Comparative in vitro and in silico analysis of the ability of basic Asp49 phospholipase A2 and Lys49-phospholipase A2-like myotoxins from Bothrops diporus venom to inhibit the metastatic potential of murine mammary tumor cells and endothelial cell tubulogenesis: Asp49 vs Lys49 phospholipases A2: Inhibition of metastasis and angiogenesis.

Snake venoms are a complex mixture of proteins and polypeptides that represent a valuable source of potential molecular tools for understanding physiological processes for the development of new drugs. In this study two major PLA2s, named PLA2-I (Asp49) and PLA2-II (Lys49), isolated from the venom of Bothrops diporus from Northeastern Argentina, have shown cytotoxic effects on LM3 murine mammary tumor cells, with PLA2-II-like exhibiting a stronger effect compared to PLA2-I. At sub-cytotoxic levels, both PLA2s inhibited adhesion, migration, and invasion of these adenocarcinoma cells. Moreover, these toxins hindered tubulogenesis in endothelial cells, implicating a potential role in inhibiting tumor angiogenesis. All these inhibitory effects were more pronounced for the catalytically-inactive toxin. Additionally, in silico studies strongly suggest that this PLA2-II-like myotoxin could effectively block fibronectin binding to the integrin receptor, offering a dual advantage over PLA2-I in interacting with the αVß3 integrin. In conclusion, this study reports for the first time, integrating both in vitro and in silico approaches, a comparative analysis of the antimetastatic and antiangiogenic potential effects of two isoforms, an Asp49 PLA2-I and a Lys49 PLA2-II-like, both isolated from Bothrops diporus venom.

RGD peptide inhibits tumor growth by affecting angiogenesis related signaling pathway of laryngeal cancer stem cells / El péptido RGD inhibe el crecimiento tumoral al afectar la vía de señalización relacionada con la angiogénesis de las células madre del cáncer de laringe

SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.

Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.

The Effects of αvß3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro.

Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvß3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvß3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvß3 integrin during tumor progression.

Alphavbeta3 integrin blocking inhibits apoptosis and induces autophagy in murine breast tumor cells.

Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The αvß3 integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of αvß3 integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, αvß3 integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.

Highly Ordered Gold Nanopatterned Indium Tin Oxide Electrodes for Simultaneous Optical and Electrochemical Probing Cell Interactions.

The formation of new types of sensitive conductive surfaces for the detection and transduction of cell-extracellular matrix recognition events in a real time, label-free manner is of great interest in the field of biomedical research. To study molecularly defined cell functions, biologically inspired materials that mimic the nanoscale order of extracellular matrix protein fibers and yield suitable electrical charge transfer characteristics are highly desired. Our strategy to achieve this goal is based on the spatial self-organization of patches of cell-adhesive molecules onto a gold-nanoparticle-patterned indium tin oxide electrode. Fibroblast adhesion response to selective ligands for integrins α5ß1 and αvß3, which are both relevant in cancer progression, is investigated by simultaneous electrochemical impedance spectroscopy and optical microscopy. Adhesive cells on α5ß1-selective nanopatterns showed enhanced membrane dynamics and tighter binding, compared with cells on αvß3-selective nanopatterns. The surface of the electrode exhibits high sensitivity to small changes in surface properties, because of the constitution of specific cell-surface interactions. Moreover, such sensitivity enables differentiation between cell types. This is exemplified by analyzing distinct features in the electrochemical readout of MCF-7 breast cancer cells versus MCF-10A mammary epithelial cells, when subjected to individual adhesive nanopatches.

Fibrin-mediated integrin signaling plays a critical role in hepatic regeneration after partial hepatectomy in mice.

UNLABELLED:  Background. The regenerative capacity of the liver is critical for proper responses to injury. Fibrin extracellular matrix (ECM) deposition is a common response to insult and contributes to inflammatory liver injury. However, the role of this matrix in hepatic regeneration has not been determined. OBJECTIVE: The purpose of this study was first to determine the role of fibrin ECM in hepatic regeneration followed by the role of the fibrin-binding αvß3 integrin in mediating this effect. MATERIAL AND METHODS: C57Bl/6J (WT) or PAI-1 knockout (KO) mice underwent 70% partial hepatectomy (PHx); plasma and histologic indices of regeneration were determined, as well as expression of key genes involved in hepatic regeneration. RESULTS: PHx promoted transient fibrin deposition by activating coagulation and concomitantly decreasing fibrinolysis. Inhibiting fibrin deposition, either by blocking thrombin (hirudin) in WT mice or by knocking out PAI-1, was associated with a decrease in hepatocyte proliferation after PHx. This strongly suggested a role for fibrin ECM in liver regeneration. To investigate if αvß3 integrin mediates this action, we tested the effects of the anti-αvß3 cyclic peptide RGDfV in animals after PHx. As was observed with inhibition of fibrin deposition, competitive inhibition of αvß3 integrin delayed regeneration after PHx, while not affecting fibrin deposition. These effects of RGDfV correlated with impaired angiogénesis and STAT3 signaling, as well as transient endothelial dysfunction. In conclusion, these data suggest that αvß3 integrin plays an important role in coordinating hepatocyte division during liver regeneration after PHx via crosstalk with fibrin ECM.

Effects of blocking αvß3 integrin by a recombinant RGD disintegrin on remodeling of wound healing after induction of incisional hernia in rats.

PURPOSE: To investigate the changes induced by DisBa-01 on repair of wound healing after induced incisional hernia (IH) in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into four experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvß3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological, biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (p<0.05). Anti- αvß3-integrin antibodies produced similar results than DisBa-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: DisBa-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutically strategy in incisional hernia.

Effects of Blocking αvß3 integrin by a recombinant RGD disintegrin on remodeling of wound healing after induction of incisional hernia in rats.

PURPOSE: Incisional hernia (IH) is characterized by defective wound healing process. Disba-01, a αvb3 integrin blocker has shown to control the rate of wound repair and therefore it could be a target for new wound healing therapies.The objective of the study was to determine the changes induced by Disba-01 on repair of wound healing after induced IH in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into 4 experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvß3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological. biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (P<0.05). Anti- αvß3-integrin antibodies produced similar results than Disba-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: These results strongly indicate that Disba-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutical strategy in IH.

Blocking αvß3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells.

Vascular endothelial growth factor (VEGF) and αvß3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvß3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvß3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.

A novel alpha(v)beta (3)-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis.

The integrin alpha(v)beta(3) is involved in multiple aspects of malignant cancer, including tumor angiogenesis and metastasis, which makes the receptor a key target for the development of anti-cancer therapies. We report here on the production, the characterization and the in vivo anti-angiogenic and anti-metastatic properties of a novel alpha(v)beta(3)-binding disintegrin, DisBa-01, isolated from a cDNA library made with RNAs from the venom gland of Bothrops alternatus. The 11,637 Da-recombinant monomeric form of DisBa-01 displayed an RGD motif and interacted with purified alpha(v)beta(3) integrin in surface plasmon resonance studies, in a dose-dependent and cation sensitive manner. A three-dimensional molecular model of DisBa-01 in complex with alpha(v)beta(3) predicted a large surface of contacts with the beta(3) subunit. DisBa-01 inhibited the adhesion of alpha(v)beta(3)-expressing human microvascular endothelial cell line-1 (HMEC-1) and murine melanoma cell line B16F10 to vitronectin (IC(50) = 555 nM and 225 nM, respectively), and transiently inhibited their proliferation without direct cell toxicity, but did not affect the binding nor the proliferation of a human breast cancer-derived cell line (MDA-MB-231) not expressing alpha(v)beta(3). In vivo, DisBa-01 dose-dependently decreased bFGF-induced angiogenesis in a matrigel plug assay in athymic nude mice (IC(50) = 83 nM). When injected intravenously to C57BL/6 mice together with B16F10 melanoma cells, DisBa-01 time- and dose-dependently inhibited lung metastasis monitored by bioluminescent imaging. We conclude that DisBa-01 is a potent new inhibitor of alpha(v)beta(3)-dependent adherence mechanisms involved in neo-vascularization and tumor metastasis processes.

Interaction of macrophages with apoptotic cells enhances HIV Type 1 replication through PGE2, PAF, and vitronectin receptor.

Phagocytosis of apoptotic cells by macrophages increases secretion of soluble mediators and generates an antiinflammatory environment. We previously reported that phagocytosis of apoptotic cells by HIV-1-infected macrophages enhances viral replication, with the participation of the cytokine transforming growth factor- beta1 and an integrin receptor. Now, we describe the role of prostaglandin E2 (PGE2), platelet-activating factor (PAF), and the integrin alphaVbeta3 (vitronectin receptor, VnR) in this phenomenon. Exacerbation of HIV-1 growth induced by phagocytosis of apoptotic cells was inhibited when HIV-1-infected macrophages were treated with a cyclooxygenase 2 inhibitor, or with a PAF receptor antagonist (BN 52021) immediately after macrophage interaction with apoptotic cells. Treatment of HIV-1-infected macrophages with BN 52021 decreased viral replication, whereas addition of PGE2 or PAF to these cells enhanced viral replication. Monoclonal antibodies (MAbs) to VnR reduced the macrophage uptake of apoptotic cells, prevented the enhancement of HIV-1 growth upon the engulfment of apoptotic cells, and potently augmented viral replication in HIV-1-infected macrophages in the absence of apoptotic cells. In conclusion, PGE2 and PAF, and ligation of VnR as well, contribute to amplify viral growth in HIV-1-infected macrophages upon uptake of apoptotic cells.

Asymmetric reduction of alpha, beta-unsaturated ketone to (R) allylic alcohol by Candida chilensis.

A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.