Effect of experimental diabetes on epidermal growth factor (EGF) receptors in the rat liver

The effect of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding was studied in microsomal membranes from rat liver. The binding of EGF in membranes from diabetic animals was significantly low, the value being about 60% of the control level. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors. Treatment of diabetic animals with insulin restored EGF receptors to control levels, whereas the treatment with triiodothyronine had no effect. Serum EGF concentrations measured were almost the same among the control, diabetic, and insulin-treated diabetic groups. These results suggest that insulin deficiency in vivo causes a decrease in hepatic EGF receptors.     

Structure-function relationships in epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha)

The solution structures of the homologous growth factors human epidermal growth factor (hEGF) and human transforming growth factor-alpha (hTGF-alpha), as determined by high resolution NMR and various computational methods, are described. Knowledge of these structures and the sequences of other homologous proteins leads to predictions about growth factor residues which may be involved in the receptor/ligand interface. Recent experiments designed to check these predictions are described briefly. These involve site-specific mutagenesis, receptor binding assays and high resolution NMR studies.     

Effect of exogenous epidermal growth factor (EGF) on nonenzymatic antioxidant capacities and MPO activity of wound tissue

Growth factors and cytokines are key regulators of the wound-healing process. Epidermal growth factor (EGF) plays a vital role during the process of wound healing. There are limited numbers of studies related to EGF implantation effects on oxidative stress in oral wound healing. The objective of this study was to investigate the effect of EGF on myeloperoxidase (MPO) activity and nonenzymatic antioxidant levels of oral wound tissue on different days. In this study, New Zealand male albino rabbits were used. After submucosal incisions, the rabbits were divided into two equal groups: untreated wounds, and EGF-implanted wounds. The levels of glutathione (GSH), ascorbic acid (AA), and MPO activity were measured spectrophotometrically. As a result, MPO activity of oral wound tissue strips was increased by exogenous EGF treatment compared with controls. In addition, GSH and AA levels of oral wound tissue strips were not changed during oral wound-healing phases for both controls and experimental groups.     

Benzo[a]pyrene and nicotine impair epidermal growth factor mediated cellular functions of buccal mucosa.

This study investigated the effect of two major ingredients in cigarette smoke, benzo[a]pyrene (BP) and nicotine, on epidermal growth factor (EGF) receptor binding and EGF-mediated cellular functions in rat buccal mucosa. Rat buccal tissue was incubated in DMEM in the absence (control) and presence of 10 microM BP or nicotine for 2.5 h at 25 degrees C. There were no significant differences in [125I]EGF binding to the buccal mucosal membranes between the control and treatment groups. Protein tyrosine kinase assay showed that EGF stimulated phosphorylation of a 170-kDa protein band in the controls, but not in the BP- and nicotine-treated samples. The basal [3H]thymidine incorporations were not significantly different between the groups. Nevertheless, addition of 5 nM EGF increased [3H]thymidine incorporation by 22% in the control, but not in the BP- or nicotine-treated group. The results demonstrate that BP and nicotine change the buccal mucosal functions associated with alteration of EGF receptor.     

Expression of epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor in thyroid tumors.

Immunohistochemical study of epidermal growth factor (EGF), epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF-alpha) expression was performed on paraffin-embedded tissue specimens of 12 follicular carcinomas, 15 papillary carcinomas and 15 adenomas of the thyroid gland. The tumors were placed in one of the following eight groups, according to the results of EGF, TGF-alpha and EGFR expression: group 1: none, group 2: only EGFR, group 3: EGFR and TGF-alpha, group 4: EGFR and EGF, group 5: TGF-alpha, and EGF, group 6: all three, group 7: only TFG-alpha and, finally, group 8: only EGF. Statistical analysis of the results revealed that the ratio of thyroid carcinomas with lymph node metastasis was significantly higher in groups 3 and 6 for follicular carcinomas (P less than 0.01) and in groups 4, 5 and 6 for papillary carcinomas (P less than 0.01). These results suggest that thyroid carcinomas expressing the systems EGF/EGFR, TGF-alpha/EGFR or TGF-alpha/EGF/EGFR display pathologic features of more aggressive disease. Furthermore, the synchronous expression of EGF, TGF-alpha and EGFR indicates that these carcinomas may regulate their growth by an autocrine and/or paracrine mechanism.     

Phosphorylation of hepatocyte growth factor receptor and epidermal growth factor receptor of human hepatocytes can be maintained in a (3D) collagen sandwich culture system.

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three-dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c-met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two-dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates epidermal growth factor (EGF) binding to basal cells from a human keratinocyte cell line

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates the proliferation, differentiation, or both, of epidermal keratinocytes in vivo and in culture. The growth of epidermal cells in culture is regulated by several biochemical mediators including epidermal growth factor (EGF). In this report the actions of TCDD on EGF binding in a basal cell population from a human keratinocyte cell line were examined. TCDD decreased the specific binding of 125I-EGF to basal cells by 40% within 96 hr. This reduction in EGF binding could not be attributed to changes in the state of differentiation as assessed by cell size and morphology, and cornified envelope competence, a marker of terminal differentiation. Modulation of EGF binding by TCDD was concentration-dependent (EC50 = 1 nM) and stereospecific, suggesting involvement of the Ah receptor. Scatchard analysis of EGF binding to the basal cells indicated a single class of high-affinity sites in both control (Kd = 0.14 nM) and treated (Kd = 0.11 nM) cultures and confirmed a decrease in the number of these sites in response to TCDD. The reduction in EGF binding correlated with a decrease in EGF-stimulated DNA synthesis and cell proliferation. Comparison of differentiation-competent squamous cell carcinoma (SCC) lines treated with TCDD supported an association between modulation of EGF binding and enhanced differentiation. The data indicate that basal cells are a target for TCDD. We propose that the modulation of EGF binding in basal keratinocytes by TCDD is one of the critical regulatory events resulting in enhanced differentiation.     

Urine acidification has no effect on peroxisome proliferator-activated receptor (PPAR) signaling or epidermal growth factor (EGF) expression in rat urinary bladder urothelium

We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR)alpha/gamma agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPARalpha, PPARdelta, PPARgamma, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPARgamma agonist pioglitazone daily for the final 3 days to directly assess the effects of diet acidification on responsiveness to PPARgamma agonism. Urothelial cell PPARalpha and gamma expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPARalpha, delta, or gamma mRNA or protein expression, PPARalpha- or gamma-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPARgamma-regulated gene expression. These results support the contention that urine acidification does not prevent PPARgamma agonist-induced bladder tumors by altering PPARalpha, gamma, or EGFR expression or PPAR signaling in rat bladder urothelium.     

Effect of cigarette smoking on human precapillary sphincters

1. Capillary filtration coefficient of human calf was measured by pressure plethysmography before and after cigarette smoking, which is known to release noradrenaline from nerve terminals of sympathetic vasoconstrictors. Calf blood flow and venous pressure-volume curves of the calf were also obtained before and after smoking.2. Capillary filtration coefficient decreased by 19% when cigarette smoke was inhaled deeply at 30 s intervals for 12-15 min, indicating the closure of precapillary sphincters.3. Calf blood flow decreased by 31% after smoking, indicating that arterioles were constricted. The degree of arteriolar constriction, however, was not strong enough to lessen the capillary hydrostatic pressure, since the absorption of tissue fluid into capillary blood vessels did not occur.4. The venous system seemed little affected by cigarette smoking, since venous pressure-volume curves were unaltered.     

Benzo(a)pyrene inhibits epidermal growth factor binding and receptor autophosphorylation in human placental cell cultures

Studies investigated the effects of benzo(a)pyrene (BP) treatment on epidermal growth factor (EGF) receptor binding and kinase activity in human placental cell cultures. Specific binding of 125I-EGF to cells from early gestation placentae was significantly decreased by 37 and 60% following exposure to 1 and 10 microM BP, respectively, for 24 hr. In contrast, cells cultured from term placentae showed no inhibitory effect of either concentration of BP. Specific binding of 125I-labeled insulin and insulin-like growth factors-I and -II to early gestation cells was decreased only 15-18% at 10 microM BP, which indicates that loss of membrane receptors appears to be selective for EGF. Scatchard analysis of early gestation cells revealed that BP was associated with a dose-dependent loss in the number of high affinity EGF binding sites. Evidence from cross-linking and autophosphorylation experiments confirmed that the Mr 170,000 binding protein was decreased in a dose-dependent manner following BP treatment. In comparison, term placental cells exhibit a 26% loss of EGF receptor autophosphorylation without alteration in binding following exposure to 10 microM BP. Thus, early gestation cells exhibit a BP-related down-regulation of EGF receptors, whereas term placental cells show receptor desensitization. No adverse effect of BP treatment was observed on the incorporation of [35S] methionine into proteins secreted by early gestation cells. Further experiments compared the effects of BP with the related poly-cyclic compounds beta-naphthoflavone, alpha-naphthoflavone, and 3-methylcholanthrene. In early gestation cells, EGF binding and receptor autophosphorylation were measurably decreased at 10 microM concentrations of these polycyclic compounds, but to a lesser extent than observed with BP. In term placental cells, however, EGF binding was unchanged or increased, whereas receptor autophosphorylation was decreased 10-26%. Thus, exposure of term placental cells to these polycyclic compounds leads to a dissociation between EGF binding and receptor protein kinase activity. Finally, aryl hydrocarbon hydroxylase activity was induced 20- to 200-fold in early placental cells exposed to BP, beta-naphthoflavone, and 3-methylcholanthrene. In summary, the direct effects of BP and related compounds observed on placental EGF receptors may indicate altered function of EGF in the regulation of cell growth and differentiation in the human placenta.     

Muscarinic type 3 receptor induces cytoprotective signaling in salivary gland cells through epidermal growth factor receptor transactivation

Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sj?gren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.     

Design and synthesis of novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) dual inhibitors bearing a pyrrolo[3,2-d]pyrimidine scaffold

Dual inhibitors of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) have been investigated for breast, lung, gastric, prostate, and other cancers; one, lapatinib, is currently approved for breast cancer. To develop novel HER2/EGFR dual kinase inhibitors, we designed and synthesized pyrrolo[3,2-d]pyrimidine derivatives capable of fitting into the receptors' ATP binding site. Among the prepared compounds, 34e showed potent HER2 and EGFR (HER1) inhibitory activities as well as tumor growth inhibitory activity. The X-ray cocrystal structures of 34e with both HER2 and EGFR demonstrated that 34e interacts with the expected residues in their respective ATP pockets. Furthermore, reflecting its good oral bioavailability, 34e exhibited potent in vivo efficacy in HER2-overexpressing tumor xenograft models. On the basis of these findings, we report 34e (TAK-285) as a promising candidate for clinical development as a novel HER2/EGFR dual kinase inhibitor.     

Effect of vascular endothelial growth factor and epidermal growth factor on iatrogenic apoptosis in human endothelial cells

To study the effect of growth factors on iatrogenic apoptosis, we examined the influence of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) on staurosporine-induced apoptosis in primary cultures of human umbilical vein endothelial cells (HUVEC). Apoptosis was evaluated by a cell viability test, the TUNEL-POD assay and the activation of the pro-apoptotic caspase-3. Staurosporine (10-100nM) caused the activation of caspase-3. This effect was manifest after 2hr of incubation and reached its maximum after 5hr. Severe loss of viability followed within 18hr. VEGF or EGF (10-100ng/mL) added together with staurosporine decreased the activation of caspase-3. The loss of viability was 24hr delayed. The action of growth factors was observed at 1% serum concentration but also at concentration optimal for HUVEC survival (10%, v/v). Furthermore, the inhibition of PI-3 kinase (PI-3K) by wortmannin or LY294002 as well as the inhibition of MEK by PD098059 or U0126 prevented the protective effect of VEGF and EGF. Western blotting analysis showed that after 3hr of incubation with staurosporine the level of the anti-apoptotic protein Mcl-1 decreased and this effect was reverted by VEGF. It is concluded that VEGF and EGF antagonize the pro-apoptotic action of staurosporine by the combined signalling of PI-3K and ERKs pathways.     

人表皮生长因子受体3靶向治疗研究进展

人表皮生长因子受体3(human epidermal growth factor receptor 3,Her3/ErbB3)是人表皮生长因子受体家族的一员,能通过配体依赖或非配体依赖的二聚化,激活并调节下游信号通路.目前Her3已成为非小细胞肺癌、乳腺癌等肿瘤治疗非常有潜力的候选靶点.但是,由于缺乏有效的生物标志物,不能针对Her3靶点筛选适应证患者,所以针对Her3的研究存在一定的局限性.此文综述了对Her3靶点的探究以及当前Her3靶向治疗的研究进展.     

6-Substituted-4-(3-bromophenylamino)quinazolines as putative irreversible inhibitors of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity

A series of new 6-substituted-4-(3-bromophenylamino)quinazoline derivatives that may function as irreversible inhibitors of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases have been prepared. These inhibitors have, at the C-6 position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solublilizing substituents. These compounds were prepared by acylation of 6-amino-4-(3-bromophenylamino)quinazoline with unsaturated acid chlorides or mixed anhydrides. We show that attaching a basic functional group onto the Michael acceptor results in greater reactivity, due to intramolecular catalysis of the Michael addition and/or an inductive effect of the protonated basic group. This, along with improved water solubility, results in compounds with enhanced biological properties. We present molecular modeling and experimental evidence that these inhibitors interact covalently with the target enzymes. One compound, 16a, was shown to have excellent oral activity in a human epidermoid carcinoma (A431) xenograft model in nude mice.     

表皮生长因子在巨细胞病毒肝炎患儿十二指肠引流液及血清中含量研究

目的研究表皮生长因子(EGF)在巨细胞病毒(CMV)肝炎中的变化。方法CMV肝炎26例,根据血生化分为胆汁淤积型组(淤胆组)和肝炎型组(肝炎组)各13例,即直接胆红素/总胆红素(DB/TB)>0.5,r-GT>ALT,大便淡白至陶土色者纳入淤胆组,反之为肝炎组,对照组26例为肝肾功能正常的功能性消化不良患儿,分别收集血和十二指肠引流液标本(空腹插鼻饲管至十二指肠降部接负压引流袋3～4小时),采用放免法测定标本中EGF含量。结果肝炎组、淤胆组、对照组血清中EGF含量分别为(0.72±0.35)μg/L、(2.35±1.19)μg/L和(0.79±0.40)μg/L,十二指肠引流液中EGF含量分别为(20.96±6.14)μg/L、((9.93±5.41)μg/L和(2.75±0.40)μg/L,所有观察对象中EGF在十二指肠引流液中浓度明显高于血清中浓度(P<0.01),肝炎组和淤胆组患儿十二指肠引流液EGF浓度均明显高于对照组(P<0.01和P<0.05),且EGF浓度和血清中ALF水平正相关,肝炎组中十二指肠液中EGF浓度高于淤胆组(P<0.05),在胆道排泄通畅时血清中EGF水平不受肝胆局部浓度变化影响。结论肝胆组织中分泌的EGF通过胆道系统排泄,CMV肝炎可使局部EGF释放增加,故十二指肠液中EGF水平是一项能灵敏反映肝胆炎性受损的指标,小婴儿行十二指肠引流是安全可行的。     

Role of conformational alteration in the epidermal growth factor receptor (EGFR) function

This mini-review addresses the effect of glycosylation and phosphorylation on the conformational alterations of the epidermal growth factor receptor (EGFR). Based on studies with full-length and truncated EGFRs, we propose a model to suggest that receptor-receptor self-association, which occurs in the truncated receptor and depends on core glycosylation, is prevented in intact receptor by a certain extracellular domain and that the function of the ligand is to remove the negative constraint. We also propose, based on works with a conformation-specific antibody directed to an unphosphorylated peptide, that the interactions among negatively charged phosphotyrosine residues in the receptor molecule result in bringing two epitopes separated by a long stretch of amino acids close to each other to form an antibody-binding site. The implications of these posttranslational modifications on receptor functions are also discussed in this article.     

Design and synthesis of pyrrolo[3,2-d]pyrimidine human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) dual inhibitors: exploration of novel back-pocket binders

To develop novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) kinase inhibitors, we explored pyrrolo[3,2-d]pyrimidine derivatives bearing bicyclic fused rings designed to fit the back pocket of the HER2/EGFR proteins. Among them, the 1,2-benzisothiazole (42m) ring was selected as a suitable back pocket binder because of its potent HER2/EGFR binding and cell growth inhibitory (GI) activities and pseudoirreversibility (PI) profile as well as good bioavailability (BA). Ultimately, we arrived at our preclinical candidate 51m by optimization of the N-5 side chain to improve CYP inhibition and metabolic stability profiles without a loss of potency (HER2/EGFR inhibitory activity, IC(50), 0.98/2.5 nM; and GI activity BT-474 cells, GI(50), 2.0 nM). Reflecting the strong in vitro activities, 51m exhibited potent tumor regressive efficacy against both HER2- and EGFR-overexpressing tumor (4-1ST and CAL27) xenograft models in mice at oral doses of 50 mg/kg and 100 mg/kg.     

表皮生长因子受体在胸腺瘤中的表达

目的探讨表皮生长因子受体(EGFR)在胸腺瘤中的表达及其意义。方法免疫组化SP法检测63例患者胸腺瘤(胸腺瘤组)和20例胸腺增生(胸腺增生组)组织中EGFR的表达,分析胸腺瘤EGFR表达与其病理分级及临床分期的相关性。结果胸腺瘤组EGFR表达阳性率高于胸腺增生组(52.4%vs.20.0%)(P<0.05)。直径>6cm的胸腺瘤EGFR表达阳性率高于直径≤6cm的胸腺瘤(P<0.05)。胸腺瘤EGFR表达水平与病理分级及临床分期呈正相关。胸腺瘤的预后和临床分期相关。结论 EGFR可能在胸腺瘤的进展和预后中发挥重要作用。EGFR可能成为胸腺瘤新的治疗靶点和预后的标志物。     

Emerging drugs targeting human epidermal growth factor receptor 2 (HER2) in the treatment of breast cancer

Introduction: Human epidermal growth factor 2 (HER2) overexpression is present in 20% of breast cancer patients. It is associated with more aggressive disease and worse clinical outcome. New drugs are thus needed. Approved and future treatments will be discussed in this review.

Areas covered: The monoclonal antibodies trastuzumab and pertuzumab, the tyrosine kinase inhibitor lapatinib and the antibody-drug conjugate trastuzmab emtansine are approved for HER2 positive breast cancer. The combination of trastuzumab, pertuzumab and docetaxel is currently the first-line treatment in the metastatic setting. New therapies are still needed due to frequent relapse and resistance. These include mammalian target of rapamycin inhibitors, heat shock protein 90 inhibitors, pan-HER2 tyrosine kinase inhibitors, antibody-drug conjugates, immunotherapy agents (antibodies and vaccines), radioimmunotherapy and HER2 specific affinity proteins. Possible developmental issues are the complexity of the molecular biology of the HER2 positive cancer cell, the occurrence of resistance, toxicity and the high cost.

Expert opinion: The determination of the right sequence of use of old and new therapies remains a challenging issue. The selection of patients who do or don’t benefit from potentially toxic chemotherapy is also difficult. Central nervous system metastases are a common problem in HER2 positive breast cancer that needs to be addressed in future trials.