Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure–activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ∼2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.     

Echinococcus granulosus: secretory activity of the rostellum of the adult cestode in situ in the dog.

Secretary activities associated with the rostellum of adult Echinococcus granulosus were studied using histological, histochemical, and ultrastructural techniques, following rapid fixation of the cestodes in situ in the small intestine of the anaesthetised dog. Studies concentrated on the host-parasite interface from 30 to 35 days postinfection. At this time, contraction of the muscular rostellar pad appeared to be associated with extension of the apical rostellum into a crypt of Lieberkühn. Crypt invasion by the apical rostellum coincided with morphological changes and secretory activity in a group of modified tegumental cells, previously referred to as the rostellar gland. Secretory material, a cystine-rich protein, was observed in the nuclei and cytoplasm of the rostellar gland cells. Release of this material into the interface was seen only following crypt invasion by the apical rostellum. Although the mechanism of release is not clear, it may be analagous to holocrine secretory mechanisms, since apparent degeneration of the rostellar gland region was associated with secretion. Possible functional activities of the secretion associated with hook formation, nutrition, regulation, adhesion, and protection are discussed.     

Application of π acceptors to the spectrophotometric and spectrofluorimetric determination of vincamine and naftidrofuryl oxalate in their pharmaceutical preparations

Three different spectrophotometric and two spectrofluorimetric methods have been developed and validated for the determination of vincamine (VN) and naftidrofuryl oxalate (NF) in tablets. The spectrophotometric methods depend on charge transfer complex formation between each of VN and NF with 7,7,8,8‐tetracyano‐quinodimethane (TCNQ), 2,6‐dichloroquinone‐4‐chloroimide (DCQ) and 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) at 843, 580 and 588 nm, respectively. The spectrofluorimetric methods are based on the formation of charge transfer complex between each of the two drugs and TCNQ, with measurement of the fluorophore formed at 312/375 and 284/612 nm, respectively, or with DDQ at 400/475 and 284/396 nm, respectively. In the spectrophotometric measurements, Beer's law was obeyed at concentration ranges of 1.5–16, 10–180 and 12–140 μg/ml for VN with TCNQ, DCQ, and DDQ, respectively. For NF, the corresponding concentrations were 2–28, 5–75 and 25–150 μg/ml with TCNQ, DCQ, and DDQ, respectively. In the spectrofluorimetric measurements, the ranges for VN were 0.05–0.9 and 0.3–4 μg/ml with TCNQ and DDQ, respectively, whereas for NF the ranges were 0.05–0.85 and 0.5–8 μg/ml with TCNQ and DDQ, respectively. The different experimental parameters affecting the development and stability of the formed color or fluorophore were studied and optimized and the molar ratios of the complexes were calculated. The proposed methods were validated according to ICH guidelines and were successfully applied for the determination of VN and NF in their tablet dosage forms.     

Focal adhesion molecules as potential target of lead toxicity in NRK-52E cell line

In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK-52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK-52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin-activated signalling. These effects appeared to be strictly associated to the Pb-induced arrest of cell cycle at G0/G1 phase also proved in this cell line.     

Responses of keratinocytes to substrate-bound vitronectin: growth factor complexes

Insulin-like growth factor-1 (IGF-I) can associate with the extracellular matrix protein vitronectin (VN) via select IGF-binding proteins, and the resulting complex stimulates responses in a variety of cell types. As VN can also associate with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we hypothesized that the multimeric nature of VN could be exploited to deliver multiple growth factors to the cell surface. We report here that VN enhances bFGF but not EGF stimulated [(3)H]-leucine incorporation in the HaCAT keratinocyte cell line, with VN synergistically enhancing cell migration in response to both EGF and bFGF when presented as a VN-bound complex. Furthermore, the addition of EGF and/or bFGF to IGF-I:IGFBP-5:VN complexes significantly enhances both [(3)H]-leucine incorporation and migration of HaCAT cells above that induced by IGF:IGFBP-5:VN complexes alone. Indeed, similar responses are observed in primary cultures of human skin keratinocytes, highlighting the potential use of these novel complexes for a wide range of tissue repair applications.     

Syndecan-1 ectodomain regulates matrix-dependent signaling in human breast carcinoma cells

Syndecan-1 was overexpressed in T47D, MCF-7, or Hs578t human breast carcinoma cell lines, mimicking overexpression observed in carcinomas in vivo. Overexpression of syndecan-1, or its ectodomain alone fused to a glycosylphosphatidylinositol anchor (GPI-mS1ED), promotes cell rounding in 2D culture. Deletions within the syndecan-1 ectodomain (S1ED) implicate an active site within the core protein between the glycosaminoglycan attachment region and the transmembrane domain. Polyclonal antibodies directed against the ectodomain, or treatment with the tyrosine kinase inhibitor genistein, block activity and revert GPI-mS1ED overexpressing cells to a normal morphology. Extracellular matrix (ECM)-dependent signaling appears to be targeted, as GPI-mS1ED cells attach and spread similarly to control cells in response to E-cadherin engagement, but fail to spread on integrin-dependent ligands. However, integrin-dependent cell attachment, and integrin activation and subsequent FAK phosphorylation are unaffected, suggesting that the syndecan regulates the integration of signaling following matrix adhesion. In 3D culture, where syndecan-1 may have a more critical role in cell behavior, the disrupted signaling leads to poorly cohesive, invasive colonies. Thus, altered matrix-dependent signaling due to increased levels of cell surface syndecan-1 may lead to epithelial cell invasion during early stages of tumorigenesis.     

Activated vitronectin as a target for anticancer therapy with human antibodies

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites. Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs). In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin. One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/ monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues. Iodine 123–radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.Supported in part by the Dutch Cancer Society (grant UU1999-2114) and the Vanderes Foundation, The Hague, The Netherlands.     

Molecular composition and function of integrin-based collagen glues—Introducing COLINBRIs

Background

Despite detailed knowledge about the structure and signaling properties of individual collagen receptors, much remains to be learned about how these receptors participate in linking cells to fibrillar collagen matrices in tissues. In addition to collagen-binding integrins, a group of proteins with affinity both for fibrillar collagens and integrins link these two protein families together. We have introduced the name COLINBRI (COLlagen INtegrin BRIdging) for this set of molecules. Whereas collagens are the major building blocks in tissues and defects in these structural proteins have severe consequences for tissue integrity, the mild phenotypes of the integrin type of collagen receptors have raised questions about their importance in tissue biology and pathology.

Scope of review

We will discuss the two types of cell linkages to fibrillar collagen (direct- versus indirect COLINBRI-mediated) and discuss how the parallel existence of direct and indirect linkages to collagens may ensure tissue integrity.

Major conclusions

The observed mild phenotypes of mice deficient in collagen-binding integrins and the relatively restricted availability of integrin-binding sequences in mature fibrillar collagen matrices support the existence of indirect collagen-binding mechanisms in parallel with direct collagen binding in vivo.

General significance

A continued focus on understanding the molecular details of cell adhesion mechanisms to collagens will be important and will benefit our understanding of diseases like tissue- and tumor fibrosis where collagen dynamics are disturbed. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Novel protein interactors of urokinase-type plasminogen activator receptor

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.     

Vitronectin promotes oligodendrocyte differentiation during neurogenesis of human embryonic stem cells

We demonstrate enhanced differentiation of oligodendrocytes during neurogenesis of human embryonic stem cells (hESCs) using an extracellular matrix protein, vitronectin (VN). We show that VN is expressed in the ventral part of the developing human spinal cord. Combined treatment of retinoic acid, sonic hedgehog, and noggin in the presence of VN allows hESCs to differentiate into O4-positive oligodendrocytes. Particularly, VN profoundly promotes the derivation of oligodendrocyte progenitors that proliferate and differentiate into oligodendrocytes in response to mitogenic and survival factors. These results support the beneficial effect of VN on oligodendrocytic differentiation of hESCs.