A novel αvβ3-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis

The integrin α_vβ₃ 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 α_vβ₃-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 α_vβ₃ 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 α_vβ₃ predicted a large surface of contacts with the β₃ subunit. DisBa-01 inhibited the adhesion of α_vβ₃-expressing human microvascular endothelial cell line-1 (HMEC-1) and murine melanoma cell line B16F10 to vitronectin (IC₅₀ = 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 α_vβ₃. In vivo, DisBa-01 dose-dependently decreased bFGF-induced angiogenesis in a matrigel plug assay in athymic nude mice (IC₅₀ = 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 α_vβ₃-dependent adherence mechanisms involved in neo-vascularization and tumor metastasis processes. Oscar H. P. Ramos and Alexandre Kauskot contributed equally to this work.     

Role of integrin alpha(v)beta3 in the early phase of liver metastasis: PET and IVM analyses

To clarify the function of integrin αvβ3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin αvβ3 cDNA (αvβ3-CHO-K1 cells). The liver accumulation of αvβ3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of αvβ3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-αvβ3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin αvβ3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of αvβ3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin αvβ3 may function to promote extravasation of integrin αvβ3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells

Early metastasis is the primary cause of death in melanoma patients. The adhesion receptor integrin αvβ3 contributes to tumor cell functions that are potentially involved in melanoma growth and metastasis. We tested whether integrin αvβ3 supports metastasis of human melanoma cells when injected into the bloodstream of immune deficient mice. Comparing variants of the same melanoma cell type that expressed either αvβ3, αIIbβ3 or no β3 integrin, we found that only αvβ3 strongly supported metastasis. Inhibition of tumor cell αvβ3 function reduced melanoma metastasis significantly and prolonged animal survival. To understand mechanisms that allow αvβ3, but not αIIbβ3 to support melanoma metastasis, we analyzed proteolytic and migratory activities of the melanoma cell variants. Melanoma cells expressing αvβ3, but not those expressing αIIbβ3 or no β3 integrin, produced the active form of metalloproteinase MMP-2 and expressed elevated mRNA levels of MT1-MMP and TIMP-2. This indicates an association between αvβ3 expression and protease processing. Furthermore, αvβ3 expression was required for efficient melanoma cell migration toward the matrix proteins fibronectin and vitronectin. The results suggest that expression of integrin αvβ3 promotes the metastatic phenotype in human melanoma by supporting specific adhesive, invasive and migratory properties of the tumor cells and that the related integrin αIIbβ3 cannot substitute for αvβ3 in this respect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Association of αvβ3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

Introduction. Expression of adhesion molecules such as α _v β ₃ integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether α _v β ₃ expression correlated with the metastatic potential of human osteosarcoma cells. Materials and methods. We developed a series of sublines (LM2–LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6–8 weeks. We quantified α _v β ₃ integrin expression using flow cytometry. Results. α _v β ₃ expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of α _v β ₃. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that α _v β ₃ was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. Conclusions. α _v β ₃ integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. α _v β ₃ integrin may therefore be a potential new target for osteosarcoma.This revised version was published online in August 2005 with a corrected cover date.     

Indium-111 labeled gold nanoparticles for in-vivo molecular targeting

The present report describes the synthesis and biological evaluation of a molecular imaging platform based on gold nanoparticles directly labeled with indium-111. The direct labeling approach facilitated radiolabeling with high activities while maintaining excellent stability within the biological environment. The resulting imaging platform exhibited low interference of the radiolabel with targeting molecules, which is highly desirable for in-vivo probe tracking and molecular targeted tumor imaging. The indium-111 labeled gold nanoparticles were synthesized using a simple procedure that allowed stable labeling of the nanoparticle core with various indium-111 activities. Subsequent surface modification of the particle cores with RGD-based ligands at various densities allowed for molecular targeting of the α_vß₃ integrin in-vitro and for molecular targeted imaging in human melanoma and glioblastoma models in-vivo. The results demonstrate the vast potential of direct labeling with radioisotopes for tracking gold nanoparticles within biological systems.     

Porcine adenovirus serotype 3 internalization is independent of CAR and alphavbeta3 or alphavbeta5 integrin

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus-adenovirus receptor (CAR) serves as a primary receptor and alphavbeta3 or alphavbeta5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, alphavbeta3 or alphavbeta5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-alphavbeta3 or anti-alphavbeta5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as alphavbeta3 or alphavbeta5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and alphavbeta3 or alphavbeta5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.     

Role of nitric oxide in the regulation of activity of proteinase inhibitors α1-antitrypsin and α2-macroglobulin by capsaicin-sensitive nerves

Regulation of activity of serine proteinase inhibitor α₁-antitrypsin and nonspecific proteinase inhibitor α₂-macroglobulin in the blood by nitric oxide was studied in intact rats and animals with damage to capsaicin-sensitive nerves. Nonselective nitric oxide synthase inhibitor L-NAME produced a dose-dependent increase in α₁-antitrypsin activity in intact animals. Neuronal NO synthase inhibitor 7-nitroindazole increased α ₂-macroglobulin activity. Deafferentation with capsaicin was followed by a decrease in α₁-antitrypsin activity. Both inhibitors of nitric oxide synthase increased activity of α₁-antitrypsin in capsaicin-receiving rats. Nitric oxide precursor L-arginine had a normalizing effect on reduced activity of α₁-antitrypsin after capsaicin deafferentation. Our results suggest that nitric oxide has a modulatory effect on activity of proteinase inhibitors and is involved in the effector influence of capsaicin-sensitive nerves on α₁-antitrypsin activity. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 9, pp. 350–354, September, 2008     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.     

Regulation of melanoma cell migration and invasion by laminin-5 and α3β1 integrin (VLA-3)

We evaluated the role of soluble factors produced from epidermal cells in melanoma cell motility by using the Boyden chamber chemoinvasion system. The migration of two melanoma cell lines, A375 and Mewo, was potentiated by conditioned media of A431 epidermoid cells in a concentration-dependent manner. The enhancement of A375 melanoma cell motility induced by the conditioned medium was blocked by antibodies against either α3 or β1 integrin subunit. The motility-stimulating activity was recovered in the same fraction as the α3 integrin-dependent adhesion-promoting activity in a high-molecular-weight (>200 kDa) fraction on Superose 12 gel chromatography, and adsorbed with an anti-laminin-5 antibody. Purified laminin-5 was capable of potentiating melanoma cell migration as measured in either the chemotaxis assay with a soluble form of laminin-5 or the haptotaxis assay with membranes coated with a mixture of laminin-5 and Matrigel. Furthermore, immobilized laminin-5 induced A375 melanoma cells to secrete matrix metalloproteinase-9 (type IV collagenase) into the culture medium. These results strongly suggest that the interaction of laminin-5 produced in the epidermis with α3β1 integrin on melanoma cells is involved in cell migration, invasion, and degradation of extracellular matrix proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Increased expression of CD146 and microvessel density (MVD) in invasive micropapillary carcinoma of the breast: Comparative study with invasive ductal carcinoma-not otherwise specified

Invasive micropapillary carcinoma (IMPC) is a rare variant of ductal carcinoma of the breast, and is characterized by a high metastatic potential and an aggressive clinical course. Studies of CD146 expression and function in breast cancer remain scarce. The aim of this study was to evaluate the role of CD146 and microvessel density (MVD) in breast IMPC. CD146 mRNA expression and immunohistochemistry for CD146 and MVD measured by CD31 were assessed in 82 cases of IMPC and 137 cases of invasive ductal carcinoma, not otherwise specified (IDC-NOS). The mRNA level of CD146 in cancer specimens was higher in IMPC than in IDC-NOS. CD146 expression in tumor cells was up-regulated in IMPC as compared with that in IDC-NOS, and was positively correlated with histological grade, ER, PR status, and P53 expression in IMPC and IDC-NOS. CD146 expression in vascular endothelial cells was significantly higher than that in IDC, and was positively correlated with tumor progression in IMPC and IDC-NOS. MVD in IMPC was significantly higher than that in IDC. CD146 expression in tumor cells was positively correlated with that in vascular endothelial cells of IMPC and IDC-NOS. The association of CD146 expression with MVD and its correlation with progression in breast carcinoma indicated that CD146 is a potentially useful prognostic marker for breast cancer. CD146 could be a new drug target in the treatment of breast cancer.     

CEACAM1和CD34蛋白表达与胃癌侵袭转移的关系

目的探讨癌胚抗原相关黏附分子1(CEACAM1)和CD34蛋白表达与胃癌侵袭转移的关系。方法免疫组化SP法检测90例胃癌组织和30名正常胃黏膜组织中CEACAM1及CD34的表达情况,分析CEACAM1和CD34标记的微血管密度(MVD)与胃癌患者临床病理特征的关系。结果 CEACAM1和CD34蛋白在胃癌中的阳性表达明显高于正常胃黏膜组织(P0.05)。CEACAM1蛋白的表达程度与肿瘤分化、侵袭深度、是否淋巴结转移及病理分期相关(P0.05),胃癌组织中MVD与肿瘤大小、分化、侵袭深度、是否淋巴结转移及病理分期相关(P0.05)。CEACAM1表达程度及CD34标记的MVD在胃癌中的表达呈正相关关系(P0.05)。结论 CEACAM1与CD34参与了胃癌的侵袭和转移,有望成为胃癌发生、发展和预后的预测指标。     

Intravenous immunoglobulin and atherosclerosis

Several inflammatory and immunological factors have been established as important contributors to atherogenesis. Among these, oxidized low-density lipoprotein (oxLDL) play a central role in the initiation and progression of atherosclerotic lesions. In atherosclerotic lesions, oxLDL was also found to co-localize with β₂-glycoprotein I (β₂-GPI). Immunoglobulin (Ig)G autoantibodies against β₂-GPI complexed with oxLDL are pro-atherogenic because they increase uptake of the complexes by macrophages. In contrast, IgM natural anti-oxLDL antibodies derived from atherosclerosis-prone apolipoprotein E (ApoE) deficient mice reduced incidence of atherosclerosis. Such anti-oxLDL antibodies have been found in humans, and the accumulating evidences seem to support the idea that anti-oxLDL antibodies have a protective role for atherogenesis. Intravenous immunoglobulins (IVIgs) contain natural anti-oxLDL antibodies and infusion of IVIg into ApoE-deficient mice has been reported to decrease atheerosclerosis. The anti-atherogenic property of IVIg may be derived from non-antigen-specific antibody binding to FCγ receptors, which blocks foam cell formation of macrophages. Several other possible mechanisms are also discussed.     

Investigation of the Presence of Apotipoprotein E,G lycosaminoglycans,Basement Membrane Proteins,and Protease inhibitors in Senile interstitial Amyloid of the Pituitary

The aim of our study was to test whether local- or organ-limited interstitial amyloid of the pituitary is associated with the presence of glycosaminoglycans, basement membrane proteins, protease inhibitors, and apolipoprotein E (apo E), as previously observed in other amyloid syndromes. Serial sections from amyloidotic and nonamyloidotic autopsy pituitaries of patients age 85 yr and over were stained with Congo red, Alcian blue, and, applying immunohistochemistry, with antibodies directed against fibronectin, collagen IV, laminin, apo E, α₁-antitrypsin and α₁-antichymotrypsin. Interstitial amyloid was deposited in the immediate vicinity of capillaries and around the acini of the anterior lobe. Glycosaminoglycans were found in capillaries and around the acini of both nonamyloidotic and amyloidotic glands and they were also related spatially to amyloid deposits. Immunostatining of nonamyloidotic and amyloidotic glands demonstrated the presence of fibronectin, collagen IV, and laminin, which was related to basement membranes (fibronectin, collagen IV, and laminin), interstitium, and serum (fibronectin only). In amyloidotic glands, each basement membrane protein presented with an additional spatial relationship to amyloid deposits. Apo E was found in amyloidotic cases only within the amyloid deposits. The results are consistent with the presence of glycosaminoglycans, basement membrane proteins, and apo E in local interstitial amyloid deposits of the pituitary, as previously described in other amyloid syndromes, such as inflammatory related AA-amyloidosis or Aβ-amyloidosis related to Alzheimer’s disease.     

Exercise training reduces cardiac angiotensin II levels and prevents cardiac dysfunction in a genetic model of sympathetic hyperactivity-induced heart failure in mice

The role of exercise training (ET) on cardiac renin-angiotensin system (RAS) was investigated in 3–5 month-old mice lacking α_2A- and α_2C-adrenoceptors (α_2A/α_2CARKO) that present heart failure (HF) and wild type control (WT). ET consisted of 8-week running sessions of 60 min, 5 days/week. In addition, exercise tolerance, cardiac structural and function analysis were made. At 3 months, fractional shortening and exercise tolerance were similar between groups. At 5 months, α_2A/α_2CARKO mice displayed ventricular dysfunction and fibrosis associated with increased cardiac angiotensin (Ang) II levels (2.9-fold) and increased local angiotensin-converting enzyme activity (ACE 18%). ET decreased α_2A/α_2CARKO cardiac Ang II levels and ACE activity to age-matched untrained WT mice levels while increased ACE2 expression and prevented exercise intolerance and ventricular dysfunction with little impact on cardiac remodeling. Altogether, these data provide evidence that reduced cardiac RAS explains, at least in part, the beneficial effects of ET on cardiac function in a genetic model of HF.     

Clinical evaluation of dual-dialyzer hemodialysis (DDHD)

A higher β₂-microglobulin (β₂MG) removal rate is possible in dual-dialyzer hemodialysis (DDHD) because of the large internal filtration provided. In this treatment, in the first dialyzer, a large amount of water moves from the blood side to the dialysate side, and in the second dialyzer, it moves from the dialysate side to the blood side. Thus, both the direction of the water shift and the amount of water change within the dialyzer from the blood inlet port to the outlet port. A blood volume monitor was used to confirm that the internal filtration rate remains stable during the course of the dialysis treatment. The β₂MG removal rate and the weekly amount of solute removal by DDHD treatment was much higher than that for conventional HD.     

Negative regulation of caspase-3 expression in the neonatal cerebral cortex by α<Subscript>2A</Subscript>-adrenoceptors

Antisense oligonucleotide to α_2A-adrenoceptors increased the levels of mRNA (reverse polymerase chain reaction) and protein for a key executioner protease of apoptosis caspase-3 (immunoblotting assay) in the cerebral cortex of newborn rats. The relationship between the observed effect and low expression of α₂-adrenoceptors was confirmed by the possibility of correcting this phenomenon by clonidine (stimulator of α₂-adrenoceptors). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 244–246, March, 2007     

Noopept efficiency in experimental Alzheimer disease (cognitive deficiency caused by β-amyloid25–35 injection into Meynert basal nuclei of rats)

Experiments on adult Wistar rats showed that injection of β-amyloid_(25–35) (2 μg) into Meynert basal nuclei caused long-term memory deficiency which was detected 24 days after this injection by the memory trace retrieval in conditioned passive avoidance reflex (CPAR). The effects of noopept, an original nootropic and neuroprotective dipeptide, on the severity of this cognitive deficiency were studied. Preventive (for 7 days before the injury) intraperitoneal injections of noopept in a dose of 0.5 mg/kg completely prevented mnestic disorders under conditions of this model. Noopept exhibited a significant normalizing effect, if the treatment was started 15 days after the injury, when neurodegenerative changes in the basal nuclei, cortex, and hippocampus were still acutely pronounced. The mechanisms of this effect of the drug are studied, including, in addition to the choline-positive effect, its multicomponent neuroprotective effect and stimulation of production of antibodies to β-amyloid_(25–35). Noopept efficiency in many models of Alzheimer disease, its high bioavailability and low toxicity suggest this dipeptide for further studies as a potential agent for the treatment of this condition (initial and moderate phases). __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 84–88, July, 2008     

Restoration of Smad4 in BxPC3 Pancreatic Cancer Cells Attenuates Proliferation without Altering Angiogenesis

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which the transforming growth factor beta (TGF-β) signal transducer, Smad4, is commonly mutated or deleted. BxPC3 human pancreatic cancer cells exhibit a homozygous deletion of the Smad4 gene, yet are growth inhibited by TGF-β1. In the present study, we sought to determine whether reintroduction of Smad4 into BxPC3 cells alters their behavior in vitro and in vivo. Sham transfected and Smad4 expressing BxPC3 cells exhibited similar responses to TGF-β1 with respect to p21 upregulation, hypophosphorylation of the RB protein, Smad2 phosphorylation, and Smad2/3 nuclear translocation. TGF-β1 did not alter p27 expression, and silencing of p21 with an appropriate siRNA markedly attenuated TGF-β1-mediated growth inhibition. Nonetheless, the presence of Smad4 was associated in vitro with a more prolonged doubling time, enhanced sensitivity to the growth inhibitory actions of exogenous TGF-β1, and a more flattened cellular morphology. In vivo, Smad4 expression resulted in delayed tumor growth and decreased cellular proliferation, without effects on either apoptosis or angiogenesis. These findings indicate that, in spite of the absence of Smad4, growth inhibition in BxPC3 cells by TGF-β1 is dependent on p21 upregulation and maintenance of RB in a hypophosphorylated, active state. Moreover, the presence of a functional Smad4 attenuates the capacity of BxPC3 cells to proliferate in vivo. However, this effect is transient, indicating that Smad4 growth inhibitory actions are circumvented in the later stages of pancreatic tumorigenicity.