Transglutaminase 2 (TG2), a cross-linking enzyme, is involved in drug resistance and in the constitutive activation of nuclear factor kappa B (NF-kappaB). We investigated the association of non-small cell lung cancer (NSCLC) treatment efficacy with TG2 and NF-kappaB expression in 120 patients: 102 with adenocarcinoma and 18 with other histologic types. All patients underwent surgery; 88 received adjuvant chemotherapy, with 28 receiving platinum-based doublet chemotherapy as first-line treatment and 29 receiving epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Patients' TG2 and NF-kappaB expression values were calculated semiquantitatively. The median TG2 value was 50 (range, 0-300) and the median NF-kappaB value was 20 (range, 0-240). Disease-free survival did not differ between the low- and high-TG2 groups. Among patients who received palliative platinum-based doublet chemotherapy, progression free survival (PFS) was longer in the low-TG2 group than in the high-TG2 group (11.0 vs. 7.0 months; P=0.330). Among those who received EGFR-TKI therapy, PFS was also longer in the low-TG2 group than in the high-TG 2 group (11.0 vs. 2.0 months; P=0.013). Similarly, in EGFR wild-type patients treated with EGFR-TKI, PFS was longer in patients with low TG2 expression (9.0 vs. 2.0 months; P=0.013). TG2 expression levels can predict PFS in patients with NSCLC treated with EGFR-TKI.
Adenocarcinoma/*drug therapy/mortality/surgery ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents/therapeutic use ; Carcinoma, Non-Small-Cell Lung/*drug therapy/mortality/surgery ; Disease-Free Survival ; Female ; GTP-Binding Proteins/*biosynthesis ; Humans ; Lung Neoplasms/*drug therapy/mortality/surgery ; Male ; Middle Aged ; NF-kappa B/biosynthesis ; Protein Kinase Inhibitors/therapeutic use ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/genetics ; Transglutaminases/*biosynthesis ; Treatment Outcome

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

The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
Antineoplastic Agents ; pharmacology ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Epidermal growth factor receptor (EGFR) and its ligands (EGF and TGFalpha) are over-expressed in a variety of tumors. Immunization EGF-carrier protein inhibits tumor growth through abrogating binding of EGF to EGFR. Here, a chimeric protein of EGF and TGFalpha (E5T) was genetically fused to Staphylococcal enterotoxin A (SEA), a bacterial superantigenic protein which promotes humoral B cell response through enhancement of Ag-specific CD4 T cells activity. The resulted fusion proteins were expressed in Escherichia coli and purified though metal chelating affinity chromatography. Immunization of E5T-mSEA fusion protein in mice induced production of high titers antibodies, which recognize both EGF and TGFalpha. Anti- E5T-mSEA serum at dilution of 1:10 significantly inhibited growth of A431 cell lines but had little effect on 293T cell lines.
Amino Acid Sequence ; Animals ; Cancer Vaccines ; biosynthesis ; immunology ; Cell Line, Tumor ; Enterotoxins ; biosynthesis ; genetics ; Epidermal Growth Factor ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunization ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Random Allocation ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transforming Growth Factor alpha ; biosynthesis ; genetics

BACKGROUND/AIMS: Mutations of the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene (KRAS) are important in the pathogenesis of lung cancer, and recent reports have revealed racial and geographical differences in mutation expression. METHODS: This study was conducted to investigate the prevalence of EGFR and KRAS mutations and their correlation with clinical variables in Korean patients with adenocarcinoma of the lung. Formalin-fixed adenocarcinoma specimens from 104 randomly selected patients diagnosed at Kosin University Gospel Hospital from October 1996 to January 2005 were used for the study. RESULTS: We found a high prevalence of EGFR mutations and a low prevalence of KRAS mutations. EGFR mutations were present in 24% (25 of 104) of the samples: one mutation in exon 18, 13 in exon 19, one in exon 20, and 10 in exon 21. The presence of an EGFR mutation was not associated with gender, smoking history, histological grade, age, bronchioalveolar components, or cancer stage in patients with adenocarcinoma of the lung. CONCLUSIONS: Mutations of KRAS were present in 9.6% (9 of 94) of the samples: eight in codon 12 and one in codon 13. EGFR mutations were never found in tumors with KRAS mutations, suggesting a mutually exclusive relationship.
Adenocarcinoma/*genetics/metabolism/mortality ; Adult ; Aged ; DNA, Neoplasm/*genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Korea/epidemiology ; Lung Neoplasms/*genetics/metabolism/mortality ; Male ; Middle Aged ; *Mutation ; Polymerase Chain Reaction ; Prognosis ; Proto-Oncogene Proteins/biosynthesis/*genetics ; Receptor, Epidermal Growth Factor/biosynthesis/*genetics ; Retrospective Studies ; Survival Rate ; ras Proteins/biosynthesis/*genetics

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

OBJECTIVETo investigate whether chemically synthesized double-stranded RNA (dsRNA) targeting epidermal growth factor receptor (EGFR) can induce gene silencing in non-small cell lung cancer (NSCLC) cells in vivo.

METHODSThe NSCLC cell line SPC-A1 was transfected with EGFR sequence-specific dsRNA formulated with Lipofectamine 2000. SPC-A1 cells (1 x 10(7)/ml) in 200 microl were injected s.c. into the left flank area of the athymic nude mice to establish a tumor-bearing nude mouse model. To calculate the tumor growth inhibition rate by measuring the diameter and the weight of the tumor. Immunohistochemistry and Western blot were used to monitor the reduction of EGFR protein production. Real-time RT-PCR was used to detect the silencing of the EGFR mRNA level.

RESULTSThe EGFR sequence specific dsRNA (dsRNA-EGFR) significantly inhibited the tumor growth in vivo. The tumor growth inhibition rate was 75.0%. The dsRNA-EGFR sequence specifically silenced EGFR with 53.6% of down-regulation of EGFR protein production and 32.3% of silencing of EGFR mRNA level.

CONCLUSIONdsRNA-EGFR show a blockbuster effect in down-regulation of EGFR mRNA level and protein production, and inhibition of tumor growth in vivo.

Animals ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Nude ; RNA Interference ; RNA, Double-Stranded ; genetics ; RNA, Messenger ; metabolism ; Random Allocation ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection ; Tumor Burden

OBJECTIVETo investigate the effect of cationic liposome-mediated RNA interference (RNAi) in silencing epidermal growth factor (EGF) receptor (EGFR) gene in breast cancer cells in vivo.

METHODSA small interfering RNA (siRNA) targeting EGFR gene was constructed and transfected into human breast cancer cell in vitro via cationic liposome. The transfected cells were inoculated into nude mice, and the tumor growth inhibition rate was calculated. The tumors were then removed for immunohistochemistry and Western blotting to examine the expression of EGFR protein. Quantitative RT-PCR was used to detect the mRNA expression of the EGF receptor gene, and enzyme-linked immunosorbent assay (ELISA) performed to assess the EGF level in both the serum and tumor extraction.

RESULTSIn athymic nude mice, MDA-MB-231 cells had obviously lower tumor formation rate than ZR-75 cells (30.00% and 88.89%). Transfection of the cells with EGFR siRNA significantly inhibited tumor formation capacity of the cells in vivo as compared with the cells transfected with empty vector or irrelevant siRNA. The results of ELISA demonstrated that in mice bearing the tumors grown from EGFR siRNA-transfected cells, the EGF levels in the serum and tumor extraction were lowered by 16.77% and 12.59%, respectively. Real-time RT-PCR showed that EGFR siRNA transfection caused a specific downregulation of EGFR mRNA expression by 21.05% in the tumor.

CONCLUSIONChemically synthesized 21-nucleotide siRNA duplexes can be effectively delivered via lipofectamine 2000 into breast cancer cells in vivo to induce a longer-lasting gene silencing effect than in vitro transfection. RNAi of EGFR gene may indicate a promising approach for management of lung cancers, especially those nodular ones with easy access.

Animals ; Breast Neoplasms ; genetics ; pathology ; therapy ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression Regulation, Neoplastic ; Mammary Neoplasms, Experimental ; genetics ; pathology ; therapy ; Mice ; Mice, Nude ; RNA Interference ; RNA, Small Interfering ; genetics ; Random Allocation ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effect of Chinese herbs for nourishing yin and removing fire (NYRF) on gene expressions of estrogen receptor alpha (ER alpha), insulin-like growth factor-1 receptor (IGF-1R) and epithelial growth factor receptor (EGFR) in the epiphyseal growth plate of the female pubertal rats.

METHODSThe rats were randomly divided into the control group and the intervened group. Immunohistochemistry and realtime-PCR methods were used to measure the gene expression of ER alpha, IGF-1R and EGFR and their protein synthesis in epiphyseal growth plate.

RESULTSAfter being intervened with NYRF, the gene expressions of ER alpha and IGF-1R were down-regulated and their protein synthesis markedly reduced, while those of EGFR were unchanged.

CONCLUSIONNYRF can modulate the development and maturation of bone by regulating the expressions of ER alpha and IGF-1R in the epiphyseal growth plate.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Gene Expression ; drug effects ; Growth Plate ; drug effects ; metabolism ; Humans ; Protein Biosynthesis ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Receptor, IGF Type 1 ; genetics ; metabolism ; Yin-Yang

OBJECTIVETo explore the effects of antisense epidermal growth factor receptor (EGF-R) oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes after EGF-R oligodeoxynucleotides transfect to HaCaT in vitro.

METHODSc-jun DNA binding activity after ultraviolet-B (UVB) irradiation and EGF-R oligodeoxynucleotides transfection were determined with a highly sensitive and specific colorimetric method. After EGF-R oligodeoxynucleotides transfection, the mRNA level of EGF-R was detected by reverse transcription polymerase chain reaction method.

RESULTSCompared with control groups, c-jun activity increased significantly in UVB (10, 20, 30 mJ/cm2) irradiation groups (P < 0.05). EGF-R mRNA and c-jun activities induced by UVB were inhibited after the keratinocytes were transfected with EGF-R antisense oligodeoxynucleotides at 2, 4 and 8 microg/ml concentrations (P < 0.01).

CONCLUSIONThe ultraviolet-induced c-jun activity of keratinocytes can be mediated by EGF-R and inhibited by EGF-R antisense oligodeoxynucleotides, which is transfected to keratinocytes and mediated by lipofectamine.

Cell Line ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Keratinocytes ; drug effects ; metabolism ; radiation effects ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection ; Ultraviolet Rays