Matrix metalloproteinases (MMP) are a family of proteolytic enzymes, the expression of which in a key step of tumor progression has recently been better defined. The overexpression of one or more MMPs is thus common among malignant tumors. It may characterize tumor progression and help predict its response to chemotherapy. Consequently, the development of a device for measuring MMP activities is an important challenge for diagnosis and prognosis. In this study, we describe an innovative supramolecular peptide/surface assembly for screening MMP activities. This sensor was used to discriminate various MMP activities and to distinguish between invasive and noninvasive cancerous cell suspensions. Our results confirm the proof-of-concept of a powerful tool for the determination of the tumor aggressiveness and a technical building block for future development of MMP lab-on-chip devices.

A triethyleneglycol (TEG) chain, a linear peptide, and a cyclic peptide labeled with 7-methoxycoumarin-3-carboxylic acid (MC) and 7-diethylaminocoumarin-3-carboxylic acid (DAC) were used to thoroughly study Förster resonance energy transfer (FRET) in inclusion complexes. (1) H NMR evidence was given for the formation of a 1:1 inclusion complex between β-cyclodextrin (β-CD) and the fluorophore moieties of model compounds. The binding constant was 20 times higher for DAC than for MC derivatives. Molecular modeling provided additional information. The UV/Vis absorption and fluorescence properties were studied and the energy transfer process was quantified. Fluorescence quenching was particularly strong for the peptide derivatives. The presence of β-CDs reduced the FRET efficiency slightly. Dye-labeled peptide derivatives can thus be used to form inclusion complexes with β-CDs and retain most of their FRET properties. This paves the way for their subsequent use in analytical devices that are designed to measure the activity of matrix metalloproteinases.

It has been widely reported that the tear film, which is crucially important as a protective barrier of the eye, undergoes biochemical changes as a result of a wide range of ocular pathology. This tends to suggest the possibility of early detection of ocular diseases on the basis of biochemical analysis of tears. However, studies of tears by conventional methods of biomolecular and biochemical analysis are often limited by methodological difficulties. Moreover, such analysis could not be applied in the clinic, where structural and morphological analyses by, mainly, slit-lamp biomicroscopy remains the recommended method. In this study, we assessed, for the first time, the potential of FTIR spectroscopy combined with advanced chemometric processing of spectral data for analysis of raw tears for diagnosis purposes. We first optimized sampling and spectral acquisition (tears collection method, tear sample volume, and preservation of the samples) for accurate spectral measurement. On the basis of the results, we focused our study on the possibility of discriminating tears from normal individuals from those of patients with different ocular pathologies, and showed that the most discriminating spectral range is that corresponding to variations of CH2 and CH3 of lipid aliphatic chains. We also report more subtle discrimination of tears from patients with keratoconus and those from patients with non-specific inflammatory ocular diseases, on the basis of variations in spectral ranges attributed notably to lipid and carbohydrate vibrations. Finally, we also succeeded in distinguishing tears from patients with early-stage and late-stage keratoconus on the basis of spectral features attributed to protein structure. Therefore, this study strongly suggests that FTIR spectral analysis of tears could be developed as a valuable and cost-saving tool for biochemical-based detection of ocular diseases, potentially before the appearance of the first morphological signs of diseases. Combined with supervised modelling methods and with use of a spectral data base acquired for representative patients, such a spectral approach could be a useful addition to current methods of clinical analysis for improvement of patient care.

The yeast Saccharomyces cerevisiae is widely used as a biological eukaryotic model and also serves as a production organism in biotechnology. One of the methods used to avoid degradation of the yeast cell content is lyophilization. The use of lyophilized yeast cells has several advantages over fresh ones: samples can be easily transported and/ or stored and variations of their metabolomic profiles do not occur during transport or storage. Fourier transform infrared (FTIR) spectroscopy is one of the most emerging approaches in modern biology that permits operation on very small quantities of whole cells without the need for extractions or purifications. This technique is very sensitive and not only allows the discrimination between different cell genotypes but also between different growth conditions. FTIR spectra provide interesting data on the metabolic status of the whole cell. Modern multivariate data processing was applied to analyse live fresh or lyophilized S. cerevisiae cells from different growth media. This study clearly demonstrates that yeast cells coming from an identical biological medium can be used indiscriminately for FTIR analysis whether they are analysed directly as live fresh cells or after lyophilization which is a freeze- drying process. Moreover, FTIR data obtained using lyophilized cells showed less variability.

Glioblastoma, the most malignant brain tumor in humans, is characterized by being severely angiogenic and an increase in vascularization generally worsens the prognosis of patients. Finding the best approach to characterize glioma blood vessels (BVs) is very important in view of helping to determine any specific biomolecular markers of these tumors. In previous work by conventional FTIR spectroscopy we were able to discriminate some molecular markers in order to differentiate between normal and tumor BVs in glioma tissue sections. The aim of the present study was to assess whether FTIR microspectroscopy using a synchrotron radiation (SR) source could provide advantages over a classical globar IR source for detailed spectral analysis on such small features like micro-BVs. Using chemometric analysis such as PCA and HCA, the results show that a high brilliant SR beam provides a very satisfying quality signal compared with the globar source to study spectral images for relevant analysis of glioma big and micro-BVs and determination of subtle molecular markers characterizing them from the surrounding tissue.

Characterization of matrix metalloprotease (MMP) activities is of increasing interest for cancer prognosis or treatment follow-up. Indeed, MMP-1, -2 and -9 are widely involved in the growth of many tumors and progression steps such as angiogenesis, invasion, and metastasis. Fluorogenic peptide MMP substrates were previously synthesized with the aim of detecting MMP activities. One of their drawbacks is their limited solubility in biological media. Grafting them onto a solid support represented a novel way to yield efficient analysis devices whilst at the same time decreasing the quantities of peptides used. Novel peptide arrays were designed in order to detect MMP activities in biological fluids. Silicon plates were used as the solid support for the design of these novel tools. These were functionalized by organic self-assembled monolayers (SAMs) on which fluorogenic peptides were covalently coupled. SAM and peptide grafting on silicon plates were confirmed by epifluorescence, ellipsometry, and FT-IR analysis. Enzymatic assays were monitored by fluorescence spectrometry and showed that immobilized linear peptides were recognized and cleaved by MMPs.

The PI3K/Akt-signaling pathway, associated with cancer development and disease progression, is recognized to be an anti-tumor drug target that could present important therapeutic benefit. However, no targeted Akt medicines have been commercialized yet, reflecting that drug selection procedures requires significant improvement from early research to clinical trials. Thus, new methods permitting both the evaluation of cytotoxic and proliferation inhibition effect on cancer cells but also to provide a global fingerprint of the drug action mechanism of new Akt inhibitor candidates are of major interest. Because it can detect very subtle molecular changes and could provide a global fingerprint of drug effects on cells, Fourier-transform infrared (FTIR) spectroscopy appears to be a promising method to develop new time- and cost-saving tools for chemical library screening improvements. In this study, we combine FTIR spectroscopy, advanced chemometrics analysis and cross-validation by standard biological assays to establish a basis of a mid-throughput methodology for rapid and automated assessment of cell response to Akt inhibitors and quantitative evaluation of their anti-proliferative effects. Our results shows that our methodology is able (1) to detect cell response to an Akt inhibitor exposure even for very low doses, (2) to provide biochemical information of interest about its effects on the cell metabolism, lipidome, and proteome, (3) to predict accurately resulting cell proliferation inhibition rate. Thus, further based on a large spectral data base, our methodology could contribute to facilitate preliminary screening of chemical libraries and improving the selection procedure of drug candidates in laboratory routine.

The complete structure of [2]benzopyrano[3,4-b]quinoxalin-5-one 1, also named isochromeno[3,4-b]quinoxalin-5-one, was established unequivocally by a single crystal X-ray analysis. Its process of formation probably included the autoxidation of the isoindole moiety of 6-chloro-isoindolo[2,1-a]quinoxaline 2 followed by a deshydratation and oxidation leading to a non isolated acid. Then a subsequent rearrangement of this adduct produces isochromeno[3,4-b]quinoxalin-5-one 1 through an intramolecular cyclization. The crystal is triclinic, space group P (l) over bar with a = 7.173 (1), b = 11.668 (2), c = 13.430 (2) angstrom, alpha = 85.56 (1)degrees, beta = 83.26 (1)degrees, gamma = 81.32 (1)degrees, V = 1101.4 (3) angstrom(3), Z = 4, C15H8N2O2, D-c = 1.497 g/cm(3), mu (MoK alpha) = 1.5418 angstrom, S = 1.017, F (000) = 512.00, R = 0.0758 and wR = 0.1840. In the unit cell, there are two independent molecules. (c) 2012 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Angiogenesis plays a critical role in tumor growth. This phenomena is regulated by numerous mediators such as vascular endothelial growth factor (VEGF). CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. We herein report the design of novel nanoparticles conjugated to CBO-P11 in order to specifically target tumor site.The conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) (PVDF) nanoparticles was investigated using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition known as "click" reaction. CBO-P11 was modified with a near-infrared cyanine dye bearing an alkyne function, allowing both "click" coupling on azido-modified nanoparticles and fluorescence labelling. Each step of this nanodevice construction was judiciously performed in aqueous solution and successfully characterized. The cytotoxicity of nanoparticles was evaluated in human brain endothelial cell line and their affinity for VEGF receptors was determined via fluorescence-based uptake assays on porcine aortic endothelial cell line.Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained.These results demonstrate the potential of these functionalized nanoparticles for targeting tumor angiogenesis and their possible use as multifunctional plateform for cancer treament if coupled with therapeutic agents.

Following our search for antimalarial compounds, novel series of ferrocenyl-substituted pyrrolo[1,2-a]quinoxalines 1-2 were synthesized from ferrocene-carboxaldehyde and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. The ferrocenic pyrrolo[1,2-a]quinoxalines 1-2 were prepared in 6 or 9 steps through a Barton–Zard reaction. Promising pharmacological results against FcB1, K1 and F32 strains were obtained with ferrocenyl pyrrolo[1,2-a]quinoxalines 1j-l linked by a bis-(3-aminopropyl)piperazine linker substituted by a nitrobenzyl moiety.

A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia and breast cancer cell lines in the 3- to 18-µM concentration range.

PET (Positron Emission Tomography) allows imaging of the in vivo distribution of biochemical compounds labeled with a radioactive tracer, mainly 18F-FDG (2-deoxy-2-[18F] fluoro-D-glucose). 18F only allows a relatively poor spatial resolution (2-3 mm) which does not allow imaging of small tumors or specific small size tissues, e.g. vasculature. Unfortunately, angiogenesis is a key process in various physiologic and pathologic processes and is, for instance, involved in modern anticancer approaches. Thus ability to visualize angiogenesis could allow early diagnosis and help to monitor the response of cancer to specific chemotherapies. Therefore, indirect analytical techniques are required to assess the localization of fluorinated compounds at a micrometric scale. Multimodality imaging approaches could provide accurate information on the metabolic activity of the target tissue. In this article, PIGE method (Particle Induced Gamma-ray Emission) was used to determine fluorinated tracers by the nuclear reaction of 19F(p,p′γ)19F in tissues. The feasibility of this approach was assessed on polyfluorinated model glucose compounds and novel peptide-based tracer designed for angiogenesis imaging. Our results describe the first mapping of the biodistribution of fluorinated compounds in both vascularized normal tissue and tumor tissue.

Malignant gliomas are very aggressive tumors, highly angiogenic and invading heterogeneously the surrounding brain parenchyma, making their resection very difficult. To overcome the limits of current diagnostic imaging techniques used for gliomas, we proposed using FTIR imaging, with a spatial resolution from 6 to 10 μm, to provide molecular information for their histological examination, based on discrimination between normal and tumor vasculature. Differentiation between normal and tumor blood vessel spectra by hierarchical cluster analysis was performed on tissue sections obtained from xenografted brain tumors of Rag-gamma mice 28 days after intracranial implantation of glioma cells, as well as for human brain tumors obtained in clinics. Classical pathological examination and immunohistochemistry were performed in parallel to the FTIR spectral imaging of brain tissues. First on the animal model, classification of FTIR spectra of blood vessels could be performed using spectral intervals based on fatty acyl (3050-2800 cm(-1)) and carbohydrate (1180-950 cm(-1)) absorptions, with the formation of two clusters corresponding to healthy and tumor parts of the tissue sections. Further data treatments on these two spectral intervals provided interpretable information about the molecular contents involved in the differentiation between normal and tumor blood vessels, the latter presenting a higher level of fatty acyl chain unsaturation and an unexpected loss of absorption from osidic residues. This classification method was further successfully tested on human glioma tissue sections. These findings demonstrate that FTIR imaging could highlight discriminant molecular markers to distinguish between normal and tumor vasculature, and help to delimitate areas of corresponding tissue.

Fmoc-protected amine derivatives of Protoporphyrin IX were synthesized by two original methods using solid-phase chemistry. The new compounds represent unsymmetrical scaffolds suitable for the generation of a large range of peptidic porphyrin derivatives through SPPS strategy.

A significant antitumor effect was previously observed with radioimmunotherapy using anti–carcinoembryonic antigen 131I-F6 monoclonal antibody in medullary thyroid cancer–bearing nude mice. Nevertheless, no complete response was observed. As seen with chemotherapy, drugs targeting the tumor microenvironment might improve radioimmunotherapy efficacy. This study evaluated the toxicity and efficacy of combining radioimmunotherapy with thalidomide or a cyclopeptidic vascular endothelial growth inhibitor (CBOP11) in mice grafted with the TT human medullary thyroid cancer cell line. Methods: Six to 10 nude mice treated with 92.5 MBq of 131I-F6 in association with 200 mg/kg/d of oral thalidomide during 20 d by force-feeding or 0.45 mg/kg/d of CBOP11 during 25 d using subcutaneous minipumps were compared with control mice receiving either treatment or naked F6 or nonspecific 131I-734. Combined therapies included 131I-F6 at day 0 followed by thalidomide between days 20 and 40, thalidomide between days 0 and 20 followed by 131I-F6 at day 25, 131I-F6 at day 0 and CBOP11 between days 0 and 25, CBOP11 between days 0 and 25 followed by 131I-F6 at day 25, and 131I-F6 at day 0 followed by CBOP11 between days 20 and 45. Animal weight, hematologic toxicity, tumor volume, and serum calcitonin were monitored for the following 3 mo. Improvement of 125I-F6 tumor biodistribution by antiangiogenic drug was studied after pretreatment by thalidomide. Follow-up of the tumor after combined antiangiogenic and radioimmunotherapy therapies was performed by histology studies. Results: Combined associations, as compared with radioimmunotherapy alone, increased leukopenia but not thrombocytopenia. Tumor volume–quadrupling time (TVQT) was 22.8 ± 3.3 d in the control group, 29.9 ± 3.6 d in the group treated with thalidomide, 34.6 ± 4.4 d in the group treated with CBOP11, and 51.0 ± 2.8 d after radioimmunotherapy alone. As compared with radioimmunotherapy, TVQT was significantly longer (P < 0.01) after thalidomide followed by radioimmunotherapy (69.83 ± 3.9), CBOP11 followed by radioimmunotherapy (71.3 ± 6.1), and CBOP11–radioimmunotherapy in concomitance (64.2 ± 6.1). Nevertheless, TVQT was not increased after radioimmunotherapy followed by thalidomide (48.8 ± 4) and radioimmunotherapy followed by CBOP11 (56.8 ± 4.8). Surprisingly, pretreatment by CBOP11 or thalidomide sensitized larger tumors (>300 mm3) to radioimmunotherapy. Change in calcitonin levels confirmed morphologic tumor response. Tumor uptake 24 h after injection of 125I-F6 was 4.5 ± 0.6 percentage injected dose per gram (%ID/g) without pretreatment and 8.7 ± 1.3 %ID/g with pretreatment by thalidomide. An increase of the antitumor effect observed using the antiangiogenic drug combined with radioimmunotherapy was correlated with a decrease of blood vessels shown by von Willebrand immunostaining. Conclusion: Pretreatment with antiangiogenic therapies improved radioimmunotherapy efficacy, with acceptable toxicity. Future investigations will be performed to understand how antiangiogenic agents sensitize large tumors to radioimmunotherapy.

Angiogenesis plays a critical role in both growth and metastasis of tumors. Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (KDR) have become critical for anti-tumor therapy. A cyclo-peptide (CBO-P11), derived from VEGF, able to inhibit the interaction between the growth factor and its receptor, was synthesized in our laboratory to provide a target for angiogenesis. We have prepared biocompatible poly(vinylidene fluoride) (PVDF) nanoparticles in order to obtain long blood circulating systems. Electron-beam (EB) irradiation was used to activate the PVDF nanoparticles. From electron paramagnetic resonance (EPR) measurements, we studied the radical stability in order to optimize the radio-grafting of acrylic acid (AA). Further functionalization of PVDF-g-PAA nanoparticles with the cyclo-peptide via a spacer arm was also possible by performing coupling reactions. High resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) and MALDI mass spectrometry allowed us to follow each chemical step of this peptide immobilization. We designed a new nanodevice suggesting a great potential for targeting angiogenesis. 7727-21-1

Matrix metalloproteinases (MMP) are a family of proteolytic enzymes, the expression of which in a key step of tumor progression has been better defined recently. The studies highlighted the ongoing need for very specific inhibitors, substrates or release devices designed to be selective for one or at least very few MMPs.

An application of ion beam microprobe analysis has been developed for the quantitative imaging of fluorine compounds in biological samples on a micrometer scale. The difficulty of imaging fluorine at trace level concentration in biological tissues has been addressed by using a combination of nuclear reaction analysis and proton resonant backscattering spectrometry. With this method, the limit of detection of fluorine in thin biological samples can be as low as 3 μg/g. Quantitative imaging of fluorodeoxyglucose distribution, the most widely used tracer for detection of solid tumors by positron emission tomography, is shown as an example of this application.