ABSTRACT
Drug nanocarriers are often derivatized with targeting moieties to achieve site specific delivery, however, the results from this approach have, as yet, not reached expectations. We have tested a new phage display based targeting moiety, the activated endothelium targeting peptide (AETP), for its vascular endothelium directed targeting efficiency, when anchored to a PEGylated liposome via maleimide chemistry. Our results have, however, not shown any evidence of improved targeting. We have hypothesized that the failure of the AETP moiety is due to its availability to target receptors being restricted, as a result of steric hindrance due to the PEG polymer, and possibly affinity for bloodstream proteins, particularly human serum albumin (HSA). In this context, molecular modeling was used to contrast the properties of the AETP moiety to those of the RGD targeting peptide, already found to be effective in previous trials. Our molecular dynamics simulation results indicate the AETP moiety is located within the PEG layer, and its hydrophobic nature causes it to be obscured by PEG to a greater extent than the more hydrophilic RGD targeting peptide. Protein-ligand docking results indicated similar affinities for HSA of both the AETP moiety and a PEG fragment, and a significantly lower affinity for the RGD peptide. We know of no means to investigate this experimentally with atomic level resolution, thus our use of computational methods to investigate this can be seen as a new tool for rational design in nanomedicine.
Subject(s)
Liposomes/administration & dosage , Liposomes/chemistry , Molecular Targeted Therapy/methods , Peptides/administration & dosage , Peptides/chemistry , Polyethylene Glycols/chemistry , Amino Acid Motifs , Animals , Cells, Cultured , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Delivery Systems/methods , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Female , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Maleimides/chemistry , Mice , Mice, Nude , Models, Chemical , Molecular Dynamics Simulation , Nanomedicine/methods , Polyethylene Glycols/administration & dosage , Protein Binding , Receptors, Cell Surface/metabolism , Tissue DistributionABSTRACT
Biopanning of tumor cells was used in order to identify matrix metalloproteinase 9 (MMP-9) targeting peptides. The tumor cell targeting peptide (TCTP-1) and two modified versions thereof were evaluated as imaging agents for positron emission tomography (PET) using a rat melanoma xenograft model. For the PET imaging purposes, the 3 peptides were 1,4,7,10-tetraazacyclo-dodecane-N',N'',N''',N''''-tetraacetic acid (DOTA) conjugated and labeled with Gallium-68 ((68)Ga) and preliminarily evaluated: (1) cyclic (68)Ga-DOTA-TCTP-1 with cystine bridge, (2) cyclic (68)Ga-DOTA-lactam-TCTP-1 with a lactam bridge, and (3) linear (68)Ga-DOTA-lin-TCTP-1. The whole-body distribution kinetics and tumor targeting of the intravenously administered (68)Ga-DOTA-peptides were evaluated in vivo by PET and ex vivo by measuring the radioactivity of excised tissues. In addition, the in vivo stability of the radiolabeled peptides in rat plasma, tumor tissue, and urine was studied. All (68)Ga-DOTA-peptides were cleared via the liver and kidneys, and approximately 44% of injected radioactivity was excreted in urine during 120 min after injection. Ex vivo biodistribution studies showed a tumor-to-muscle ratio of 5.5 ± 1.3 (mean ± SD) for (68)Ga-DOTA-TCTP-1, 3.2 ± 0.2 for (68)Ga-DOTA-lactam-TCTP-1, and 3.2 ± 0.6 for (68)Ga-DOTA-lin-TCTP-1 at 120 min after injection. The (68)Ga-DOTA-lactam-TCTP-1 peptide appeared to be the most stable in vivo. The fraction of intact (68)Ga-DOTA-lactam-TCTP-1 in tumor was 59 ± 4.2% at 120 min after injection. The stability was moderate for (68)Ga-DOTA-TCTP-1 and poor for (68)Ga-DOTA-lin-TCTP-1. The possibility of imaging tumors that overexpress MMP-9, such as melanoma, by using radiolabeled TCTP peptides in PET imaging makes these peptides highly attractive for diagnostic and therapeutic applications. However, further modifications to improve the stability and affinity of the peptides are needed.
Subject(s)
Drug Evaluation , Gallium Radioisotopes , Matrix Metalloproteinase 9 , Melanoma/diagnosis , Peptides/chemical synthesis , Animals , Cell Line, Tumor , Cystine/chemistry , Disease Models, Animal , Gallium Radioisotopes/chemistry , Heterocyclic Compounds, 1-Ring/chemistry , Kinetics , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase 9/pharmacokinetics , Melanoma/diagnostic imaging , Peptides/pharmacokinetics , Positron-Emission Tomography , Rats , Tissue Distribution , Transplantation, Heterologous , Tumor Protein, Translationally-Controlled 1ABSTRACT
The crystal structure of full-length Csk (C-terminal Src kinase) molecules shows a hydrophobic interaction between the SH2-kinase linker residue Phe183 and the alphaC-helix of the catalytic domain. To study the possible involvement of this contact in the regulation of the activity of Csk and CHK (Csk homologous kinase), a Csk SH2-kinase linker deletion mutant, Csk Phe183 and CHK Leu223 point mutants were analyzed. It was observed that a residue with a long hydrophobic side chain in position 183 (Csk) and 223 (CHK) is required to sustain the catalytic activity of Csk and CHK. These results suggest that Csk Phe183 and CHK Leu223 stabilize the movement of the alphaC-helix of these protein tyrosine kinases.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins pp60(c-src) , src Homology Domains , Alanine/chemistry , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , CSK Tyrosine-Protein Kinase , Catalysis , Catalytic Domain , Crystallography, X-Ray , Gene Deletion , HeLa Cells , Humans , Leucine/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenylalanine/chemistry , Plasmids/metabolism , Point Mutation , Precipitin Tests , Protein Structure, Tertiary , Transfection , Tyrosine/chemistry , Valine/chemistry , src-Family KinasesABSTRACT
c-Myc is an oncogenic transcription factor involved in the regulation of cell proliferation, differentiation and apoptosis. The direct targets of c-Myc mediating these various processes are slowly being unravelled. This study indicates that the ornithine decarboxylase (ODC) gene is a physiological transcriptional target of c-Myc in association with induction of cell proliferation and transformation, but not with induction of apoptosis. In addition to the two conserved CACGTG c-Myc-binding sites in the first intron, the CATGTG motif in the 5'-flanking region of the murine odc is also shown to be a functional c-Myc response element. odc is thus a c-Myc target with three binding sites a distance apart. Transient transfection studies with different c-Myc, Max and Mad constructs in COS-7 cells showed that the balance between c-Myc/Max, Max/Max and Max/Mad complexes is crucial for the regulation, resulting in either transactivation or transrepression of an ODC-CAT reporter gene. Transcription of both ODC-CAT and endogenous odc was strongly induced in HeLa cells expressing tetracycline-regulated c-Myc, concomitant with c-Myc promoting the S-phase entry of the cells. Transformation of NIH3T3 cells by c-Ha-ras-(Val12) oncogene was reversed by expression of transcriptionally inactive c-Myc, which was associated with repression of ODC-CAT expression. Further, the c-Myc-induced transactivation of ODC-CAT in COS-7 cells was suppressed by co-expression of the retinoblastoma tumor suppresser pRb, evidently as a result of pRb directly or indirectly interacting with c-Myc. Importantly, the endogenous c-Myc and pRb proteins were also found to associate in Colo 320HSR cells under physiological conditions. These results suggest that c-Myc and pRb can interact in vivo, and may in part control some aspects of cell proliferation and transformation through modulation of odc expression.
Subject(s)
Genes, myc/genetics , MADS Domain Proteins/metabolism , Ornithine Decarboxylase/genetics , Retinoblastoma Protein/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Binding Sites , COS Cells , Cells, Cultured , Chlorocebus aethiops , DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , HeLa Cells , Humans , Mice , Ornithine Decarboxylase/biosynthesis , Transcription Factors/metabolism , Transcriptional Activation/geneticsABSTRACT
PURPOSE: Barrett's esophagus (BE), a precancerous condition for Barrett's adenocarcinoma, is classically characterized by flames of salmon-colored mucosa extending into normal pale esophageal mucosa. This flaming is thought to be a consequence of continuous erosis of mucosa caused by chronic reflux. Another characteristic feature of Barrett's adenocarcinoma patients is the frequent development of lymph node metastases. We addressed whether onset of angiogenesis occurs in BE and if the lymphatic system might provide a route for Barrett's adenocarcinoma cells to infiltrate regular lymph nodes. PATIENTS AND METHODS: Fifteen surgically resected Barrett's dysplasia or adenocarcinoma patients were included. Immunohistochemistry and a modified whole mount analysis were used. RESULTS: The incipient angiogenesis originates from the pre-existing vascular network in the lamina propria and infiltrates Barrett's epithelium, giving its ominous salmon-red color. Barrett's epithelium-specific goblet cells express vascular endothelial growth factor (VEGF)-A. The immature blood vessels show a relative absence of smooth muscle actin (SMA)-positive mural cells and express VEGF receptor (VEGFR)-2 and matrix metalloproteinase (MMP)-9 on their exterior. Coexpression of VEGF-C and its receptor VEGFR-3 on lymphatic vessels is demonstrated. CONCLUSION: BE is strongly neovascularized not eroded. This novel concept of a molecular mechanism of the origin of BE might emphasize why precancerous BE can give rise to the more cancerous dysplasia and Barrett's adenocarcinoma stages. In addition, adenocarcinoma cells induce lymphangiogenesis. The new lymphangiogenic vessels might provide a systemic route for adenocarcinoma cells to invade circulation and induce lymph node metastasis.
