Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

A photocleavable peptide-tagged mass probe for chemical mapping of epidermal growth factor receptor 2 (HER2) in human cancer cells

Human epidermal growth factor receptor 2 (HER2) testing has great value for cancer diagnosis, prognosis and treatment selection. However, the clinical utility of HER2 is frequently tempered by the uncertainty regarding the accuracy of the methods currently available to assess HER2. The development of novel methods for accurate HER2 testing is in great demand. Considering the visualization features of in situ imaging and the quantitative capability of mass spectrometry, integration of the two components into a molecular mapping approach has attracted increasing interest. In this work, we reported an integrated chemical mapping approach using a photocleavable peptide-tagged mass probe for HER2 detection. The probe consists of four functional domains, including the recognition unit of an aptamer to catch HER2, a fluorescent dye moiety (FITC) for fluorescence imaging, a reporter peptide for mass spectrometric quantification, and a photocleavable linker for peptide release. After characterization of this novel probe (e.g., conjugation efficiency, binding affinity and specificity, and photolysis release efficiency), the probe binding and photolysis release conditions were optimized. Then, fluorescence images were collected, and the released reporter peptide after photolysis was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A limit of quantification (LOQ) of 25 pM was obtained, which very well meets the requirements for clinical laboratory testing. Finally, the developed assay was applied for HER2 testing in four breast cancer cell lines and 42 pairs of human breast primary tumors and adjacent normal tissue samples. Overall, this integrated approach based on a photocleavable peptide-tagged mass probe can provide chemical mapping including both quantitative and visual information of HER2 reliably and consistently, and may pave the way for clinical applications in a more accurate manner.

An integrated approach based on a photocleavable peptide tagged mass probe provides chemical mapping including quantitative and visual information of HER2.     

Single-molecule force spectroscopy study of interaction between transforming growth factor beta1 and its receptor in living cells

Transforming growth factor beta1 (TGF-beta1) regulates many important cellular processes such as cell proliferation, differentiation, and apoptosis, etc. Its signaling is initiated by binding to and bringing together TGF-beta type II receptor (TbetaRII) and type I receptor (TbetaRI). However, it is not fully understood how the TGF-beta1 ligand-receptor interaction occurs in living cells and what is the molecular mechanism of the signaling complex TGF-beta1/TbetaRII/TbetaRI formation. In this study, we have investigated the interaction between TGF-beta1 and its receptors in living cells with single-molecule force spectroscopy for the first time. By positioning TGF-beta1-modified atomic force microscope (AFM) tips on the cells expressing fluorescent protein tagged TGF-beta receptors, the living-cell force measurement was realized with a combined fluorescence microscope and AFM. We found that coexpression of TbetaRI with TbetaRII enhanced the binding force of TGF-beta1 with its receptors, whereas the expressed TbetaRI itself exhibited no binding affinity to TGF-beta1. Moreover, the unbinding dynamics of TGF-beta1/TbetaRII and TGF-beta1/TbetaRI/TbetaRII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-beta1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-beta1 with the receptor after TbetaRI is recruited and the important contribution of TbetaRI to the signaling complex formation during TGF-beta1 signaling.     

Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGF alpha-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.     

Molecular dynamics simulation reveals structural and thermodynamic features of kinase activation by cancer mutations within the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a major target for drugs in treating lung carcinoma as it promotes cell growth and tumor progression. Structural studies have demonstrated that EGFR exists in an equilibrium between catalytically active and inactive forms, and dramatic conformational transitions occur during its activation. It is known that EGFR mutations promote such conformational changes that affect its activation and drug efficacy. The most common point mutation in lung cancer patients is a leucine to arginine substitution at amino acid 834 (L834R). In a recent article, we have studied changes in drug binding affinities due to cancer mutations of EGFR using ensemble molecular dynamics (MD) simulations. Here, we address an enhanced activation mechanism thought to be associated with this mutation. Using extended timescale MD simulations, the structural and energetic properties are studied for both active and inactive conformations of EGFR. The thermodynamic stabilities of these two conformations are characterized by free energy landscapes estimated from molecular mechanics/Poisson-Boltzmann solvent area calculations. Our study reveals that the L834R mutation introduces conformational changes in both states, adjusting the relative stabilities of active and inactive conformations and hence the activation of the EGFR kinase.     

A far-red fluorescent contrast agent to image epidermal growth factor receptor expression

Recent developments in optical technologies have the potential to improve the speed and accuracy of screening and diagnosis of curable precancerous lesions and early cancer, thereby decreasing the costs of detection and management of epithelial malignancies. The development of molecular-specific contrast agents for markers of early neoplastic transformation could improve the detection and molecular characterization of premalignant lesions. In the oral cavity, epidermal growth factor receptor (EGFR) overexpression has been identified in early stages of premalignant lesions of the oral squamous cell carcinoma; therefore, real-time assessment of EGFR expression could serve as a biomarker for oral neoplasia. The purpose of our study was to develop a molecular-specific optical contrast agent targeted against EGFR for in vivo assessment of epithelial neoplasia using a monoclonal antibody and the far-red fluorescent dye, Alexa Fluor 660 streptavidin. In addition to demonstrating the specificity of the contrast agent for EGFR in cell lines, we document the ability to achieve penetration through 500 microm thick epithelial layers using multilayer tissue constructs and permeability-enhancing agents. Finally, using the fluorescence intensity of the contrast agent on fresh oral cavity tissue sections, we were able to distinguish abnormal from normal oral tissue. This contrast agent should have important clinical applications for use in conjunction with fluorescence spectroscopy or imaging (or both) to facilitate tumor detection and demarcation.     

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.     

Effects of the expression level of epidermal growth factor receptor on the ligand-induced restructuring of focal adhesions: a QCM-D study

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity.

A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces

A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4 × 10⁶ cells mL⁻¹ with a detection limit of 40 cells mL⁻¹ was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35 × 10⁵ with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening.     

Proteome alterations in human hepatoma cells transfected with antisense epidermal growth factor receptor sequence

The epidermal growth factor (EGF) is a member of the growth factor superfamily that can stimulate the proliferation of many types of cells. Overexpression of EGF receptor (EGFR) was observed in many types of cancer cells. Anti-EGFR antibodies or antisense nucleic acid sequences of EGFR can suppress the growth of hepatoma cells. In order to further investigate the proteome alterations associated with malignant growth of the human hepatoma cells and the influence of EGFR signal pathway on the cellular proteome, we have comparatively analyzed the proteomes of human hepatoma cells transfected with antisense EGFR sequence (cell strain JX-1) and its control cells (cell strain JX-0) by two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 40 protein spots showed significant expression changes in JX-1 cells compared to JX-0 cells. Three of them, including the tumor suppressor protein maspin, changed with tendency to the normal levels. Two protein spots were identified as HSP27 in the same gel, and one of them had a reduced level in JX-1 cells. The apparent alterations of HSP27 in expression level might be the results from their differential chemical modifications, suggesting the effect of dynamic post-translational modifications of proteins on the growth of hepatoma cells. Other proteins such as glutathione peroxidase (GPX-1) and 14-3-3-sigma also exhibited altered expression in JX-1 cells, and their functional implications are discussed.     

UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival

We have recently shown that UVB radiation activates epidermal growth factor receptor (EGFR)/extracellular regulated kinase 1 and 2 (ERK1/2) and p38 signaling pathways in keratinocytes. However, the functional relevance of these processes for downstream signaling and cell survival remains to be determined. The specific EGFR inhibitor PD153035 markedly decreased UVB-induced phosphorylation of EGFR, ERK1/2 and shc, whereas p38 activation was unaffected. PD153035 pretreatment followed by UVB reduced clonogenic potential and enhanced peroxide production, apoptosis and cell death. Our data suggest that ligand-independent phosphorylation of EGFR and likely dependent downstream signaling pathways regulate cellular defense mechanisms important for cell survival following oxidative stress.     

Chemical characterization of recombinant human epidermal growth factor

Human epidermal growth factor (EGF) produced from yeast and purified by ion-exchange and gel filtration chromatography was analysed by reversed-phase liquid chromatography (RP-LC) and showed two main peaks (EGF-1 and EGF-2) with different relative yields from batch to batch and similar biological activities (radioreceptor analysis). Each component isolated by RP-LC was reduced, carboxymethylated and digested with endopeptidase Lys-C and endopeptidase Glu-C separately. Each digest was analysed by fast atom bombardment mass spectrometry (FAB-MS) without further purification. It was verified that both EGF-1 and EGF-2 have the same amino acid sequence of human EGF except at the C-terminal end. EGF-2 lacks the C-terminal arginine and EGF-1 lacks the C-terminal leucine- arginine residues. The location of the SS bonds was also verified by FAB-MS of the peptide mixture obtained by 2% acetic acid hydrolysis. The fermentation and purification processes were followed by RP-LC and FAB-MS and allowed the amount of EGF-1 to be reduced to 10–15%, but intact EGF was never detected.     

Natural products derived from traditional chinese medicine as novel inhibitors of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) has become an important molecular target in cancer therapy. Various small molecules and therapeutic antibodies targeting EGFR family members have been developed during recent years and are established in clinical oncology. However, increasing clinical application of EGFR tyrosine kinase inhibitors has resulted in the development of resistance to EGFR-targeting drugs due to the selection of EGFR-mutated variants. This phenomenon forced the search for novel EGFR inhibitors with activity towards EGFR-mutant tumors. This review describes recent achievements in natural products derived from medicinal plants as novel EGFR inhibitors.     

Immobilized glucose-6-phosphate dehydrogenase as a substrate for solubilized epidermal growth factor receptor tyrosine kinase

Based on our previously reported solution assay protocol, a solid-phase assay for the tyrosine kinase activity of the epidermal growth factor receptor has been developed. Glucose-6-phosphate dehydrogenase, immobilized noncovalently on microtiter plates, was used as the substrate in the solid-phase assay. Phosphorylation of the immobilized substrate takes place in the presence of ATP and a solubilized epidermal growth factor receptor preparation. After washing off the soluble reaction mixture, the phosphotyrosine-containing dehydrogenase produced on the well surface is quantitated by an ELISA method using a polyclonal antiphosphotyrosine antibody, a second antibody conjugated with horseradish peroxidase, and finally theo-phenylenediamine reaction. The absorbance at 492 nm developed in the wells is a measure of the kinase activity of the solubilized receptor preparation. Putative inhibitors of receptor kinase can be conveniently incorporated in this assay system to test for potential inhibitory activity. This assay, being rapid and convenient, is useful in drug screening programs where a high through-put rate is required.     

Targeting cells that overexpress the epidermal growth factor receptor with polyethylene glycolated BPD verteporfin photosensitizer immunoconjugates

Photoimmunotherapy was introduced two decades ago but has been studied infrequently in vivo and is virtually untested clinically. Progress has been limited because high-quality, well-characterized photosensitizer immunoconjugates (PICs) have been difficult to make. Here, we describe the development of an innovative conjugation method for producing water-soluble PICs that are free of insoluble aggregates and free of unacceptable amounts of noncovalently associated photosensitizer impurities. The method exploits two procedures previously untried in this research area. First, a small number of antibody lysines (<3 per antibody) are polyethylene glycolated (PEGylated) using a 10 kDa branched polyethylene glycol (PEG), which dramatically enhances PIC solubility and reduces PIC aggregation. Second, a 50% dimethyl sulfoxide-50% aqueous two-solvent system is used to prevent photosensitizer aggregation and noncovalent interactions. These measures allow efficient covalent linkage of the photosensitizer BPD Verteporfin (BPD) to antibody lysines, thorough purification of the resulting PICs (verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), maintenance of PIC antigen-binding activity (verified by cellular binding-uptake assays) and reduction of nonspecific cellular uptake (e.g. macrophage capture) of the PICs. Loading levels could be varied controllably in the range < or = 11 BPD/antibody. PICs of the C225 anti-epidermal growth factor receptor (EGFR) chimeric monoclonal antibody killed EGFR-overexpressing A-431 cells photodynamically but did not significantly affect EGFR-negative NR6 cells. Although fluorescence measurements demonstrated that the PICs were quenched by as much as an order of magnitude compared with free BPD, an impressive 90% reduction in A-431 cell viability was achieved using 20 J/cm2 of 690 nm light after a 40 h incubation with the C225 PICs. The results suggest that PEGylated BPD-C225 PICs merit further investigation in animal models of EGFR-overexpressing cancers.     

Selective separation of active inhibitors of epidermal growth factor receptor from Caragana jubata by molecularly imprinted solid-phase extraction

A feasibility research was performed to study the possibilities of using a molecularly imprinted polymer as sorbent material in solid-phase extraction for the separation of active inhibitors of epidermal growth factor receptor (EGRF) from Caragana Jubata, a Chinese traditional Tibetan medicine. A molecularly imprinted polymer using quercetin, an active anti-EGFR inhibitor (IC50 = 15 microM), as the template and acrylamide as the functional monomer was prepared. The polymer was evaluated as a selective sorbent in molecularly imprinted solid-phase extraction. The EtOAc extract of Caragana Jubata was loaded on the polymer, and two novel active anti-EGFR inhibitors were found to be selectively retained after washing the polymer with appropriate solvent to disrupt the non-specific interactions occurring between the sample and the polymer matrix, which were identified as (E)-piceatannol (IC50 =4.9 microM) and butein (IC50 = 10 microM). The present work affords us a new potential method for selective separation of bioactive components from herb by using molecularly imprinted polymer as a solid-phase extraction adsorbent.     

Variation of hydrophobicity of human urinary epidermal growth factor.

Epidermal growth factor is present in human urine in large amounts, but its biological significance is not known. The results of this study indicate that the predominant 6000-dalton form of epidermal growth factor in human urine is divided by hydrophobic interaction chromatography into four fractions; only 3% of the total 6000-dalton epidermal growth factor coeluted with the biosynthetic epidermal growth factor and the rest was separated into three different peaks. These different forms may lack one or two amino or carboxy terminal amino acids from the 53 amino acids present in epidermal growth factor, or they may be products of deamidation or oxidation of amino acid(s). Further knowledge of these micromodifications of epidermal growth factor secreted in urine may reveal the origin and function of epidermal growth factor in humans.