ABSTRACT
Materials for delivery of oligonucleotides need to be simple to produce yet effective in vivo to be considered for clinical applications. Formulations of biomaterials based on combinations of existing demonstrated polymeric gene carriers with targeted derivatives are potential candidates for rapid translation but have not been fully explored for siRNA applications. Here we investigated formulations based on derivatised PEI for delivery of siRNA to gastrointestinal cancer cells. siRNA was complexed with linear PEI alone or with a mixture of linear PEI and transferrin-conjugated branched PEI (TfPEI), and knockdown of reporter genes was investigated. Overall, the in vitro use of complexes containing TfPEI resulted in up to 93% knockdown at 72 h post-transfection. Sustained knockdown was also achieved in a bioluminescent xenograft model. When complexes were delivered intratumorally, a 43% reduction in luminescence was achieved in the treated group compared with the control group 48 h after treatment. For systemic administration, only the intraperitoneal route, and not the intravenous route was effective, with 49% knockdown achieved at 72 h and sustained up to 144 h (44%) after a single administration of TfPEI-complexed siRNA. No toxicity or induction of the interferon response was observed. These findings demonstrate that simple formulations of transferrin-conjugated PEI with a 'parent' polymer such as linear PEI have potential as a method for therapeutic delivery of siRNA when administered either intratumorally or systemically.
Subject(s)
Polyethyleneimine/administration & dosage , RNA, Small Interfering/chemistry , Transferrin/administration & dosage , Cell Line, Tumor , Drug Carriers , Gene Silencing , Humans , Polyethyleneimine/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transferrin/chemistryABSTRACT
VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.
Subject(s)
Carcinoma, Non-Small-Cell Lung/blood supply , Carcinoma, Non-Small-Cell Lung/metabolism , Gene Expression Regulation, Neoplastic , Hypoxia/metabolism , Lung Neoplasms/blood supply , Lung Neoplasms/metabolism , Neoplasm Proteins/biosynthesis , Sp1 Transcription Factor/biosynthesis , Vascular Endothelial Growth Factor A/biosynthesis , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Hypoxia , Cell Line, Tumor , Dactinomycin/pharmacology , Female , Histones/genetics , Histones/metabolism , Humans , Hypoxia/genetics , Hypoxia/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Neoplasm Proteins/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Nucleic Acid Synthesis Inhibitors/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding , Sp1 Transcription Factor/genetics , Vascular Endothelial Growth Factor A/genetics , p300-CBP Transcription Factors/genetics , p300-CBP Transcription Factors/metabolismABSTRACT
The ability to deliver genetic material for therapy remains an unsolved challenge in medicine. Natural gene carriers, such as viruses, have evolved sophisticated mechanisms and modular biopolymer architectures to overcome these hurdles. Here we describe synthetic multicomponent materials for gene delivery, designed with features that mimic virus modular components and which transfect specific cell lines with high efficacy. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery process. The experimentally facile assembly suggests these materials could form a generic class of carrier systems that could be customized for many different therapeutic settings.
Subject(s)
Biomimetic Materials/chemistry , Capsid Proteins/chemistry , Gene Transfer Techniques , Nanoparticles/chemistry , Neoplasms/metabolism , Nucleic Acids/chemistry , Polymers/chemistry , Biomimetic Materials/adverse effects , Capsid Proteins/metabolism , Endocytosis , Ethylene Oxide/adverse effects , Ethylene Oxide/chemistry , Gene Transfer Techniques/adverse effects , HCT116 Cells , HL-60 Cells , Hemolysis , Humans , Ligands , Nanoparticles/adverse effects , Nanoparticles/ultrastructure , Neoplasm Proteins/metabolism , Neoplasms/pathology , Neoplasms/therapy , Nucleic Acids/metabolism , Peptides/adverse effects , Peptides/chemistry , Polyamines/adverse effects , Polyamines/chemistry , Polyelectrolytes , Polyethylene Glycols/adverse effects , Polyethylene Glycols/chemistry , Polymers/adverse effects , Receptors, Transferrin/metabolism , Surface Properties , Transferrin/chemistry , Transferrin/metabolismABSTRACT
BACKGROUND AND AIMS: Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines. METHODS: Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT. RESULTS: The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins. CONCLUSION: The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.
Subject(s)
Helicobacter Infections/metabolism , Helicobacter pylori/pathogenicity , Neoplasm Proteins/biosynthesis , Stomach Neoplasms/microbiology , Animals , Cell Transformation, Neoplastic/genetics , Coculture Techniques , Disease Models, Animal , Epithelial Cells/pathology , Gastrins/biosynthesis , Gastrins/genetics , Gastrins/physiology , Gene Expression Regulation, Neoplastic , Helicobacter Infections/pathology , Heparin-binding EGF-like Growth Factor , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/physiology , Matrix Metalloproteinase 7/biosynthesis , Matrix Metalloproteinase 7/genetics , Matrix Metalloproteinase 7/physiology , Mesenchymal Stem Cells/pathology , Mice , Mice, Transgenic , Neoplasm Proteins/genetics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Tumor Cells, Cultured , Up-Regulation , VirulenceABSTRACT
UNLABELLED: Gastrin/cholecystokinin subtype 2 receptors (CCK-2Rs) are overexpressed in several tumor types and are, thus, a potential target for peptide receptor radionuclide therapy (PRRT) of cancer. To improve the in vivo performance of CCK-2R binding peptides, we have previously synthesized and screened a series of divalent gastrin peptides for improved biochemical and biologic characteristics. In this study, we explore in more detail the most promising of these compounds and compare its performance with a previously described monomeric peptide. METHODS: From six (111)In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-conjugated divalent gastrin peptides based on the C-terminal sequence of minigastrin, the maleimide-linked compound DOTA-GSC(succinimidopropionyl-EAYGWNleDF-NH(2))-EAYGWNleDF-NH(2) (MGD5) was selected. The in vitro stability, receptor binding, and internalization of (111)In-MGD5 were studied and compared with those of monomer compound (111)In-APH070. In vivo biodistribution and imaging using a SPECT/CT camera were also performed. RESULTS: More than 90% of the labeled divalent peptide remained intact after 20 h of incubation in plasma. The inhibitory concentration of 50% of the divalent peptide was 1.0 versus 5.6 nM for the monomer, and the dissociation constant was 0.7 versus 2.9 nM. The rate of internalization of the divalent peptide was twice that of the monomer. Tumor uptake of the divalent peptide in vivo was about 6 times that of the monomer. The rate of washout of the divalent peptide from the tumor was lower than that of the monomer. CONCLUSION: Dimerization of the CCK-2R binding site results in an increase in binding affinity and an increase in tumor uptake both in vitro and in vivo. It is likely that these increases would result in improved tumor-targeting efficiency in patients with CCK-2R-positive tumors.
Subject(s)
Gastrins/chemistry , Gastrins/metabolism , Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Receptor, Cholecystokinin B/metabolism , Amino Acid Sequence , Animals , Cell Line, Tumor , Female , Gastrins/pharmacokinetics , Heterocyclic Compounds, 1-Ring/chemistry , Isotope Labeling , Mice , Neoplasms/diagnostic imaging , Neoplasms/genetics , Protein Stability , Protein Transport , Rats , Tissue Distribution , Tomography, Emission-Computed, Single-Photon , Tomography, X-Ray ComputedABSTRACT
Angiomyolipomas are benign mesenchymal tumours of smooth muscle, blood vessels and fat which occur sporadically or associated with tuberous sclerosis and lymphangioleiomyomatosis (LAM), a rare cystic lung disease. Angiomyolipoma and LAM are caused by loss of function of either the tuberous sclerosis-1 or -2 genes resulting in activation of p70S6kinase (S6K1) and uncontrolled cellular proliferation. LAM and angiomyolipoma can be exacerbated by oestrogens but how this occurs is not understood. To address this question, we created a xenograft tumour system in nude mice using immortalised angiomyolipoma cells. Angiomyolipoma xenografts had active S6K1, p38, p42/44 MAPK and Akt; they grew more rapidly and had greater Akt phosphorylation after oestrogen treatment of tumour-bearing mice. Transcriptional profiling showed oestrogen induced 300 genes including extracellular matrix proteins, proteases, cell cycle regulatory proteins and growth factors including platelet derived growth factor-C (PDGF-C). Biologically active PDGF-C was produced by primary angiomyolipoma cells in culture and PDGF-C protein was present in the neoplastic smooth muscle cells of 5/5 human angiomyolipoma and 4/5 LAM tissues examined by immunohistochemistry. These findings suggest that the response to oestrogen in this model is mediated by activation of Akt and transcriptional events. This model may prove useful for studying the biology and effect of drugs on angiomyolipoma and diseases related to TSC.
Subject(s)
Angiomyolipoma/metabolism , Estrogens/pharmacology , Lung Neoplasms/metabolism , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Angiomyolipoma/genetics , Angiomyolipoma/physiopathology , Animals , Cell Line, Transformed , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , Lung Neoplasms/physiopathology , Lymphangioleiomyomatosis/genetics , Lymphangioleiomyomatosis/metabolism , Lymphangioleiomyomatosis/physiopathology , Lymphokines/genetics , Lymphokines/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Nude , Multiprotein Complexes , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Proteins , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , TOR Serine-Threonine Kinases , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Xenograft Model Antitumor AssaysABSTRACT
All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-beta1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was approximately 10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-beta1 revealed that RB200 bound HER-1 ligands, including transforming growth factor-alpha and heparin-binding EGF, and HER-3 ligands NRG1-alpha and NRG1-beta3. RB200 inhibited EGF- and NRG1-beta1-stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid-stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3.
Subject(s)
Immunoglobulin Fc Fragments/metabolism , Immunoglobulin Fc Fragments/pharmacology , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Xenograft Model Antitumor Assays , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Cell Proliferation/drug effects , Cloning, Molecular , Dimerization , Drug Synergism , Epidermal Growth Factor/metabolism , Humans , Immunoglobulin Fc Fragments/chemistry , Ligands , Mice , Neuregulin-1/metabolism , Phosphoserine/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Receptor, ErbB-2/chemistry , Receptor, ErbB-3/chemistry , TrastuzumabABSTRACT
The Shh pathway has been implicated in gastric carcinogenesis, and inhibition of this pathway has been shown to inhibit tumour growth in gastric cell lines. Assessing the in vivo efficacy of Shh pathway antagonists in blocking Shh signaling in the stomach is important for clinical trial design, but has not been previously investigated. We investigated the in vivo efficacy of a Shh antagonist, cyclopamine, in correlation to the secondary effects induced by this treatment on gastrin levels and acid secretion. Gastrin has been shown to induce Shh production, processing and activity, which is believed to be mediated by acid secretion. We tested this hypothesis and showed that hypergastrinaemia induces Shh production in vivo, and confirmed that this effect on Shh is mediated by acid secretion. We showed that cyclopamine treatment induces both hypergastrinaemia and Shh, and does not inhibit Gli-1. Inhibition of the effect of hypergastrinaemia on the Shh pathway, in cyclopamine-treated mice, was demonstrated by use of lansoprazole which concomitantly inhibited Gli-1, and did not increase Shh production. Therefore, this evidence suggests that hypergastrinaemia, via increased acid secretion, may increase expression of Shh and that Shh antagonists may require concomitant acid inhibition to successfully inhibit a pathway known to be up-regulated in gastric carcinogenesis.
Subject(s)
Gastric Acid/metabolism , Gastric Mucosa/metabolism , Gastrins/metabolism , Hedgehog Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Veratrum Alkaloids/pharmacology , 2-Pyridinylmethylsulfinylbenzimidazoles/pharmacology , Animals , Anti-Ulcer Agents/pharmacology , Gastrins/blood , Hedgehog Proteins/metabolism , Lansoprazole , Mice , Mice, Transgenic , Stomach/drug effectsABSTRACT
PURPOSE: Gastrin is known to enhance the growth of pancreatic carcinoma via the cholecystokinin (CCK)-2/gastrin receptor. We investigated the anti-tumor effect of Z-360 (calcium bis [(R)-(-)-3-[3-{5-cyclohexyl-1-(3,3-dimethyl-2-oxo-butyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl}ureido]benzoate]), a novel orally active CCK-2 receptor antagonist alone or combined with the chemotherapeutic agent, gemcitabine in human pancreatic adenocarcinoma cell lines. RESULTS: Z-360 potently inhibited specific binding of [3H]CCK-8 to the human CCK-2 receptor, with a Ki value of 0.47 nmol/l, and showed antagonistic activity for this receptor. The anti-tumor effect of Z-360 alone or combined with gemcitabine was assessed using subcutaneous xenografts of MiaPaCa2 and PANC-1 and an orthotopic xenograft model (PANC-1). Oral administration of Z-360 significantly inhibited the growth of MiaPaCa2 (41.7% inhibition at 100 mg/kg, P<0.01). Combined administration of Z-360 and gemcitabine significantly inhibited subcutaneous PANC-1 tumor growth compared with either agent alone (27.1% inhibition compared to effect with gemcitabine, P<0.05), and significantly prolonged survival compared with the vehicle control (median survival of 49 days in vehicle compared to 57 days in the combination group, P<0.05). In vitro studies showed that Z-360 significantly inhibited gastrin-induced proliferation of human CCK-2 receptor-expressing cells, and also significantly reduced gastrin-induced PKB/Akt phosphorylation to the level of untreated controls. CONCLUSION: In the present study, we have shown that Z-360 combined with gemcitabine can inhibit pancreatic tumor growth and prolong survival in a pancreatic carcinoma xenograft model, on a possible mode of action being the inhibition of gastrin-induced PKB/Akt phosphorylation through blockade of the CCK-2 receptor. Our results suggest that Z-360 may be a useful adjunct to gemcitabine for the treatment of pancreatic carcinoma and a therapeutic option for patients with advanced pancreatic cancer.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/pharmacology , Benzodiazepinones/pharmacology , Pancreatic Neoplasms/drug therapy , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzodiazepinones/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Disease Models, Animal , Female , Gastrins , Humans , Mice , Mice, Nude , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Cholecystokinin B/antagonists & inhibitors , Survival Rate , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
Gastrin gene expression is upregulated in a number of pre-malignant conditions and established cancer through a variety of mechanisms. Depending on the tissue where it is expressed and the level of expression, differential processing of the polypeptide product leads to the production of different biologically active peptides. In turn, acting through the classical CCK-2R receptor, CCK-2R isoforms and alternative receptors, these peptides trigger signalling pathways which influence the expression of downstream genes that affect cell survival, angiogenesis and invasion. Here we review this network of events, highlighting the importance of cellular context for interpreting the role of gastrin peptides and a possible role for gastrin in supporting the early stage of carcinogenesis.
Subject(s)
Cell Transformation, Neoplastic , Gastrins/chemistry , Gene Expression Regulation, Neoplastic , Peptides/chemistry , Animals , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Progression , Electron Transport Complex IV/metabolism , Gastrins/metabolism , Humans , Matrix Metalloproteinases/metabolism , Neoplasm Invasiveness , Neovascularization, PathologicABSTRACT
UNLABELLED: The gastrin/cholecystokinin-2 (CCK-2) receptor has been identified as a possible target for peptide receptor radionuclide imaging and therapy. Several radiolabeled peptides binding to this receptor have been explored in animal models and clinical trials but either low tumor uptake or high renal retention has been found. The aim of this study was to identify a peptide with improved tumor-to-kidney pharmacodynamics when compared with current candidates. METHODS: A small peptide-chelator library of 34 compounds based on the C-terminal sequences of CCK-8 or minigastrin was constructed. The peptides were radiolabeled with (111)In with high labeling efficiency (>90%), as determined by high-performance liquid chromatographic analysis. The labeled peptides were screened by assessing tumor and kidney uptake in pancreatic xenograft nude mouse models, including AR42J. An extensive biodistribution analysis was performed on the lead candidate from the library. RESULTS: Minigastrin analogs containing a pentaglutamate sequence showed the highest tumor uptake but very high renal retention. CCK analogs showed the lowest tumor and renal uptake. Deletion of the pentaglutamate sequence in the gastrin analogs lowered the tumor uptake by a factor of 3 but decreased the kidney uptake by a factor of 20. Insertion of histidine residues in the sequence reduced kidney uptake by a further factor of almost 2-fold. In AR42J tumor-bearing mice, the peptide with the sequence DOTA-HHEAYGWMDF-NH(2) (DOTA is tetraazacyclododecane tetraacetic acid) showed the highest tumor-to-kidney ratio of all peptides studied, with saturable uptake in target organs and low uptake by nontarget tissues other than the kidney. CONCLUSION: This peptide is a worthwhile candidate for clinical studies to determine whether it is suitable for use in peptide receptor-targeted radionuclide therapy.
Subject(s)
Gastrins/chemistry , Neoplasms/drug therapy , Receptors, Peptide/chemistry , Amino Acid Sequence , Animals , Cell Line, Tumor , Humans , Kidney/metabolism , Male , Mice , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , Rats , Receptor, Cholecystokinin B/chemistryABSTRACT
A DOTA-gastrin analogue (APH070) which, when labelled with (111)In, has high affinity for the gastrin/CCK-2 receptor (3nM) and low tumour to kidney ratio in animal models, has been formulated and manufactured for a clinical study. Oxidation of the peptide methionine residue greatly reduces receptor affinity, therefore development work focused on producing a stable intermediate drug product (iDP) whilst ensuring that the formulation, container, closure and manufacturing process did not inhibit the extremely sensitive radiolabelling reaction (itself a source of oxidation). Stress testing revealed that APH070 was stable at 2-8 degrees C at pH 6-9. Addition of an antioxidant (monothioglycerol) to the peptide formulation reduced stability when compared to buffer alone. Use of FluroTec (4023/50) stoppers (rather than FluroTec Plus (4110/40)) increased both the stability and radiolabelling efficiency of APH070. Long term stability (6 months) of the final formulation (1mg/ml APH070 in 0.01 M pH 7.2 phosphate buffer) stored at 5 degrees C in type I glass vials with FluroTec (4023/50) stoppers was 98.6+/-0.2% and 98.4+/-0.1% for upright and inverted samples, respectively. Clinical scale radiolabelling of the final formulation routinely achieves the specification of >85% (111)In-APH070 (unoxidised) stable for up to 2h after dilution with 0.9% w/v saline solution. Specific uptake of the radiopharmaceutical in CCK-2R-expressing AR42J tumours in nude mice has been demonstrated.
Subject(s)
Drug Packaging , Gastrins/chemical synthesis , Indium Radioisotopes , Isotope Labeling , Oligopeptides/chemical synthesis , Radiopharmaceuticals/chemical synthesis , Receptor, Cholecystokinin B/metabolism , Technology, Pharmaceutical/methods , Animals , Cell Line, Tumor , Chemistry, Pharmaceutical , Chromatography, High Pressure Liquid , Drug Stability , Drug Storage , Gastrins/metabolism , Hydrogen-Ion Concentration , Male , Methionine/chemistry , Mice , Mice, Nude , Neoplasms, Experimental/metabolism , Oligopeptides/metabolism , Oxidation-Reduction , Radiopharmaceuticals/metabolism , Reproducibility of Results , Solubility , Solutions , Temperature , Time FactorsABSTRACT
Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.
Subject(s)
Anilides/pharmacology , Apoptosis/drug effects , Capsaicin/pharmacology , Cinnamates/pharmacology , Diterpenes/pharmacology , Mitochondria, Heart/drug effects , TRPV Cation Channels/antagonists & inhibitors , Anilides/administration & dosage , Animals , Capsaicin/analogs & derivatives , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cells, Cultured , Cinnamates/administration & dosage , Diterpenes/administration & dosage , Dose-Response Relationship, Drug , Humans , Hydrogen Peroxide/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Membrane Potentials/drug effects , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Oxygen Consumption/drug effects , Rats , TRPV Cation Channels/drug effects , TRPV Cation Channels/metabolismABSTRACT
We have developed an improved and reliable method for stereoselective functionalization at C4 of naturally occurring (+)-catechin. Our method utilizes DDQ oxidation followed by trapping of the quinonemethide intermediate with allyl alcohol. The quinonemethide intermediate can be regenerated from the allyl ether by exposure to boron trifluoride diethyl etherate. This reactive intermediate can be trapped with a wide range of external nucleophiles. NBS bromination, lithium halogen exchange, and alkylation gave access to C8-allyl derivatives of (+)-catechin, and this allyl group was used in a series of cross-metathesis experiments to prepare novel dimeric catechin-derived products. Gallate ester derivatives of the novel C4- and C8-substituted catechins were prepared, and these materials were screened for potential anticancer activity in a range of human cancer cell lines. From these preliminary cytotoxicity assays (MTT) we found that C8-propyl-catechin gallate was more active (IC50 = 31 microM) than catechin gallate (CG, IC50 = 53 microM) or epicatechin gallate (ECG, IC50 = 76 microM) against the colorectal adenocarcinoma cell line HCT116. Differential sensitivity in pancreas (Pan1), bladder (RT112), stomach (MGLVA1), liver (HepG2), and fibroblasts (46Br.1G1) cell lines was also observed.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Catechin/analogs & derivatives , Antineoplastic Agents/chemistry , Catechin/chemical synthesis , Catechin/chemistry , Catechin/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , In Vitro Techniques , Molecular Conformation , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Gastrin is a growth factor for both gastrointestinal and non-gastrointestinal tumours. Endocytosis of gastrin has been demonstrated in tumour cell lines expressing cholecystokinin-B/gastrin receptor (CCK-BR); this has raised the possibility of receptor targeted therapy. The aim of this study was to examine endocytosis of gastrin and CCK-BR in tumour cell lines. A small gastrin analogue, RG-G7, and the anti-CCK-BR antibody, anti-GRE1, were fluorescently labelled and uptake by cancer cell lines including AR42J, HepG2, and C170HM2 as well as transfected NIH3T3 fibroblast cells was assessed using standard and confocal fluorescence microscopy. CCK-BR expression of cell lines was assayed by reverse transcription-polymerase chain reaction and Western blotting. Apoptosis was detected using a fluorescent TUNEL method. RG-G7 and anti-GRE1 antibody were specifically taken up by all cell lines expressing CCK-BR. In addition to cytoplasmic uptake with RG-G7 and anti-GRE1 the latter also showed specific uptake into the nucleus. A coincidence of anti-GRE1 and apoptosis was seen. Targeting CCK-BR by peptide or antibody may offer therapeutic opportunities for some cancers.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Colonic Neoplasms/metabolism , Liver Neoplasms/metabolism , Pancreatic Neoplasms/metabolism , Receptor, Cholecystokinin B/metabolism , Animals , Apoptosis/physiology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Endocytosis , Humans , Immunoblotting/methods , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Microscopy, Confocal/methods , NIH 3T3 Cells , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Receptor, Cholecystokinin B/biosynthesis , Receptor, Cholecystokinin B/geneticsABSTRACT
Gastrin is a pro-proliferative, anti-apoptotic hormone with a central role in acid secretion in the gastric mucosa and a long-standing association with malignant progression in transgenic mouse models. However, its exact role in human gastric malignancy requires further validation. Gastrin expression is tightly regulated by two closely associated hormones, somatostatin and gastrin-releasing peptide, and aspects of their interaction may be deregulated during progression to gastric adenocarcinoma. Furthermore, agonists and antagonists of the receptors for all three hormones have shown modest clinical efficacy against gastric adenocarcinoma, which might provide useful information on the future combined use of these agents.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Gastrins/metabolism , Precancerous Conditions/metabolism , Stomach Neoplasms/etiology , Stomach Neoplasms/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Antineoplastic Agents, Hormonal/therapeutic use , Apoptosis , Cancer Vaccines/therapeutic use , Cell Differentiation , Cell Movement , Cell Transformation, Neoplastic/pathology , Gastrin-Releasing Peptide/metabolism , Gastrins/antagonists & inhibitors , Gastrins/genetics , Gene Expression Regulation, Neoplastic , Helicobacter Infections/complications , Helicobacter Infections/metabolism , Humans , Mice , Neoplasm Invasiveness , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic/metabolism , Precancerous Conditions/pathology , Risk Factors , Somatostatin/metabolism , Stomach Neoplasms/blood supply , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathologyABSTRACT
Both gastrin and Helicobacter pylori have been shown capable of up-regulating gene expression and protein shedding of heparin-binding epidermal growth factor (HB-EGF). Furthermore, the bacteria have previously been shown to induce serum hypergastrinemia in infected individuals. The aim of this work was to assess the extent to which the ability of H. pylori to up-regulate expression of HB-EGF can be attributed to its effect on gastrin. Gastric cells, transfected with either gastrin small interfering RNA or antisense plasmid or the gastrin/cholecystokinin-2 receptor (CCK-2R), were cultured for 24 hours with H. pylori(+/-), a CCK-2R antagonist. Gene expression levels were measured using reverse transcription-PCR, whereas protein changes were measured using ELISA, Western blotting, and immunofluorescence. H. pylori induced significantly higher levels of HB-EGF gene expression and ectodomain shedding in the CCK-2R-transfected cells than the vector control (P < 0.01). Addition of the CCK-2R inhibitor significantly decreased gene and shedding up-regulation. Gastrin down-regulation reduced the effect of the bacteria on HB-EGF gene and protein expression levels. Endogenous gastrin and CCK-2R expression were also found to be significantly up-regulated in all cell lines as a result of exposure to H. pylori (P < 0.02). Gastric mucosal tissue from H. pylori-infected individuals had significantly higher CCK-2R expression levels than noninfected (P < 0.003), and in hypergastrinemic mice, there was an increase in HB-EGF-expressing cells in the gastric mucosa and colocalization of HB-EGF with CCK-2R-positive enterochromaffin-like cells. In conclusion, gastrin and the CCK-2R play significant roles in the induction of HB-EGF gene and protein expression and ectodomain shedding by H. pylori.
Subject(s)
Epidermal Growth Factor/biosynthesis , Gastrins/physiology , Helicobacter Infections/metabolism , Helicobacter pylori/physiology , Receptor, Cholecystokinin B/physiology , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/microbiology , Animals , Cell Line, Tumor , DNA, Antisense/genetics , Disease Models, Animal , Enterochromaffin Cells/metabolism , Epidermal Growth Factor/genetics , Gastrins/genetics , Gastrins/pharmacology , Helicobacter Infections/genetics , Heparin-binding EGF-like Growth Factor , Humans , Intercellular Signaling Peptides and Proteins , Mice , Plasmids/genetics , RNA, Small Interfering/genetics , Receptor, Cholecystokinin B/antagonists & inhibitors , Receptor, Cholecystokinin B/biosynthesis , Receptor, Cholecystokinin B/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Stomach Neoplasms/microbiology , Transfection , Up-RegulationABSTRACT
This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycine-extended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC(Min) mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin-2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC(Min) mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK-2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy.
Subject(s)
Endothelial Cells/drug effects , Epidermal Growth Factor/physiology , Gastrins/pharmacology , Animals , Cell Differentiation/drug effects , Chick Embryo , Colonic Neoplasms/blood , Colonic Neoplasms/blood supply , Endothelial Cells/cytology , Endothelial Cells/metabolism , Epidermal Growth Factor/biosynthesis , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , Gastrins/antagonists & inhibitors , Gastrins/blood , Gene Expression/drug effects , Heparin-binding EGF-like Growth Factor , Humans , Immune Sera , Intercellular Signaling Peptides and Proteins , Isoenzymes/metabolism , Metalloendopeptidases/metabolism , Mice , Neovascularization, Physiologic/drug effects , Omeprazole/pharmacology , Receptor, Cholecystokinin B/antagonists & inhibitorsABSTRACT
Targeted drug delivery to selected sites allows reduced toxicity, enhanced efficiency and interchangeable target potential [Langer, R. (2001) Science 293, 58-59 and Molema, G. & Meijer, D. K. F., eds. (2001) Drug Targeting (Wiley-VCH, Weinheim, Germany)]. We describe a bipartite drug-delivery system that exploits (I) endogenous carbohydrate-to-lectin binding to localize glycosylated enzyme conjugates to specific, predetermined cell types followed by (II) administration of a prodrug activated by that predelivered enzyme at the desired site. The carbohydrate structure of an alpha-L-rhamnopyranosidase enzyme was specifically engineered through enzymatic deglycosylation and chemical reglycosylation. Combined in vivo and in vitro techniques (gamma scintigraphy, microautoradiography and confocal microscopy) determined organ and cellular localization and demonstrated successful activation of alpha-L-rhamnopyranoside prodrug. Ligand competition experiments revealed enhanced, specific localization by endocytosis and a strongly carbohydrate-dependent, 60-fold increase in selectivity toward target cell hepatocytes that generated a >30-fold increase (from 0.02 to 0.66 mg) in protein delivered. Furthermore, glycosylation engineering enhanced the serum-uptake rate and enzyme stability. This created enzyme activity (0.2 units in hepatocytes) for prodrug therapy, the target of which was switched simply by sugar-type alteration. The therapeutic effectiveness of lectin-directed enzyme-activated prodrug therapy was shown through the construction of the prodrug of doxorubicin, Rha-DOX, and its application to reduce tumor burden in a hepatocellular carcinoma (HepG2) disease model.
Subject(s)
Prodrugs/therapeutic use , Animals , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/therapeutic use , Doxorubicin/analogs & derivatives , Doxorubicin/pharmacokinetics , Doxorubicin/therapeutic use , Drug Delivery Systems , Enzyme Activation , Enzyme Stability , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Glycosylation , Humans , Kidney/metabolism , Lectins , Liver/metabolism , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Nude , Prodrugs/pharmacokinetics , Protein Engineering , Rabbits , Rats , Rats, Wistar , Tissue DistributionABSTRACT
Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells. MMPs and TIMPs were examined using quantitative real-time RT-PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity. The most abundant MMPs were pro-MMP-2, pro- MMP-3, active MMP-3 and MT1-MMP. TIMP-1 and TIMP-2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM-conditioned medium to inhibit recombinant MMP-2 in a QF assay. Thrombin increased MMP activity by activation of pro-MMP-2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4. In conclusion, ASM cells express pro-MMP-2, pro and active MMP-3, MMP-9 and MT1-MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP-2 can be activated by thrombin which may contribute to airway remodelling.