Breast Neoplasms ; metabolism ; Female ; Humans ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Receptors, Progesterone ; metabolism

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

OBJECTIVETo investigate the effects of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the biological activity of glioblastoma cell line GL15.

METHODSGlioblastoma GL15 cells were cultured and transfected with LRIG3-EGFP plasmid. The location of LRIG3 in GL15 cells was observed with confocal microscopy. The proliferation and invasiveness of GL15 cells were detected with methyl thiazolyl tetrazolium (MTT) and Transwell methods respectively; the expression of epidermal growth factor receptor (EGFR) and LRIG3 mRNA and protein were detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot respectively.

RESULTAfter transfection with the plasmid LRIG-EGFP, LRIG3 fusion protein was found in cytoplasm of GL15 cells and cell proliferative and invasiveness were reduced. The expression of EGFR and LRIG3 varied with the duration of EGF treatment (100 ng/ml): the expression of EGFR decreased while the expression of LRIG3 increased as time prolonged.

CONCLUSIONLRIG3 can inhibit the proliferation and invasiveness of glioblastoma cells and may be used as a target gene in gene therapy of glioblastoma.

Brain Neoplasms ; pathology ; Cell Proliferation ; Epidermal Growth Factor ; genetics ; Glioblastoma ; pathology ; Humans ; Membrane Proteins ; genetics ; metabolism ; Neoplasm Invasiveness ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured

A DNA fragment encoding extracellular domain of mouse epidermal growth factor receptor (EGFR) was obtained by PCR from a previous recombinant plasmid. The DNA fragment was then ligated into prokaryotic expression vector, and expressed in Escherichia Coli. The recombinant protein was purified under denature conditions by affinity chromatography, and refolded with gradient dialysis. The recombinant protein could produce antibodies to recognize extracellular domain and full-length of mouse EGFR, and form homodimer in the presence of EGF detected by western blot analysis. These findings provide evidence that the renatured recombinant extracellular domain of mouse epidermal growth factor receptor is immunogenetic and may be important for further application of this protein in functional and immunological research.
Animals ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Mice ; Plasmids ; Receptor, Epidermal Growth Factor ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Transfection

OBJECTIVETo probe the mechanism of electroacupuncture at points "Weibingfang" in treatment of acute gastric mucosal lesion.

METHODSForty Wistar rats of sanitary degree were randomly divided into 4 groups, normal group, model group, sham-model group and Weibingfang group, 10 rats in each group. The acute gastric mucosal lesion model was made by intragastric perfusion of anhydrous alcohol. The Weibingfang group were treated by electroacupuncture at "Neiguan" (PC 6), "Zhongwan" (CV 12) and "Zusanli" (ST 36) with sparse-dense wave, frequency of 10-30 Hz, current intensity of 2 mA, for 20 min. One hour after the treatment, the blood from the abdominal aorta and the gastric mucosa tissue were taken, and serum epidermal growth factor (EGF) level and epidermic growth factor receptor mRNA (EGFR mRNA) expression in the gastric mucosa were detected with enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively.

RESULTSThere were significant differences in blood EGF level (41.62 +/- 12.58) ng/L and EGFR mRNA expression (0.78 +/- 0.03) in the model group were significantly different from [(60.37 +/- 12.01) ng/L and 0.55 +/- 0.04] in the normal group and [(61.21 +/- 13.46) ng/L and 0.53 +/- 0.05] in the sham-model group (P < 0.05); after electroacupuncture, blood EGF level (70.59 +/- 10.14) ng/L increased and the EGFR mRNA expression (1.18 +/- 0.02) in the gastric mucosa was up-regulated with a significant differences as compared with those in the model group (P < 0.05).

CONCLUSIONPoint "Weibingfang" can promote proliferation, differentiation and migration of the gastric mucosal epidermic cells to repair the gastric mucosal lesion.

Acupuncture Points ; Animals ; Electroacupuncture ; Epidermal Growth Factor ; blood ; genetics ; Female ; Gastric Mucosa ; metabolism ; Gene Expression ; Humans ; Male ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Stomach Diseases ; genetics ; metabolism ; therapy

OBJECTIVETo observe the effect of Tanshinone II A on the expression of epidermal growth facter (EGF) and epidermal growth facter recepter (EGFR) in human hepatocellular carcinoma cell line SMMC-7721.

METHODSThe human hepatocellular carcinoma SMMC-7721 cells cultured in vitro was treated with different concentrations of Tanshinone II A. The proliferation of the cells was measured by MTT assay, and the apoptosis of the cells was investigated by flow cytometry and cytochemical staining with Hoechst 33342. The expression of EGF and EGFR was detected by immunocytochemistry method. The levels of EGF in medium were measured by radioimmunoassay.

RESULTTanshinone II A inhibited the growth of SMMC-7721 cells remarkably in a dose-dependent manner. The inhibitory rate reached the peak (72.5%) after 0.5 microg/ml Tanshinone II A was used for 48 h, which was significantly higher than that in the controls (P<0.05). FCM analysis showed that when SMMC-7721 cells were treated with 0.5 microg/ml Tanshinone II A, the apoptosis rates for 24 h, 48 h and 72 h were (4.06+/-0.27)%, (7.58+/-0.56)% and (5.23+/-0.13)%, respectively which were markedly higher than those in the controls (all P<0.01). SMMC-7721 cell apoptosis with cell shrinkage, nuclear chromatin concentration and fragmentation as well as the formation of apoptotic bodies were observed by cytochemical staining when treated with Tanshinone II A. The immunocytochemistry showed that the expressions of EGF and EGFR were down regulated while the concentration of Tanshinone II A was increasing. The high expression rates for EGF and EGFR were 10%, 20%, respectively, and the gray scale was 181.52+/-1.63, 179.37+/-1.59, which were markedly higher than those in the controls (all P<0.05). The levels of EGF in medium measured by radioimmunoassay were decreased significantly after Tanshinone II A treatment.

CONCLUSIONTanshinone II A can inhibit cell proliferation and induce apoptosis in hepatocellular carcinoma cell line SMMC-7721, which may be related to the down-regulation of EGF and EGFR protein expression.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Diterpenes, Abietane ; Down-Regulation ; drug effects ; Epidermal Growth Factor ; genetics ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Phenanthrenes ; pharmacology ; Receptor, Epidermal Growth Factor ; genetics ; metabolism

Animals ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; Molecular Imaging ; Mutation ; Positron-Emission Tomography ; RNA, Messenger ; metabolism ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics

The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2. The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21 (DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni2+ -NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni2+ -NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.
Escherichia coli ; genetics ; metabolism ; Humans ; Inclusion Bodies ; genetics ; metabolism ; Protein Folding ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUNDDecorin is a small leucine-rich proteoglycan and it plays an important role in regulation of cell growth and migration in various tumor cell lines. Decorin was found down-regulated in non-small cell lung cancer tissue and may be involved in regulation of lung cancer development.

METHODSIn this study, lentivirus-mediated RNA interference and over expression were employed to change the expression levels of decorin in lung cancer A549 cells. We tested the cell cycle of A549 cells and the expression of transforming growth factor (TGF)-β, cyclin D1, epidermal growth factor receptor (EGFR), P53, and P21.

RESULTSWe found that up-regulation of decorin could inhibit proliferation, block cell cycle at G1 and decrease invasive activity of A549 cells. Moreover, we also show that up-regulation of decorin induced significant decreases of TGF-β1, cyclin D1 expression, phosphorylation of EGFR, and increases of P53 and P21 expression. Opposite results were observed in A549 cells with down-regulation of decorin.

CONCLUSIONOur results suggest that decorin is a key regulator involved in proliferation and migration of A549 cells.

Cell Cycle ; genetics ; physiology ; Cell Movement ; genetics ; physiology ; Cell Proliferation ; Cyclin D1 ; genetics ; metabolism ; Decorin ; genetics ; metabolism ; Humans ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo investigate the regulation of epithelium growth factor receptor (EGFR), pan-Ras, and extracellular regulated protein kinase (ERK) with both a ras homologue member I (ARHI) suppression and epithelium growth factor (EGF) stimulation.

METHODSAfter identification and implication, the constructed plasmid pIRES2-EGFP-ARHI was transfected into Panc-1. The untransfected cell was also explored as controls. The growth curve was drawn to indicate the proliferation effect of ARHI. EGFR-ELISA was performed to investigate the expression of EGFR. Western blot analysis was used to investigate the expression of protein MAPK/ERK1/2, pan-Ras in Panc-1.

RESULTSThe proliferation rate of Panc-1 was inhibited by ARHI compared with both empty plasmid and untransfected cell. The amount of EGFR was parallel in both transfected and untransfected cell but affected by EGF stimulation. The amount of pan-Ras was decreased after ARHI transfection. The optimum concentration of EGF effect on P-ERK was 50 ng/ml.

CONCLUSIONBoth ARHI and EGF play roles in the EGF-EGFR-Ras-Raf-MAPK/ERK1/2 pathway.

Cell Proliferation ; Epidermal Growth Factor ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Transfection ; Tumor Cells, Cultured ; ras Proteins ; metabolism ; rho GTP-Binding Proteins ; genetics