Triazolopeptides Inhibiting the Interaction between Neuropilin-1 and Vascular Endothelial Growth Factor-165.

Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 µM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 µM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure-activity relationships.

Structure-activity relationship study of a small cyclic peptide H-c[Lys-Pro-Glu]-Arg-OH: a potent inhibitor of Vascular Endothelial Growth Factor interaction with Neuropilin-1.

Inhibition of angiogenesis is one of the most promising approaches in anticancer therapy. It was recently suggested that Neuropilin-1 (NRP-1) in tumour cells may serve as a separate receptor for Vascular Endothelial Growth Factor-165 (VEGF165) which is one of the main pro-angiogenic agents in the organism. Therefore molecules inhibiting VEGF165 binding to NRP-1 could be potential candidates for new antiangiogenic and anticancer drugs. Here we present a structure-activity relationship study of the peptide H-c[Lys-Pro-Glu]-Arg-OH which showed high inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18µM) in our previous study. We report the design, synthesis, in vitro assays and docking analysis of four small cyclic peptides (14-,15-membered ring) and one bigger cyclic compound (30-membered ring). Our study shows that both the ring size and configuration of amino acid residues present in the structure are crucial for high inhibitory effect.

Conformational latitude - activity relationship of KPPR tetrapeptide analogues toward their ability to inhibit binding of vascular endothelial growth factor 165 to neuropilin-1.

Neuropilin-1 has been found to be overexpressed in several kinds of malignant tumors, and it is postulated that its interaction with the vascular endothelial growth factor 165 leads to progression of tumor vascularization and growth. Several analogues (KxxR) with various conformational latitudes have been synthesized and found as inhibitors of NRP-1. Detailed insight provided by molecular dynamics simulation allowed forming a clear relationship between flexibility of xx part of the molecule and its inhibitory activity.

Discovery of novel inhibitors of vascular endothelial growth factor-A-Neuropilin-1 interaction by structure-based virtual screening.

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A165 to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A165 binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A165 binding to recombinant NRP-1 with Ki of about 10 µM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.

Transcriptome analysis and in vivo activity of fluvastatin versus zoledronic acid in a murine breast cancer metastasis model.

Statins and bisphosphonates are two distinct classes of isoprenoid pathway inhibitors targeting downstream enzyme to HMG-CoA reductase (upstream enzyme) and farnesyl-pyrophosphate synthase, respectively. Here, we studied fluvastatin (Fluva) and zoledronate (Zol), representative molecules of each class, respectively. In vivo metastatic potentials of both molecules were assessed. For the first time, we observed a significant reduction in progression of established metastases with Fluva treatment. Treatment with both Zol at 100 µg/kg and Fluva at 15 mg/kg inhibited 80% of the metastasis bioluminescence signal and increased survival of mice. The Zol and Fluva transcriptomic profiles of treated MDA-MB-231 cells revealed analogous patterns of affected genes, but each of them reached with different kinetics. The observable changes in gene expression started after 24 h for Fluva IC(50 72 h) and only after 48 h for Zol IC(50 72 h). To obtain early changes in gene expression of Zol-treated cells, a 3 times higher dose of Zol IC(50 72 h) had to be applied. Combining Fluva and Zol in vivo showed no synergy, but a benefit of several days in survival of mice. This study demonstrated that Zol or Fluva is of potential clinical use for the treatment of established metastasis.

HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165.

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.

Duration of adjuvant chemotherapy for patients with non-metastatic colorectal cancer.

BACKGROUND: Surgery of primary tumour is the backbone of colorectal cancer treatment (CRC). But in stage III cancer, metastatic or local relapse is often observed (50%). So, adjuvant treatment is always considered in this setting. The best treatment duration of hypothetic disease is not easy to define. Adjuvant chemotherapy for CRC actually lasts 6 months. The choice of optimal duration is based upon old studies using 5-fluorouracil (5FU). During the last ten years, results of major randomized controlled studies (RCTs) comparing different durations of treatments and different schedules in adjuvant setting were published. Several studies compared a 6-month chemotherapy with a longer treatment. Conversely, a single study by Chau et al compared a 6 month chemotherapy with continuous treatment lasting 3 months. But the optimal duration of these chemotherapies could be challenged. Even though the optimal duration of chemotherapy in CRC is a major issue, it has never been answered adequately. OBJECTIVES: To evaluate the optimal duration of adjuvant treatment, we performed a meta-analysis of all RCTs comparing two durations of adjuvant treatment, 6 months versus 9 to 12 months. SEARCH STRATEGY: Publications were identified from PubMed (February 28th, 2009), Embase, and the Cochrane Database of Clinical Controlled Trials (CENTRAL) in the Cochrane Library 2009 issue 1. Reviews and books were also scrutinized. Abstracts were reviewed from ASCO annual meetings proceedings from 1998 to 2009. SELECTION CRITERIA: Patients with surgically resected colorectal cancer with high risk of recurrence. DATA COLLECTION AND ANALYSIS: Several RCTs compared shorter versus longer durations of chemotherapy, 6 studies for overall survival (OS) and 7 studies for relapse free survival (RFS), for a total of 10326 patients, mean age 63.1 years, including 9826 colon and 500 rectum cancers. MAIN RESULTS: Treatments were always based on 5-FU. Two studies were excluded, an epidemiological study and a study comparing continuous treatment during 3 months with conventional chemotherapy during 6 months. The later because it compared 2 durations less than or equal to 6 months. Shorter duration of chemotherapy (3-6 months) compared with longer duration (9-12 months) was not associated to poorer RFS (RR =0.96, 95% CI : 0.90-1.02) and OS (RR = 0.96 ; 95% CI : 0.91-1.02). AUTHORS' CONCLUSIONS: The present meta-analysis confirmed that adjuvant chemotherapy of CRC should not last for more than 6 months. Prolonged duration would result in lower benefit to risk ratio. However, the results do not make it possible to favour either 3 or 6 month durations. They should help design a future RCT comparing different durations of continuous treatment.

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC).

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.

Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells.

We have recently reported that the CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 induces proliferation, migration, and invasion of the Huh7 human hepatoma cells through its G-protein-coupled receptor CXCR4 and that glycosaminoglycans (GAGs) are involved in these events. Here, we demonstrate by surface plasmon resonance that the chemokine binds to GAG mimetics obtained by grafting carboxylate, sulfate or acetate groups onto a dextran backbone. We also demonstrate that chemically modified dextrans inhibit SDF-1/CXCL12-mediated in vitro chemotaxis and anchorage-independent cell growth in a dose-dependent manner. The binding of GAG mimetics to the chemokine and their effects in modulating the SDF-1/CXCL12 biological activities are mainly related to the presence of sulfate groups. Furthermore, the mRNA expression of enzymes involved in heparan sulfate biosynthesis, such as exostosin-1 and -2 or N-deacetylase N-sulfotransferases remained unchanged, but heparanase mRNA and protein expressions in Huh7 cells were decreased upon GAG mimetic treatment. Moreover, decreasing heparanase-1 mRNA levels by RNA interference significantly reduced SDF-1/CXCL12-induced extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Therefore, we suggest that GAG mimetic effects on SDF-1/CXCL12-mediated hepatoma cell chemotaxis may rely on decreased heparanase expression, which impairs SDF-1/CXCL12's signaling. Altogether, these data suggest that GAG mimetics may compete with cellular heparan sulfate chains for the binding to SDF-1/CXCL12 and may affect heparanase expression, leading to reduced SDF-1/CXCL12 mediated in vitro chemotaxis and growth of hepatoma cells.

Microsatellite instability does not predict the efficacy of chemotherapy in metastatic colorectal cancer. A systematic review and meta-analysis.

BACKGROUND: Microsatellite Instability (MSI) status is a good prognostic factor for colorectal cancer (CRC) but its predictive value for chemosensitivity remains controversial. A previous meta-analysis (MA) in the adjuvant setting showed that MSI-high (H) status did not predict the efficacy of chemotherapy. The predictive value of MSI status on the effect of metastatic chemotherapy was investigated by MA. PATIENTS AND METHODS: Studies were identified by electronic search through PubMed, Embase and ASCO proceedings online databases, using several key words (colorectal cancer, chemotherapy, microsatellite instability). For each study, the ratio of response rate (RR), complete (CR) and partial response (PR) divided by stable disease and progression was calculated. From 190 articles and 100 abstracts, only eight independent studies were selected. The data were analysed with a random-effect model (due to heterogeneity between studies) using EasyMA software. Statistical calculations were performed on six studies representing 964 patients (mean age 63 years; 91 MSI-H; 873 microsatellite stable (MSS) tumours). A total of 287 patients received 5-fluorouracil (5FU)-based chemotherapy, whereas 678 patients received combinations of 5FU or capecitabine with oxaliplatin and/or irinotecan. RESULTS: No benefit of metastatic chemotherapy in terms of RR for MSI-H patients compared with MSS patients was found. The global hazard ratio (HR) for RR was 0.82 (95% confidence interval, CI: 0.95; 0.65-1.03; p=0.09). CONCLUSION: MSI status does not predict the effect of chemotherapy which is similar in MSI-H and MSS metastatic CRC tumours.

Glycosaminoglycans and their synthetic mimetics inhibit RANTES-induced migration and invasion of human hepatoma cells.

The CC-chemokine regulated on activation, normal T-cell expressed, and presumably secreted (RANTES)/CCL5 mediates its biological activities through activation of G protein-coupled receptors, CCR1, CCR3, or CCR5, and binds to glycosaminoglycans. This study was undertaken to investigate whether this chemokine is involved in hepatoma cell migration or invasion and to modulate these effects in vitro by the use of glycosaminoglycan mimetics. We show that the human hepatoma Huh7 and Hep3B cells express RANTES/CCL5 G protein-coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans. Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells and to a lesser extent that of Hep3B cells. RANTES/CCL5 also stimulates the tyrosine phosphorylation of focal adhesion kinase and activates matrix metalloproteinase-9 in Huh7 hepatoma cells, resulting in increased invasion of these cells. The fact that RANTES/CCL5-induced migration and invasion of Huh7 cells are both strongly inhibited by anti-CCR1 antibodies and heparin, as well as by beta-d-xyloside treatment of the cells, suggests that CCR1 and glycosaminoglycans are involved in these events. We then show by surface plasmon resonance that synthetic glycosaminoglycan mimetics, OTR4120 or OTR4131, directly bind to RANTES/CCL5. The preincubation of the chemokine with each of these mimetics strongly inhibited RANTES-induced migration and invasion of Huh7 cells. Therefore, targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for human hepatocellular carcinoma.

Branched pentapeptides as potent inhibitors of the vascular endothelial growth factor 165 binding to Neuropilin-1: Design, synthesis and biological activity.

The demonstrated involvement of VEGF165/NRP-1 complex in pathological angiogenesis has catalyzed interest in blocking this interaction to combat angiogenesis dependent diseases. It was shown before that Lys-Pro-Pro-Arg is a fairly strong inhibitor of the VEGF165/NRP-1 interaction. Our current findings suggest that the side chain elongation of the Lys1 by branching it with additional homoarginine (Har) residue, to obtain Lys(Har)-Pro-Pro-Arg, allows more effective inhibition. Moreover, increasing the flexibility of the middle part of molecule, in particular with simultaneous introduction of additional interacting elements at the second or third position, produced compounds up to 30-fold more active (IC50 = 0.2 µM) than the heptapeptide ATWLPPR (A7R) which is one of the first peptide known as an effective antagonist of the VEGF165 binding to NRP-1 and in vivo decreases breast cancer angiogenesis and growth. Herein, we present also the structure-activity study of Lys(Har)-Pro-Pro-Arg, discussing the design, synthesis, inhibitory activity, proteolytic stability and molecular modeling of the prepared derivatives. For two of the most active analogs the high proteolytic stability was also observed. These studies provide the next step for elucidating the optimal structure of the small peptidic inhibitors of VEGF165/NRP-1 interaction that could serve as research tools or be prospective drug candidates.

Effects of statins on bone mineral density: a meta-analysis of clinical studies.

CONTEXT: Statins inhibit HMG-CoA reductase, preventing synthesis of mevalonate but also of isoprenoids, which affect osteoclast activity. Amino-bisphosphonates share this effect. In vitro and in vivo, statins show convincing anabolic and anti-resorptive bone effects. However, in a clinical meta-analysis (MA), they did not prevent hip fractures. OBJECTIVE AND DESIGN: Our meta-analysis studied the impact of statins on bone mineral density (BMD) at various sites and compared the effects of lipophilic and more hydrophilic statins. DATA SOURCES: Our PubMed and Embase queries using two keywords (statins, BMD) were updated to October 2006. DATA COLLECTION: Two readers independently collected BMDs from studies. DATA SYNTHESIS: Twenty-one studies, mostly observational (three randomized controlled trials and one pseudo-randomized study), were assessed. Two studies were excluded (no control groups). Three studies could not be analyzed. The sixteen studies analyzed mainly included postmenopausal osteopenic women (2971 patients under statins). Statins significantly increased BMD at total hip (TH) and femoral neck (FN). Effect sizes (ESs) were modest: 0.21 at TH (95% confidence interval [CI]: 0.16-0.25) and 0.20 at FN (CI: 0.08-0.28). Among women, statins acted similarly (ES: 0.20 for TH and 0.18 for FN; CI: 0.14-0.25 and 0.06-0.31 respectively); lipophilic statins (simvastatin, lovastatin) almost entirely caused this effect, at both TH (ES: 0.20; CI: 0.15-0.26) and FN (ES: 0.22; CI: 0.06-0.37). CONCLUSION: Our findings of modest but statistically significant beneficial effects of statins on hip BMD should promote large double-blind randomized controlled trials on their bone effects, in view of their major beneficial cardiovascular effects with excellent safety profile.

Expression of ghrelin in fundus is increased after gastric banding in morbidly obese patients.

BACKGROUND: Ghrelin, a 28 amino-acid acylated orexigenic peptide secreted by the stomach, acts on the hypothalamic arcuate nucleus which stimulates feeding behavior. Serum ghrelin level increases during fasting and decreases after a meal. Serum ghrelin is low in obese patients.Whether ghrelin is implicated in weight loss in obese patients after laparoscopic adjustable gastric banding (LAGB) is still debated. In this study, we assessed serum ghrelin level and gastric fundus expression before and 1 year after LAGB. METHODS: Gastric fundus expression of ghrelin was assessed by immunohistochemistry using a rabbit anti-human ghrelin antibody simultaneously with serum total ghrelin levels (RIA) in 13 obese patients (2 men and 11 women) after an overnight fast, before LAGB and 1 year after. Immunostaining was "blindly" analyzed by a single pathologist, measuring the density of stained fundic cells near muscularis mucosa. RESULTS: Mean age of the 13 patients was 41 years, and mean baseline BMI was 46 kg/m2. Pre- and post-LAGB gastric expression of ghrelin was analyzable in 11 patients. It was always identified, mostly with moderate or intense staining. Mean density of stained cells significantly increased 1 year after LAGB: 31/mm2 (21-38) before vs 38/mm2 (27-57) after surgery (P<0.01). This increase did not correlate with changes in BMI, nor did pre- or postoperative gastric expression of ghrelin correlate with the corresponding serum values. CONCLUSION: We showed for the first time that ghrelin expression assessed by immunohistochemistry was present in the fundus of all 11 obese patients and that it was significantly increased 1 year after LAGB, which would exclude a pathogenetic role of ghrelin in weight loss after LAGB.

Structure-function analysis of the antiangiogenic ATWLPPR peptide inhibiting VEGF(165) binding to neuropilin-1 and molecular dynamics simulations of the ATWLPPR/neuropilin-1 complex.

Heptapeptide ATWLPPR (A7R), identified in our laboratory by screening a mutated phage library, was shown to bind specifically to neuropilin-1 (NRP-1) and then to selectively inhibit VEGF(165) binding to this receptor. In vivo, treatment with A7R resulted in decreasing breast cancer angiogenesis and growth. The present work is focused on structural characterization of A7R. Analogs of the peptide, obtained by substitution of each amino acid with alanine (alanine-scanning) or by amino acid deletion, have been systematically assayed to determine the relative importance of the side chains of each residue with respect to the inhibitory effect of A7R on VEGF(165) binding to NRP-1. We show here the importance of the C-terminal sequence LPPR and particularly the key role of C-terminal arginine. In solution, A7R displays significant secondary structure of the backbone adopting an extended conformation. However, the functional groups of arginine are very flexible in the absence of NRP-1 pointing to an induced fit upon binding to the receptor. A MD trajectory of the A7R/NRP-1 complex in explicit water, based on the recent tuftsin/NRP-1 crystal structure, has revealed the hydrogen-bonding network that contributes to A7R's binding activity.

Physicochemical properties and in vitro cytotoxicity of iron oxide-based nanoparticles modified with antiangiogenic and antitumor peptide A7R.

Superparamagnetic iron oxide-based nanoparticles (SPIONs) are promising carriers as targeted drug delivery vehicles, because they can be guided to their target with the help of an external magnetic field. Functionalization of nanoparticles' surface with molecules, which bind with high affinity to receptors on target tissue significantly facilitates delivery of coated nanoparticles to their targeted site. Here, we demonstrate conjugation of an antiangiogenic and antitumor peptide ATWLPPR (A7R) to SPIONs modified with sebacic acid (SPIONs-SA). Successful conjugation was confirmed by various analytical techniques (FTIR, SERS, SEM-EDS, TEM, TGA). Cell cytotoxicity studies, against two cell lines (HUVEC and MDA-MB-231) indicated that SPIONs modified with A7R reduced HUVEC cell viability at concentrations higher than 0.01 mg Fe/mL, in comparison to cells that were exposed to either the nanoparticles modified with sebacic acid or A7R peptide solely, what might be partially caused by a process of internalization.

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1.

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence. Here, we present a structure-activity relationship study of the systematic optimization of amino acid residues in positions 1-3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP-1.

Delivery of the bacterial nitroreductase gene into endothelial cells prolongs the survival of tumour-bearing mice by bystander mechanisms.

A current target of cancer gene therapy is tumour vasculature. We present a gene-directed enzyme prodrug therapy (GDEPT) approach to target tumours in vivo by modifying endothelial cells (ECs) with the Escherichia coli nitroreductase (ntr) gene. Firstly, we isolated two ntr-transfected clones of the human umbilical vein endothelial cell line (HUV-EC-C/ntr+) that showed a differential sensitivity in vitro to the prodrug, dinitroaziridinylbenzamide (CB1954), with respect to untransfected HUV-EC-C cells (HUV-EC-C/ntr-). Then, these cells were injected subcutaneously into nude mice, either in association with the murine melanoma cell line, B16-F10 ('co-injected' groups), or into tumour-bearing animals ('post-injected' groups). After intratumoural injection, we demonstrated, using PCR analysis, that human ECs resided in the site of the injection without spreading to other organs, such as the liver or lung. After the treatment of mice with CB1954, we observed a prolonged survival of animals carrying the HUV-EC-C/ntr+ clones with respect to control animals injected with HUV-EC-C/ntr- cells. Significant differences in tumour growth were also observed and, after immuno-histological analysis, tumours carrying HUV-EC-C/ntr+ clones showed large areas of tumour necrosis, probably due to tumour ischemia, as well as the presence of major histocompatibility complex class-II (MHC-II) positive cells. Collectively, our data indicate that targeting of the tumour vasculature by this GDEPT strategy may be an efficient approach for cancer treatment in vivo, depending on two possible bystander mechanisms based on tumour ischemia and immune cell activation.

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.

Serum procalcitonin in uncomplicated falciparum malaria: a preliminary study.

BACKGROUND: Procalcitonin (PCT) has been found elevated in complicated forms of Plasmodium falciparum malaria. Its usefulness has almost never been assessed in uncomplicated falciparum malaria. METHOD: We assessed diagnostic and prognostic value of PCT in a prospective series of 25 adults with uncomplicated P. falciparum malaria. Patients originated mainly from western Africa and were infected during a stay back in their native country (19 semi-immune and 6 non-immune subjects; 11 had not received any chemoprophylaxis). RESULTS: Parasitaemia ranged from 0.01 to 3%. Eighteen patients had their first PCT determined at admission or within 24h thereafter (mean +/- SD: 3.0 +/- 4.6 ng/ml; range: 0.1-19.7). PCT was higher than 0.5 ng/ml in 14 patients (78%), higher than 2 ng/ml in 7 (39%). PCT correlated with parasitaemia (r = 0.53; p = 0.027), not with C-reactive protein (CRP). Delay between first symptoms and diagnosis was much longer among patients with PCT higher than 2 ng/ml than among those with a lower PCT. CONCLUSION: PCT was often elevated in uncomplicated malaria, especially when delay between first symptoms and diagnosis was long or parasitaemia was high (prognostic marker).