ngr-peptide and Neoplasms

Angiogenesis is an active process, regulating new vessel growth, and is crucial for the survival and growth of tumours next to other complex factors in the tumour microenvironment. We present possible molecular imaging approaches for tumour vascularisation and vitality, focusing on radiopharmaceuticals (tracers). Molecular imaging in general has become an integrated part of cancer therapy, by bringing relevant insights on tumour angiogenic status. After a structured PubMed search, the resulting publication list was screened for oncology related publications in animals and humans, disregarding any cardiovascular findings. The tracers identified can be subdivided into direct targeting of angiogenesis (i.e., vascular endothelial growth factor, laminin, and fibronectin) and indirect targeting (i.e., glucose metabolism, hypoxia, and matrix metallo-proteases, PSMA). Presenting pre-clinical and clinical data of most tracers proposed in the literature, the indirect targeting agents are not 1:1 correlated with angiogenesis factors but do have a strong prognostic power in a clinical setting, while direct targeting agents show most potential and specificity for assessing tumour vascularisation and vitality. Within the direct agents, the combination of multiple targeting tracers into one agent (multimers) seems most promising. This review demonstrates the present clinical applicability of indirect agents, but also the need for more extensive research in the field of direct targeting of angiogenesis in oncology. Although there is currently no direct tracer that can be singled out, the RGD tracer family seems to show the highest potential therefore we expect one of them to enter the clinical routine.

Topics: Animals; Biomarkers, Tumor; Cell Hypoxia; Glucose; Humans; Integrins; Matrix Metalloproteinases; Medical Oncology; Molecular Imaging; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Positron-Emission Tomography; Tomography, Emission-Computed, Single-Photon; Vascular Endothelial Growth Factor A

Besides the common issue of drug-resistance, the conventional approaches for cancer diagnostics and treatment are constantly challenged by poor selectivity and limited access to neoplastic cells, which not only lead to the dose-limiting effect on the tumor region, but also bring side-effects to healthy cells/tissues. In recent years, a novel strategy has arisen to target the vasculature of tumors for drug-delivery and molecular imaging, based on the success of anti-angiogenic therapy. In addition to being easily accessible, the endothelial cells of tumor vasculature are also genetically stable and thus do not develop drug-resistance, making them ideal targets for chemotherapeutics and biomedical imaging. Among various ligands identified so far, the Asn-Gly-Arg (NGR) tripeptide can specifically target the neovasculature via interaction with the aminopeptidase N (APN/CD13) receptor which is highly up-regulated in the membranes of endothelial tumor cells. NGR-directed drug delivery as well as molecular imaging have therefore been undergone development, and appear to be intriguing approaches in current cancer research. Herein we highlight some recent developments of the NGR peptide based cancer therapy including drug-delivery and imaging studies, with future perspectives. Some of these agents have been under clinical trials, indicating promising future for the NGR-based drugs.

Topics: Animals; Antineoplastic Agents; Drug Delivery Systems; Humans; Molecular Imaging; Neoplasms; Neovascularization, Pathologic; Oligopeptides

In the last decades, NGR-containing peptides have been proved useful for ligand-directed targeted delivery of various chemotherapeutic drugs to tumor vasculature. Aminopeptidase N (APN; CD13) has been demonstrated to be a key binding site for NGR peptides on tumor vasculature. For drug targeting, chemical means have been applied to couple NGR-peptides to small molecule drugs, such as cytokines, antiangiogenic compounds, viral particles, contrast agents, DNA complexes and other biologic response modifiers. Some products have shown impressive results in preclinical animal models, such as NGR-TNF which was currently tested in Phase III trials. In this article we will review the process of NGR-to-isoDGR transition and provide suggestions for the design of the diverse NGR peptide-chemotherapeutics conjugates.

Topics: Angiogenesis Inhibitors; Animals; Clinical Trials as Topic; Drug Delivery Systems; Drug Evaluation, Preclinical; Humans; Neoplasms; Neovascularization, Pathologic; Oligopeptides

The enzyme aminopeptidase N (APN, also known as CD13) is a Zn(2+) dependent membrane-bound ectopeptidase that degrades preferentially proteins and peptides with a N-terminal neutral amino acid. Aminopeptidase N has been associated with the growth of different human cancers and suggested as a suitable target for anti-cancerous therapy. Different approaches have been used to develop new drugs directed to this target, including enzyme inhibitors as well as APN-targeted carrier constructs. This review discusses the prevalence and possible function of APN in malignant diseases, mainly solid tumors, as well as its "drugability" evaluated in preclinical in vivo models, and also provides a brief overview of current clinical trials focused on APN.

Topics: CD13 Antigens; Cell Proliferation; Clinical Trials as Topic; Glycine; Humans; Hydroxamic Acids; Leucine; Neoplasm Invasiveness; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Prognosis; Protease Inhibitors

Various peptide sequences have been discovered by selecting peptide-phage display libraries in vitro against specific receptors or in vivo in tumor-bearing animals. One class of these peptides is characterized by the presence of Asn-Gly-Asp (NGR), a structural motif that can recognize the endothelium and other cells of neoangiogenic vessels. Because of this property these peptides have been used by several investigators to deliver a variety of drugs, cytokines, nanoparticles, viruses and imaging agents to tumor blood vessels. Here we review the reports on these conjugates and discuss the structural, functional and stability properties of NGR embedded into different molecular scaffolds.

Topics: Angiogenesis Inhibitors; Animals; Cytokines; Drug Delivery Systems; Humans; Neoplasms; Oligopeptides

We induced thrombosis of blood vessels in solid tumors in mice by a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA, targeting aminopeptidase N (CD13) and the integrin alpha(v)beta(3) (CD51/CD61) on tumor vascular endothelium. The designed fusion protein tTF-NGR retained its thrombogenic activity as demonstrated by coagulation assays. In vivo studies in mice bearing established human adenocarcinoma (A549), melanoma (M21), and fibrosarcoma (HT1080) revealed that systemic administration of tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels as shown by histologic analysis. tTF-NGR, but not untargeted tTF, induced significant tumor growth retardation or regression in all 3 types of solid tumors. Thrombosis induction in tumor vessels by tTF-NGR was also shown by contrast enhanced magnetic resonance imaging (MRI). In the human fibrosarcoma xenograft model, MRI revealed a significant reduction of tumor perfusion by administration of tTF-NGR. Clinical first-in-man application of low dosages of this targeted coagulation factor revealed good tolerability and decreased tumor perfusion as measured by MRI. Targeted thrombosis in the tumor vasculature induced by tTF-NGR may be a promising strategy for the treatment of cancer.

Topics: Adult; Angiogenesis Inhibitors; Animals; Cells, Cultured; Drug Delivery Systems; Embolism; Female; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Salvage Therapy; Thromboplastin; Xenograft Model Antitumor Assays

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.

Topics: Angiogenesis Inhibitors; Cell Line, Tumor; Crystallography, X-Ray; Human Umbilical Vein Endothelial Cells; Humans; Models, Molecular; Neoplasms; Oligopeptides; Recombinant Fusion Proteins; Signal Transduction; Tumor Necrosis Factor-alpha

The biological behavior of

Topics: Acetates; Cell Line, Tumor; Chelating Agents; Drug Delivery Systems; Ethylenediamines; Gallium Radioisotopes; Heterocyclic Compounds, 1-Ring; Humans; Molecular Imaging; Neoplasms; Oligopeptides; Radiopharmaceuticals

As current tumor chemotherapy faces many challenges, it is important to develop drug delivery systems with increased tumor-targeting ability, enhanced therapeutic effects and reduced side effects. In this study, a pH-sensitive liposome was constructed containing CHEMS-anchored PEG2000 for extended circulation and NGR peptide as the targeting moiety. The NGR-modified docetaxel-loaded pH-sensitive extended-circulation liposomes (DTX/NGR-PLL) prepared possess suitable physiochemical properties, including particle size of approximately 200nm, drug encapsulation efficiency of approximately 70%, and pH-sensitive drug release properties. Experiments performed in vitro and in vivo on human fibrosarcoma cells (HT-1080) and human breast adenocarcinoma cells (MCF-7) verified the specific targeting ability and enhanced antitumor activity to HT-1080 cells. The results of intravenous administration demonstrated that NGR-modified liposomes can significantly and safely accumulate in tumor tissue in xenografted nude mice. In conclusion, the liposomes constructed hold promise as a safe and efficient drug delivery system for specific tumor treatment.

Topics: Animals; Cell Line, Tumor; Cholesterol Esters; Delayed-Action Preparations; Docetaxel; Drug Delivery Systems; Drug Liberation; Humans; Hydrogen-Ion Concentration; Liposomes; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Oligopeptides; Polyethylene Glycols; Taxoids; Xenograft Model Antitumor Assays

Previously it was shown that the recombinant EGFR/CD13 bi-targeted fusion protein ER(Fv)‑LDP-NGR which consists of an anti‑EGFR scFv antibody fragment, a tri‑cyclic NGR peptide, and a lidamycin-derived apoprotein, inhibited the proliferation of cancer cells and markedly suppressed tumor growth of breast carcinoma MCF-7 xenografts in athymic mice. This study investigated the mechanism of action of the fusion protein. Human breast cancer MCF-7 cells, lung adenocarcinoma A549 cells, and microvascular endothelial HMEC‑1 cells were used for a series of assays and determinations. ER(Fv)‑LDP-NGR downregulated the transcription and expression of the target proteins EGFR and CD13, and interfered with the intracellular EGFR signaling pathway, cell cycle signaling pathway and apoptotic pathway. It induced apoptosis, inhibited proliferation, caused cell cycle G2/M phase arrest, and suppressed cell migration. Accompanied by weakening the capability to degrade extracellular matrix, ER(Fv)‑LDP-NGR depressed the invasion capacity of cancer cells. In addition, ER(Fv)‑LDP-NGR prevented microvascular endothelial cells from tube formation, which is closely related to angiogenesis. In conclusion, the EGFR/CD13 bi-targeted fusion protein ER(Fv)‑LDP-NGR displays multi-functional characteristics, acting on both cancer cells and endothelial cells. It might be an effective agent for targeted cancer therapy.

Topics: A549 Cells; Apoptosis; CD13 Antigens; Cell Cycle; Cell Line; Cell Movement; Cell Proliferation; Down-Regulation; ErbB Receptors; Humans; MCF-7 Cells; Neoplasms; Oligopeptides; Recombinant Fusion Proteins; Signal Transduction; Single-Chain Antibodies; Transcription, Genetic

The enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors.. Both yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by an Escherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs.. This study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cytosine Deaminase; Drug Delivery Systems; Flucytosine; Human Umbilical Vein Endothelial Cells; Humans; Neoplasms; Oligopeptides; Prodrugs; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Near-infrared fluorescence (NIRF) imaging is an emerging imaging technique for studying diseases at the molecular level. Optical imaging with a near-infrared emitting fluorophore for targeting tumor angiogenesis offers a noninvasive method for early tumor detection and efficient monitoring of tumor response to anti-angiogenesis therapy. CD13 receptor, a zinc-dependent membrane-bound ectopeptidase, plays important roles in regulating tumor angiogenesis and the growth of new blood vessels. In this chapter, we use CD13 receptor as an example to demonstrate how to construct CD13-specific NGR-containing peptides via bioorthogonal click chemistry for visualizing and quantifying the CD13 receptor expression in vivo by means of NIRF optical imaging.

Topics: Animals; CD13 Antigens; Cell Line, Tumor; Click Chemistry; Fluorescent Dyes; Humans; MCF-7 Cells; Mice; Molecular Imaging; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Tomography, Optical

Antimicrobial peptides (AMPs) with Asn-Gly-Arg (NGR) motif have potent cytotoxicity, preferably against tumor cells due to their binding to CD13 on tumor cells. However, the importance of αvβ3 expression for antitumor activity of AMPs containing NGR has not been clarified. This study was aimed at designing a new AMP containing NGR and testing their biological activity against different types of tumor cells with varying CD13 and αvβ3 expression. We first synthesized the new AMP containing NGR motif (CK21), which effectively entered into CD13+ HT-1080, but less into CD13- αvβ3+ MDA-MB-435 and further less into stable αvβ3-silencing MDA-MB-435 cells. Furthermore, CK21 displayed dose-dependent antiproliferation against these tumor cells and induced cell cycling arrest at G2/M phases and apoptosis of these tumor cells. In addition, CK21 inhibited the invasion of these tumor cells in vitro and inhibited the growth of implanted tumor cells in vivo. Particularly, the antitumor effect of CK21 in CD13+ HT-1080 was stronger than that of CD13- αvβ3+ MDA-MB-435 and much stronger than that of stable αvβ3-silencing MDA-MB-435. Our data indicated that the new AMPs containing NGR had potent antitumor activity against CD13+ or αvβ3+ tumor cells, preferably against CD13+ tumor cells, possibly through binding to CD13 or αvβ3 on tumor cells.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Blotting, Western; CD13 Antigens; Cell Movement; Cell Proliferation; Flow Cytometry; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Oligopeptides; Peptide Fragments; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

tTF-NGR retargets the extracellular domain of tissue factor via a C-terminal peptide GNGRAHA, a ligand of the surface protein aminopeptidase N (CD13) and upon deamidation of integrin αvβ3, to tumor vasculature. tTF-NGR induces tumor vascular infarction with consecutive antitumor activity against xenografts and selectively inhibits tumor blood flow in cancer patients. Since random PEGylation resulted in favorable pharmacodynamics of tTF-NGR, we performed site-directed PEGylation of PEG units to the N-terminus of tTF-NGR to further improve the antitumor profile of the molecule. Mono-PEGylation to the N-terminus did not change the procoagulatory activity of the tTF-NGR molecule as measured by Factor X activation. Experiments to characterize pharmacokinetics in mice showed a more than 1 log step higher mean area under the curve of PEG20k-tTF-NGR over tTF-NGR. Acute (24 h) tolerability upon intravenous application for the mono-PEGylated versus non-PEGylated tTF-NGR compounds was comparable. PEG20k-tTF-NGR showed clear antitumor efficacy in vivo against human tumor xenografts when systemically applied. However, site-directed mono-PEGylation to the N-terminus does not unequivocally improve the therapeutic profile of tTF-NGR.

Topics: Animals; Cell Line, Tumor; Cloning, Molecular; Humans; Mass Spectrometry; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Polyethylene Glycols; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins; Thromboplastin

The asparagine-glycine-arginine (NGR) peptide sequence found by phage display, was radiolabeled with technetium-99m and tested in different tumor models. Similar uptake occurred with ovarian and lung tumor cells. Biodistribution of the radiotracer revealed predominant renal excretion with more substantial uptake in animals bearing ovarian tumor cells. In contrast imaging studies indicated better visualization for lung tumor. NGR peptide was characterized as a promising diagnostic candidate, particularly for lung cancer. Improvements are envisaged using NGR combined with RGD as a heterodimer molecule.

Topics: Animals; Cell Line, Tumor; Female; Lung Neoplasms; Male; Mice; Neoplasms; Oligopeptides; Ovarian Neoplasms; Peptide Library; Radiopharmaceuticals; Technetium

Cell-penetrating peptides (CPPs) mediated tumor-oriented nanocarriers have been widely studied by researchers recently. However, applications of CPPs in vivo were usually hampered by their loss in untargeted tissues and enzymatic degradation. These shortfalls required strategies to camouflage CPPs before their arrival at the targeted site. In this work, we constructed a thermosensitive liposome (TSL) containing Asparagines-Glycine-Arginine (NGR) peptide as the targeting moiety and heat-activable cell-penetrating peptide-doxorubicin conjugate for enhancing specific cancer therapy. Different to the masking strategies of CPPs reported, CPPs existing in conjugation form of CPPs and doxorubicin (CPP-Dox) were hidden in TSL to cloak and protect CPPs. Meanwhile, NGR moiety and local tumor hyperthermia were utilized to achieve specific targeting of CPPs to the tumor. The nanocarrier (CPP-Dox/NGR-TSL) prepared in this work possessed suitable physiochemical properties such as small particle size of about 90 nm, high drug encapsulation efficiency of approximately 95%, good stability in the medium containing 10% fetal bovine serum (FBS) and so on. In vitro experiments on Human fibrosarcoma cells (HT-1080) and human breast adenocarcinoma cells (MCF-7) verified the specific targeting ability and enhanced intracellular drug delivery of the liposomes to HT-1080 cells. Furthermore, comparing with NGR-targeted TSL containing Dox (Dox/NGR-TSL), the results of intravenous administration showed CPP-Dox/NGR-TSL significantly inhibited tumor growth in nude mice xenografted HT-1080 tumors and excellent body safety. In conclusion, the nanocarrier constructed in this study would be a safe and efficiently drug delivery system for specific cancer treatment.

Topics: Animals; Antineoplastic Agents; Cattle; Cell Line, Tumor; Cell Proliferation; Cell-Penetrating Peptides; Doxorubicin; Endocytosis; Female; Flow Cytometry; Humans; Ligands; Mice; Mice, Nude; Neoplasms; Oligopeptides; Phosphatidylethanolamines; Polyethylene Glycols; Temperature

Pathological angiogenesis is crucial in tumor growth, invasion and metastasis. Previous studies demonstrated that the vascular endothelial growth inhibitor (VEGI), a member of the tumor necrosis factor superfamily, can be used as a potent endogenous inhibitor of tumor angiogenesis. Molecular probes containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 receptor which is overexpressed on neovasculature and several tumor cells. Near-infrared fluorescence (NIRF) optical imaging for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The aim of this study was to develop a new NIRF imaging probe on the basis of an NGR-VEGI protein for the visualization of tumor vasculature. The NGR-VEGI fusion protein was prepared from prokaryotic expression, and its function was characterized in vitro. The NGR-VEGI protein was then labeled with a Cy5.5 fluorophore to afford Cy5.5-NGR-VEGI probe. Using the NIRF imaging technique, we visualized and quantified the specific delivery of Cy5.5-NGR-VEGI protein to subcutaneous HT-1080 fibrosarcoma tumors in mouse xenografts. The Cy5.5-NGR-VEGI probe exhibited rapid HT-1080 tumor targeting, and highest tumor-to-background contrast at 8 h post-injection (pi). Tumor specificity of Cy5.5-NGR-VEGI was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR-VEGI (20 mg/kg). Ex vivo NIRF imaging further confirmed in vivo imaging findings, demonstrating that Cy5.5-NGR-VEGI displayed an excellent tumor-to-muscle ratio (18.93 ± 2.88) at 8 h pi for the non-blocking group and significantly reduced ratio (4.92 ± 0.75) for the blocking group. In conclusion, Cy5.5-NGR-VEGI provided highly sensitive, target-specific, and longitudinal imaging of HT-1080 tumors. As a novel theranostic protein, Cy5.5-NGR-VEGI has the potential to improve cancer treatment by targeting tumor vasculature.

Topics: Animals; Apoptosis; Carbocyanines; Drug Delivery Systems; Female; Fluorescent Dyes; Humans; Mice; Mice, Nude; Molecular Imaging; Molecular Probes; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Spectroscopy, Near-Infrared; Tumor Necrosis Factor Ligand Superfamily Member 15

The aim of this study was to prepare a new neovascularity targeting antitumor drug delivery system mediated by single-walled carbon nanotubes (SWNTs).. In this study, antiangiogenesis agent 2-methoxyestradiol was loaded by SWNTs via π~π accumulation. The SWNTs were then linked with NGR (Asn-Gly-Arg) peptide, which could target tumor angiogenesis. This drug delivery system was characterized by transmission electron microscope, scanning electron microscopy, and atomic force microscope analysis. The suppression efficacy of tumor growth in cultured breast cancer cell line was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The in vivo antitumor activity was evaluated on the Sarcoma (S180) tumor-bearing mice model.. The characteristics of this drug delivery system showed that the particle of complex was 190 ± 4.3 nm in size distribution and 23.56 ± 2.03 mV in zeta potential. The inhibition ratio of this SWNTs drug delivery system at 24, 48, and 72 h was about 57.7%, 83.6%, and 88.2%. Compared with normal saline group, the relative tumor volumes in the 2ME, SWNTs-2ME, and NGR-SWNTs-2ME groups were decreased 1 week after administration.. This novel neovascularity targeting drug delivery system containing NGR-SWNTs-2ME may be beneficial to improve treatment efficacy and minimize side effects in future cancer therapy.

Topics: 2-Methoxyestradiol; Animals; Cell Line, Tumor; Drug Delivery Systems; Estradiol; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Nanotubes, Carbon; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Tissue Distribution; Tubulin Modulators

Single-walled carbon nanotubes (SWNTs) are special nano-materials which exhibit interesting physical and chemical properties, presenting new opportunities for biomedical research and applications. In this study, we have successfully adopted a novel strategy to chemically functionalize SWNTs with polyethylenimine (PEI) through purification, oxidation, amination and polymerization, which were then bound by DSPE-PEG2000-Maleimide for further conjugation with the tumor targeting NGR (Cys-Asn-Gly-Arg-Cys-) peptide via the maleimide group and sulfhydryl group of cysteine, and finally hTERT siRNA was loaded to obtain a novel tumor targeting siRNA delivery system, designated as SWNT-PEI/siRNA/NGR. The results showed that SWNT-PEI/siRNA/NGR could efficiently cross cell membrane, induced more severe apoptosis and stronger suppression in proliferation of PC-3 cells in vitro. Furthermore, in tumor-bearing mice model the delivery system exhibited higher antitumor activity due to more accumulation in tumor without obvious toxicity in main organs. The combination of RNAi and near-infrared (NIR) photothermal therapy significantly enhanced the therapeutic efficacy. In conclusion, SWNT-PEI/siRNA/NGR is a novel and promising anticancer system by combining gene therapy and photothermal therapy.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Drug Delivery Systems; Endocytosis; Gene Expression Regulation, Neoplastic; Humans; Hyperthermia, Induced; Lasers; Male; Mice; Mice, Inbred BALB C; Nanotubes, Carbon; Neoplasms; Oligopeptides; Organ Specificity; Phototherapy; Polyethyleneimine; RNA Interference; RNA, Messenger; RNA, Small Interfering; Spectroscopy, Fourier Transform Infrared; Telomerase; Transfection

Poor penetration of antitumor drugs into the extravascular tumor tissue is often a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvβ3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here, we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Delivery Systems; Drug Design; Humans; Mice; Neoplasms; Oligopeptides; Protein Binding

The NGR-containing peptides have been shown to bind specifically to CD13/aminopeptidase N (APN) receptor, one of the attractive tumor vasculature biomarkers. In this study, we evaluated (64)Cu-labeled monomeric and dimeric NGR peptides for microPET imaging of CD13 receptor expression in vivo. Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. NGR-containing peptides were conjugated with 1,4,7,10-tetraazadodecane-N,N',N″,N‴-tetraacetic acid (DOTA) and labeled with (64)Cu (t(1/2) = 12.7 h) in ammonium acetate buffer. The resulting monomeric ((64)Cu-DOTA-NGR1) and dimeric ((64)Cu-DOTA-NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal micorPET, and biodistribution studies. In vitro studies demonstrated that CD13 receptors are overexpressed in human fibrosarcoma HT-1080 cells and negative in human colon adenocarcinoma HT-29 cells. The binding affinity of (64)Cu-DOTA-NGR2 to HT-1080 cells was measured to be within low nanomolar range and about 2-fold higher than that of (64)Cu-DOTA-NGR1. For small animal microPET studies, (64)Cu-DOTA-NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to (64)Cu-DOTA-NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 in CD13-negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a coinjected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. We concluded that both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. (64)Cu-DOTA-NGR2 showed higher tumor uptake and better tumor retention than (64)Cu-DOTA-NGR1, presumably due to bivalency effect and increase in apparent molecular size. (64)Cu-DOTA-NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expression in vivo.

Topics: Animals; CD13 Antigens; Cell Line, Tumor; Copper Radioisotopes; Female; HT29 Cells; Humans; Mice; Mice, Nude; Neoplasms; Oligopeptides; Positron-Emission Tomography; Radiopharmaceuticals; Tissue Distribution

Cancer is responsible for millions of immature deaths every year and is an economical burden on developing countries. One of the major challenges in the present era is to design drugs that can specifically target tumor cells not normal cells. In this context, tumor homing peptides have drawn much attention. These peptides are playing a vital role in delivering drugs in tumor tissues with high specificity. In order to provide service to scientific community, we have developed a database of tumor homing peptides called TumorHoPe.. TumorHoPe is a manually curated database of experimentally validated tumor homing peptides that specifically recognize tumor cells and tumor associated microenvironment, i.e., angiogenesis. These peptides were collected and compiled from published papers, patents and databases. Current release of TumorHoPe contains 744 peptides. Each entry provides comprehensive information of a peptide that includes its sequence, target tumor, target cell, techniques of identification, peptide receptor, etc. In addition, we have derived various types of information from these peptide sequences that include secondary/tertiary structure, amino acid composition, and physicochemical properties of peptides. Peptides in this database have been found to target different types of tumors that include breast, lung, prostate, melanoma, colon, etc. These peptides have some common motifs including RGD (Arg-Gly-Asp) and NGR (Asn-Gly-Arg) motifs, which specifically recognize tumor angiogenic markers. TumorHoPe has been integrated with many web-based tools like simple/complex search, database browsing and peptide mapping. These tools allow a user to search tumor homing peptides based on their amino acid composition, charge, polarity, hydrophobicity, etc.. TumorHoPe is a unique database of its kind, which provides comprehensive information about experimentally validated tumor homing peptides and their target cells. This database will be very useful in designing peptide-based drugs and drug-delivery system. It is freely available at http://crdd.osdd.net/raghava/tumorhope/.

Topics: Carrier Proteins; Databases, Protein; Humans; Information Storage and Retrieval; Neoplasm Proteins; Neoplasms; Oligopeptides; Peptides; Software

Blood vessels in tumors express higher level of aminopeptidase N (APN) than normal tissues. Evidence suggests that the CNGRC motif is an APN ligand which targets tumor vasculature. Increased expression of APN in tumor vascular endothelium, therefore, offers an opportunity for targeted delivery of NGR peptide-linked drugs to tumors.. To determine whether an additional cyclic CNGRC sequence could improve endothelial cell homing and antitumor effect, human plasminogen kringle 5 (hPK5) was modified genetically to introduce a CNGRC motif (NGR-hPK5) and was subsequently expressed in yeast. The biological activity of NGR-hPK5 was assessed and compared with that of wild-type hPK5, in vitro and in vivo. NGR-hPK5 showed more potent antiangiogenic activity than wild-type hPK5: the former had a stronger inhibitory effect on proliferation, migration and cord formation of vascular endothelial cells, and produced a stronger antiangiogenic response in the CAM assay. To evaluate the tumor-targeting ability, both wild-type hPK5 and NGR-hPK5 were (99 m)Tc-labeled, for tracking biodistribution in the in vivo tumor model. By planar imaging and biodistribution analyses of major organs, NGR-hPK5 was found localized to tumor tissues at a higher level than wild-type hPK5 (approximately 3-fold). Finally, the effects of wild-type hPK5 and NGR-modified hPK5 on tumor growth were investigated in two tumor model systems. NGR modification improved tumor localization and, as a consequence, effectively inhibited the growth of mouse Lewis lung carcinoma (LLC) and human colorectal adenocarcinoma (Colo 205) cells in tumor-bearing mice.. These studies indicated that the addition of an APN targeting peptide NGR sequence could improve the ability of hPK5 to inhibit angiogenesis and tumor growth.

Topics: Amino Acid Motifs; Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chick Embryo; Female; Humans; Mice; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Peptide Fragments; Plasminogen; Recombinant Fusion Proteins

We have designed a PEGylated LPD (liposome-polycation-DNA) nanoparticle for systemic, specific, and efficient delivery of small interfering RNA (siRNA) into solid tumors in mice by modification with NGR (aspargine-glycine-arginine) peptide, targeting aminopeptidase N (CD13) expressed in the tumor cells or tumor vascular endothelium. LPD-PEG-NGR efficiently delivered siRNA to the cytoplasm and downregulated the target gene in the HT-1080 cells but not CD13(-) HT-29 cells, whereas nanoparticles containing a control peptide, LPD-PEG-ARA, showed only little siRNA uptake and gene silencing activity. LPD-PEG-NGR efficiently delivered siRNA into the cytoplasm of HT-1080 xenograft tumor 4 hours after intravenous injection. Three daily injections (1.2 mg/kg) of c-myc siRNA formulated in the LPD-PEG-NGR effectively suppressed c-myc expression and triggered cellular apoptosis in the tumor, resulting in a partial tumor growth inhibition. When doxorubicin (DOX) and siRNA were co-formulated in LPD-PEG-NGR, an enhanced therapeutic effect was observed.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; CD13 Antigens; Cell Line, Tumor; Doxorubicin; Drug Delivery Systems; Endothelium, Vascular; Genes, myc; HT29 Cells; Humans; Liposomes; Mice; Mice, Nude; Nanoparticles; Neoplasms; Oligopeptides; RNA, Small Interfering; Xenograft Model Antitumor Assays

To induce tumor cell apoptosis, a modified 15 kDa actin linked with a peptide NGR "homing" into tumor or tumor vessels was expressed in Escherichia coli. After refolding and purification, this fusion protein NGR-15actin was labeled with FITC to testify whether NGR-15actin could integrate into the cytoskeleton. It was found that this targeted peptide could induce HepG2 and HeLa cells apoptosis through its effect on the cytoskeleton function by binding to cytoskeleton protein. Thus, targeted NGR-15actin could be a candidate molecule for the therapy of cancer.

Topics: Actins; Apoptosis; Escherichia coli; Gene Expression; HeLa Cells; Hep G2 Cells; Humans; Neoplasms; Oligopeptides; Recombinant Fusion Proteins

tTF-NGR consists of the extracellular domain of the (truncated) tissue factor (tTF), a central molecule for coagulation in vivo, and the peptide GNGRAHA (NGR), a ligand of the surface protein aminopeptidase N (CD13). After deamidation of the NGR-peptide moiety, the fusion protein is also a ligand for integrin αvβ3 (CD51/CD61). Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR showed binding to specific binding sites on endothelial cells in vitro as shown by flow cytometry. Subcutaneous injection of tTF-NGR into athymic mice bearing human HT1080 fibrosarcoma tumors induced tumor growth retardation and delay. Contrast enhanced ultrasound detected a decrease in tumor blood flow in vivo after application of tTF-NGR. Histological analysis of the tumors revealed vascular disruption due to blood pooling and thrombotic occlusion of tumor vessels. Furthermore, a lack of resistance was shown by re-exposure of tumor-bearing mice to tTF-NGR after regrowth following a first cycle of treatment. However, after subcutaneous (s.c.) push injection with therapeutic doses (1-5 mg/kg bw) side effects have been observed, such as skin bleeding and reduced performance. Since lethality started within the therapeutic dose range (LD10 approximately 2 mg/kg bw) no safe therapeutic window could be found. Limiting toxicity was represented by thrombo-embolic events in major organ systems as demonstrated by histology. Thus, subcutaneous injection of tTF-NGR represents an active, but toxic application procedure and compares unfavourably to intravenous infusion.

Topics: Angiogenesis Inhibitors; Animals; Blood Vessels; Cell Line, Tumor; Cells, Cultured; Drug Delivery Systems; Humans; Infarction; Injections, Subcutaneous; Mice; Mice, Nude; Neoplasms; Neovascularization, Pathologic; Oligopeptides; Recombinant Fusion Proteins; Thromboplastin; Xenograft Model Antitumor Assays

Adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. This strategy provided Ad with a very efficient entry pathway in both endothelial cells and tumor cells, with the highest efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, AdHNGR and AdFNGR were shown to bind not only to CD13 receptor, but also to alphavbeta3 integrins. Both vectors were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells, thus leading to a dramatic inhibition of their proliferation and increased cell death. Although AdHNGR and Adwt were found to display similar gene transduction efficacy in Lewis lung carcinoma (LLC), pseudotyping AdHNGR with an Ad3-fiber unmasked the ability of NGR-peptide to target these tumors. As a result, delivery of angiostatin K1-5 cDNA into highly aggressive tumors translated into a stronger inhibition of their growth. Altogether, our results suggest that NGR-bearing Ad are valuable tools to realize the potential of this anti-angiogenic approach to anti-tumor therapy.

Topics: Adenoviridae; Angiostatins; Animals; Capsid; Carcinoma, Lewis Lung; CD13 Antigens; Cell Line, Tumor; Cytokine Receptor gp130; Endothelial Cells; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Mice; Neoplasms; Oligopeptides; Transduction, Genetic