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.     

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 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.     

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.     

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.     

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.     

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.     

Single-molecule study of lateral mobility of epidermal growth factor receptor 2/HER2 on activation

The transmembrane protein HER2, a member of the epidermal growth factor receptor family of tyrosine kinase, plays important roles in many fundamental cellular processes as well as the pathogenesis of many cancers. In this work, we have applied the single-molecule fluorescence microscopic method to study lateral mobility change of HER2 on activation by imaging and tracking individual GFP-tagged HER2 molecules on the membrane of living cells. The single HER2 molecules displayed different diffusion rates and modes. It was interesting to find that the mobility of HER2 increased upon stimulation by heregulin beta1, the specific ligand of HER3. The faster diffusion was related to the tyrosine phosphorylation of HER2 or EGFR. The results provided new information for the understanding of HER2 activation and molecular mechanism of signal transduction through HER2/HER3 heterodimerization.     

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.     

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.     

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.     

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.     

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.     

Stability of recombinant human epidermal growth factor in various solutions

The stability of recombinant human epidermal growth factor (hEGF) in various solutions was examined. hEGF degraded spontaneously and temperature-dependently to several degradation products in phosphate buffered saline or in 0.1 N acetic acid. The enzymatic degradation was observed in human serum or in pepsin/HCl solution. The structure and biological activities of these compounds were examined. The results suggest that the Asp11 and Trp50 residues are important for the receptor binding.     

Observation of phosphorylation site-specific dissociation of singly protonated phosphopeptides

In ultraviolet photodissociation of phosphopeptide ions with a basic residue (arginine, lysine, or histidine) at the N-terminus, intense a_n − 97 peaks were observed. These ions were formed by cleavage at phosphorylated residues only. For multiply phosphorylated peptides, this site-specific cleavage occurred at every phosphorylated residue. H/D exchange studies showed that a_n − 97 was formed by H₃PO₄ loss from a_n+1 radical cations. The site-specificity of phosphate loss observed here is in contrast to the nonspecific phosphate loss from b_n and y_n reported previously. Characteristics of the reaction and its potential utility for phosphopeptide analysis are discussed.     

Synthesis of Modified Tripeptides and Tetrapeptides as potential bisubstrate inhibitors of the epidermal growth factor receptor protein tyrosine kinase

The synthesis of a series of bisubstrate inhibitors of the epidermal growth factor receptor protein kinase (EGF-R PTK) consisting of small pep tides linked covalently to adenosine via appropriate triphosphate substitutes is described. Boc-Glu(O^tBu)-Tyr-Leu-OBzl ( 5 ) and Ac-Glu(O^tBu)-Tyr-Leu-Arg(Pmc)-NH₂ ( 8 ; Pmc = 2,2,5,7,8-pentamethylchroman-6-sulfonyl) were prepared by standard peptide chemistry, (Scheme 1), then modified at the OH group of tyrosine either with adipic anhydride or with 4-(chlorosulfonyl)benzoic acid, 4-(chlorosulfonyl)-2-hydroxybenzoic acid, or benzene-1,4-disulfonyldichloride (Scheme 2), and finally coupled with the 5′-OH group of 2′,3′-O-isopropylideneadenosine (Scheme 3). In addition, N⁶-[(benzyloxy)carbonyl]-2′,3′-O-isopropylidene-adenosine 5′-(hydrogenhexanedioate) ( 26 ), an ATP substitute, was coupled with the morpholide of 5 (Scheme 4). Removal of the protecting groups gave the bisubstrate analogs 23, 24 , and 28. The compounds synthesized were tested as inhibitors of the EGF-R PTK. The most active bisubstrate-type inhibitor was 24 , composed of the tripeptide sequence H-Glu-Tyr-Leu-OBzl, the 2-hydroxy-4-sulfonylbenzoyl moiety, and adenosine; it showed an IC₅₀ value of 33 μM.     

Novel diphenylamine 2,4'-dicarboxamide based azoles as potential epidermal growth factor receptor inhibitors: synthesis and biological activity

Several hybrid molecules of diphenylamine-2,4'-dicarboxamide with various azolidinones and related heterocyclic rings have been synthesized and explored as epidermal growth factor receptor (EGFR) kinase inhibitors. Most of them displayed promising in vitro tyrosine kinase inhibition as well as potent cellular antiproliferative activity in the EGFR over-expressing breast cancer cell line (MCF-7). Compounds 12b and 13b that exhibited the highest inhibition in the kinase assay (89, 81% inhibition at 10 μM, respectively), showed potent antiproliferative effect against MCF-7 tumor cell line (IC(50) 1.04, 0.91 μM respectively). Molecular docking studies revealed that these compounds can bind to ATP binding site of the EGFR kinase domain and were involved in H-bonding with Met 793, in analogy to the known EGFR tyrosine kinase inhibitors. Moreover, compounds 15a-c possessed profound antitumor activity (IC(50) 0.59-0.73 μM) and significant EGFR-TK inhibition, making them of particular interest. In summary, the newly synthesized compounds provide promising new lead for the future design and development of anticancer agents of potential EGFR-TK inhibitory activity.     

Screening epidermal growth factor receptor antagonists from Radix et Rhizoma Asari by two‐dimensional liquid chromatography

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti‐inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two‐dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose‐dependent manner in the range of dose from 0.10 to 102.4 μM. This two‐dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates.