both Integ. and Onco. were 2.5 ng. CT was approximately 20 ng. In Alzheimer patient, abnormally high beta-Amyloid (1-42) of 7-12 ng markedly reduced to within normal value of less than 1.5 ng by 20-30 min TES. Stimulation beyond 30 minutes gradually reduced NC telomeres. TES pulse rate of 4 pulses/sec for the same patient initially increased NC telomere up to 750-950 ng BDORT units within 20 minutes, but when stimulation continued more than 20 min, NC telomeres rapidly reduced to -150 ng in less than 10 min of TES with reduced beneficial effects.

Effects of Himatanthus lancifolius on human leukocyte chemotaxis and their adhesion to integrins.

The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.

Low-energy helium-neon laser induces locomotion of the immature melanoblasts and promotes melanogenesis of the more differentiated melanoblasts: recapitulation of vitiligo repigmentation in vitro.

Helium-neon laser (He-Ne Laser, 632.8 nm) is a low-energy laser that has therapeutic efficacy on various clinical conditions. Our previous study has demonstrated efficacy of He-Ne laser on vitiligo, a disease characterized by skin depigmentation. To regain skin tone on vitiligo lesions, the process began by the migration of the immature melanoblasts (MBs) to the epidermis, which was followed by their functional development to produce melanin. In this study, we investigated the physiologic effects of He-Ne laser irradiation on two MB cell lines: the immature NCCmelb4 and the more differentiated NCCmelan5. The intricate interactions between MBs with their innate extracelluar matrix, fibronectin, were also addressed. Our results showed that He-Ne laser irradiation enhanced NCCmelb4 mobility via enhanced phosphorylated focal adhesion kinase expression and promoted melanogenesis in NCCmelan5. In addition, He-Ne laser decreased the affinity between NCCmelb4 and fibronectin, whereas the attachment of NCCmelan5 to fibronectin increased. The alpha5beta1 integrin expression on NCCmelb4 cells was enhanced by He-Ne laser. In conclusion, we have demonstrated that He-Ne laser induced different physiologic changes on MBs at different maturation stages and recapitulated the early events during vitiligo repigmentation process brought upon by He-Ne laser in vitro.

Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells.

Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues.

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen.

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.

Analysis of IL-2, IFN-gamma and TNF-alpha production, alpha5 beta1 integrins and actin filaments distribution in peritoneal mouse macrophages treated with homeopathic medicament.

The newer forms of immune modulatory therapy are aimed at specific cells or cytokines that contribute to the immune response. These forms of immunotherapy have been referred to as 'biological response modifiers'. Our lab was interested in investigating if a homeopathic medicament 'Metodo Canova' (MC), sold in homeopathic drugstores, does enhance immunological system responses acting through macrophages pathway. Mice peritoneal macrophages were cultivated with or without homeopathic medicament for 24 h for alpha5, beta1 and actin filaments distribution analyses through immunolabelling for confocal microscopy. To detect the IL-2, IFN-gamma and TNF-alpha production these cells were cultivated for 48 h with or without medicament, followed by analyses of these cytokines in supernatant culture with ELISA kits. It was observed differences in morphology and molecular distribution (alpha5 and beta1 integrins, actin filaments and Fc receptors) between the groups control and treated with MC. In control group macrophages had the morphology of resident cells and in MC treated group macrophages were more spread, had many cellular projections and a substantial increase in cytoplasmic volume. In addition, macrophages culture with two doses of MC showed that TNF-alpha production decreased when compared with control group.