Receptor-mediated Endocytosis Of Epidermal Growth Factor Research Articles

Variable-Angle Nanoplasmonic Fluorescence Microscopy: An Axially Resolved Method for Tracking the Endocytic Pathway.

The study of endocytosis, which encompasses diverse mechanisms in biology, requires the utilization of high axial resolution to monitor molecular behavior on both the cell surface and interior of the cell. We have designed a novel axially resolved fluorescence microscopic technique, termed variable-angle nanoplasmonic fluorescence microscopy. The proof-of-principle of this approach is achieved by selectively following the events in the vicinity of a cell membrane or in a cell. We use a 30 nm Au-coated semitransparent coverslip as the nanoplasmonic chip to achieve both surface plasmon resonance excitation and critical angle excitation by tuning the incident angles. This approach leads to improved axial resolution compared to total internal reflection fluorescence microscopy, which is a common imaging technique in cell biology. It offers a unique opportunity to semiquantitatively determine fluorophore axial distributions in the cell. Observing the epidermal growth factor receptor-mediated endocytosis in Caski cells clearly demonstrates the potential application of this new method for cell biology studies.

GE11 peptide modified and reduction-responsive hyaluronic acid-based nanoparticles induced higher efficacy of doxorubicin for breast carcinoma therapy.

Novel breast carcinoma dual-targeted redox-responsive nanoparticles (NPs) based on cholesteryl-hyaluronic acid conjugates were designed for intracellular delivery of the antitumor drug doxorubicin (DOX). A series of reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety (HA-ss-Chol) and GE11 peptide conjugated HA-ss-Chol (GE11–HA-ss-Chol) were synthesized. The obtained conjugates showed attractive self-assembly characteristics and high drug loading capacity. GE11–HA-ss-Chol NPs were highly stable under conditions mimicking normal physiological conditions, while showing a fast degradation of the vehicle’s structure and accelerating the drug release dramatically in the presence of intracellular reductive environment. Furthermore, the cellular uptake assay confirmed GE11–HA-ss-Chol NPs were taken up by MDA-MB-231 cells through CD44- and epidermal growth factor receptor-mediated endocytosis. The internalization pathways of GE11–HA-ss-Chol NPs might involve clathrin-mediated endocytosis and macropinocytosis. The intracellular distribution of DOX in GE11–HA-ss-Chol NPs showed a faster release and more efficient nuclear delivery than the insensitive control. Enhanced in vitro cytotoxicity of GE11–HA-ss-Chol DOX-NPs further confirmed the superiority of their dual-targeting and redox-responsive capacity. Moreover, in vivo imaging investigation in MDA-MB-231 tumor-bearing mice confirmed that GE11–HA-ss-Chol NPs labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide, a near-infrared fluorescence dye, possessed a preferable tumor accumulation ability as compared to the single-targeting counterpart (HA-ss-Chol NPs). The antitumor efficacy showed an improved therapy efficacy and lower systemic side effect. These results suggest GE11–HA-ss-Chol NPs provide a good potential platform for antitumor drugs.

Genetically Engineered Epidermal Growth Factor Conjugate Crosses Cell Membrane

Epidermal growth factor (EGF) is a well-known pleiotropic growth factor in mammal, and has been attempted to be used in many different fields. However, the application of EGF is limited because of its poor cell permeation. In order to increase membrane permeation ability of EGF, a genetically modified recombinant EGF (GST-TAT-EGF) was prepared through conjugation of EGF with two protein transduction domains, glutathione-S-transferase and TAT47-57. The results showed that the GST-TAT-EGF fusion protein exhibited higher ability in biomembrane penetration than that of the EGF alone. The results also implied that two different mechanisms (EGF receptor-mediated endocytosis and direct penetration) might be involved in GST-TAT-EGF transmembrane delivery.

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.

Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15.

Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.

Efficient Gene Transfer to EGF Receptor Overexpressing Cancer Cells by Means of EGF-Labeled Cationic Liposomes

Epidermal growth factor (EGF)-labeled cationic liposomes (EGF-liposomes) were prepared for efficient gene transfer vector to EGF receptor expressing cells. Transfection activity of EGF-liposomes associating plasmid PGV-C which encodes luciferase showed a 2-fold increase in EGF receptor expressing cells, HEC-1-A, compared to that of EGF-non-labeled liposomes (N-liposomes). In EGF receptor deficient HRA cells, however, both EGF- and N-liposomes exhibited low transfection efficiency and no difference was observed between them. Furthermore, by the addition of anti EGF receptor antibody, transfection efficiency of EGF-liposomes was suppressed, suggesting EGF receptor-mediated endocytosis of EGF-liposomes. Transfection activity of EGF-liposomes was strongly dependent on the concentrations of fusogenic lipid, dioleoylphosphatidylethanolamine in liposomes. By X-gal staining 6–8 % of GCH-1(m) cells which also had EGF receptor expressed β-galactosidase activity following the transfection by EGF-liposomes associating pSV-β-galactosidase. These findings indicate that EGF-liposomes could be a preferable vector for EGF-receptor expressing cells.

Kinetic analysis of receptor-mediated endocytosis (RME) of proteins and peptides: use of RME as a drug delivery system

Receptor-mediated endocytosis (RME) which plays an important role in the elimination of biologically active polypeptide can be utilized as a target of the drug delivery system (DDS) which aims to deliver the drug with a limited membrane permeability into the cell. To estimate the efficiency of DDS, it is essential to quantify the cellular uptake of the drug and carriers. Kinetic analysis is an effective tool in understanding targeting efficiency. We have been kinetically analyzing the RME of epidermal growth factor (EGF) as a model compound. Based on the kinetic model representing RME, a desirable carrier system and the target have been described. We also tried to deliver antisense oligonucleotides to hepatocytes utilizing the asialoglycoprotein receptor and analyzed its uptake kinetically to predict the improvement of oligonucleotide uptake.

A novel gene delivery system using EGF receptor-mediated endocytosis

A monoclonal antibody to the human epidermal growth factor (EGF) receptor was conjugated with polylysine and the resulting conjugate was affinity-linked with DNA (gene). This novel gene delivery system utilizes receptor-mediated endocytosis and would be especially suitable for gene therapy for EGF receptor-overproducing squamous cell carcinomas.

Transforming growth factor beta 1 inhibits epidermal growth factor receptor endocytosis and down-regulation in cultured fetal rat hepatocytes

Incubation of fetal rat hepatocytes (FRH) with transforming growth factor beta 1 (TGF-beta 1) resulted in growth arrest and a biphasic effect on epidermal growth factor (EGF) receptor. After 2 h of exposure, EGF receptor (EGFR) was reduced by 43%. From 6 to 24 h, TGF-beta 1 exposure resulted in progressive increase in EGFR up to 74% over control. The increased binding was due to increase in high affinity EGF binding sites. FRH grown in medium containing EGF exhibited down-regulated EGFR with loss of high affinity EGF binding sites. With TGF-beta 1 exposure, high affinity EGFR was not down-regulated by EGF. Since down-regulation of EGFR involves internalization, the kinetics of EGF receptor-mediated endocytosis were examined. In TGF-beta 1-exposed FRH, EGF endocytosis was inhibited, with a reduction in the first order rate constant for the process from 0.078 to 0.043 min-1. Despite inhibition of growth, receptor down-regulation, and EGF endocytosis after TGF-beta 1 exposure, EGF-induced receptor autophosphorylation was preserved as demonstrated by [32P]phosphate-labeling of immunoprecipitated EGFR. These observations provide direct evidence that TGF-beta 1 regulates growth of fetal cells. Further, they suggest that TGF-beta 1 regulates endocytosis of EGF and possibly of other ligands.

生理活性ペプチドの体内動態を支配するReceptor-Mediated Endocytosis過程の速度論 : 肝及び腎への取り込み過程の解析

A remarkable feature of the disposition of polypeptide hormones and their analogues is the contribution of specific binding sites (receptors) to the polypeptides distribution and clearance in the body. The concept of "clearance receptor" is now well established, and receptor-mediated endocytosis (RME) is known as a general mechanism in the uptake of biologically important polypeptides. This review article focuses on the kinetic analysis of the RME of epidermal growth factor (EGF) based mainly on our observations of EGF handling by the liver and kidney. Pharmacokinetic analysis of the tissue distribution of EGF in rats performed in vivo clarified that the uptake of EGF by the liver and kidney exhibited clear saturation with Km values of 7 nM and 0.4 nM, respectively, and that both tissues could account for more than 80% of the total plasma clearance of EGF in the distribution phase. The hepatic and renal handlings of EGF by isolated perfused organs were also analyzed to obtain the kinetic parameters with respect to RME. In the overall RME process in the liver, the EGF-receptor association and dissociation processes are rapid (mean time less than 1 min), the degradation process is much slower (mean time 4-5 h) and the internalization process is intermediate (mean time 4-5 min). The mean time required for the recovery of receptors after being down-regulated in the liver is approximately 30 min, and was much shorter than those in other tissues. Such rapid recovery from the receptor down regulation in the liver reflects the process of recycling of internalized EGF receptors to the cell surface. The kinetic parameters obtained from the perfused livers and from isolated hepatocytes were compared, and a significant difference was observed only for the association rate constant (kon). A hypothesis accounting for this difference is proposed, in which the association rate of EGF to the cell surface receptor is considered to be limited by the diffusion in the unstirred water layer in the interstitial space of the intact liver such as the perfused liver. The comparison of EGF handling between the filtering and nonfiltering perfused kidneys demonstrated that the bulk of receptor-mediated EGF uptake takes place via the antiluminal plasma membrane with very high affinity (Kd = 0.1 nM).

Inhibition and Recovery of Macromolecular Synthesis, Membrane Transport, and Lysosomal Function Following Exposure of Cultured Cells to Hyperthermia

Iodine-125 epidermal growth factor (EGF) was used as a molecular probe to investigate the effects of hyperthermia on its binding, internalization, and degradation in rat fibroblasts. The decreased affinity of the receptor for EGF showed no evidence of recovery 12 hr after exposure to 45°C for 30 min. Receptor-mediated endocytosis of EGF was refractory to heating at 45°C for 30 min; there was no evidence of delayed effects of heating for at least 14 hr. In contrast, degradation of internalized EGF (a lysosomal function) was sensitive to heating and showed only partial recovery up to 14 hr after exposure to 45°C; the internalized EGF was incompletely degraded to products larger than iodotyrosine. Synthesis of protein and RNA, as determined by incorporation of [3 H]leucine and [3 H]uridine into acid-insoluble material, was decreased to very low values after heating to 45°C for 30 min. Only incorporation of [3 H]leucine showed any recovery over the 14 hr following heating. In contrast, transport of [3 H]leuci...