Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo investigate the effects of adenovirus-delivered angiopoietin-1 siRNA (Ad. Ang-1siRNA) on the expression of matrix metalloproteinase-2, 9 (MMP-2, 9) and tissue inhibitor of metallopro-teinase-1 (TIMP-1) in rats with acute lung injury (ALI) induced by phosgene (Psg).

METHODSWe first established a rat model of Psg-induced acute lung injury (ALI). The rats were randomly divided into 6 groups: air control group with exposure to air, air+adenovirus (air+Ad) group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after air exposure, air+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after air exposure, Psg group with exposure to 8.33 mg/L Psg (purity 100%, of the same volume as the inhaled air in the air control group) for 5 min, Psg+Ad group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after exposure to the same dose of Psg, and Psg+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after exposure to the same dose of Psg. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected 36 h after exposure. The protein expression of Ang-1, MMP-2, 9, and TIMP-1 in serum and BALF was determined by double-antibody sandwich ELISA. RT-PCR was used to determine the mRNA levels of Ang-1, MMP-2, 9, and TIMP-1 in lung tissue. The protein expression of MMP-2, 9 and TIMP-1 in lung tissue was determined by Western blot.

RESULTSA rat model of Psg-induced ALI was successfully established. The levels of MMP-2, 9 in serum, BALF, and lung tissue were significantly increased in the Psg group and Psg+Ad/Ang1 group as compared with the control group (P<0.01); no significant change was observed in serum TIMP-1 protein expression (P>0.05); interestingly, TIMP-1 protein expression in BALF and lung tissue was significantly increased (P<0.01). Compared with the Psg group, the Psg+Ad/Ang1 group showed a significant decrease in MMP-2, 9 expression in BALF, serum, and lung tissue (P<0.05), but no significant change in protein expression of TIMP-1 was discovered (P>0.05).

CONCLUSIONAd.Ang-1siRNA has a potential beneficial effect in rats with Psg-induced ALI through inhibition of MMP-2, 9 expression, but has no significant effect on the expression of TIMP-1.

Acute Lung Injury ; chemically induced ; metabolism ; Adenoviridae ; genetics ; Angiopoietin-1 ; physiology ; Animals ; Bronchoalveolar Lavage Fluid ; Chemical Warfare Agents ; toxicity ; Disease Models, Animal ; Lung ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; Matrix Metalloproteinases ; metabolism ; Phosgene ; toxicity ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

OBJECTIVETo study the influence of hepatitis B virus (HBV) replication and expressions of different viral genes on CDC37 level in hepatocytes.

METHODSWe amplified and cloned 6 HBV genes (P, preS1, preS2, S, C and X) into pCMV expression vectors, which were transfected in Huh7 and HepG2 hepatoma cell lines, and CDC37 expression level in the cells was detected using Western blotting. Wealso cloned the promoter sequence of CDC37 into pGL3 vector, and co-transfected pGL3 with pCMV recombinant plasmids into Huh7 and HepG2 cells and the fluorescent signals were detected. To study the influence of HBV replication on CDC37 expression, we constructed 1.28-copy overlength genomes of HBV genotypes B, C, D and CD recombinant. The overlength HBV genomes were transformed into Adeasier-1 cells for recombination and into 293 cells for packaging. Huh7 and HepG2 cell lines infected with the packaged HBV recombinant adenoviruses were examined for CDC37 expression with Western blotting.

RESULTSWestern blotting showed that the expression of different HBV genes did not obviously affect the protein level of CDC37 in the hepatocytes. The protein expression of HBV genes had no effect on the activity of CDC37 promoter. Huh7 and HepG2 cells infected with 1.28-copy HBV replicon showed no significant changes in the expression level of CDC37.

CONCLUSIONHBV replication and its gene expression have no effect on the level of CDC37 in hepatocytes in vitro.

Adenoviridae ; Cell Cycle Proteins ; metabolism ; Chaperonins ; metabolism ; Gene Expression Regulation, Viral ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; virology ; Humans ; Transfection ; Virus Replication

Oncolytic adenoviruses (Ads), which are live, replication-competent viruses that can selectively replicate in tumor cells and lead to cell lysis, have been used in tumor therapy. But due to the complexity and high mutability of human tumors, it becomes a major strategy to improve the selectivity, efficacy, and safety of oncolytic Ads. The oncolytic Ads that can express short hairpin RNA, cytokines, suicide gene, and matrix-modulating proteins have higher antitumor activity than the wild type. Tumor-specific promoters, especially hTERT and HRE promoters, increase the selectivity of oncolytic Ads for tumor cells. Moreover, oncolytic Ads surface-modified by polyethylene glycol (PEG), liposomes, biodegradable nanoparticles, and polypeptides have reduced immunogenicity and hepatotoxicity and improved antitumor activity when systemically administered, and the selectivity of oncolytic Ads can be significantly increased when linking PEG to antibodies, small peptides, cytokines, and ligands. Therefore, engineered oncolytic Ads combining the advantages of viral and non-viral vectors, as well as immunotherapy, are a promising strategy for improving the efficacy of targeted virotherapy.
Adenoviridae ; genetics ; physiology ; Animals ; Humans ; Neoplasms ; therapy ; virology ; Oncolytic Virotherapy ; trends ; Virus Replication

Adenovirus remains a significant threat to public health. Recent studies showed that bats can harbor diverse adenoviruses. To further investigate the distribution and genetic diversity of bat adenoviruses in China, we collected throat and anal swab samples of 11 bat species from 6 provinces of China, including Beijing, Hunan, Jiangxi, Yunnan, Guizhou and Hainan. Nested PCR was used to identify potential bat adenoviruses from the samples, and positive results were cloned and sequenced for genetic diversity study. In addition, nucleotide sequence alignments based on corresponding amino acid sequence similarities were used for phylogenetic analyses. Our results showed that about 20% of bat species in China are positive to adenoviruses, and Myotis ricketti is likely to be the most important host of bat adenoviruses in all locations. Moreover, we identified two diverse sequences of bat adenoviruses from the same sample of Ia io in Guizhou province of China. In general, the average nucleotide and amino acid sequence similarities of the conserved region of DNA polymerases of bat adenoviruses are 66.6% and 74.7%, respectively. The differences between bat species and their residences environments may have driven the adaptive evolution of the viruses, leading to the genetic diversity of the bat adenoviruses.
Adenoviridae ; classification ; genetics ; isolation & purification ; physiology ; Animals ; China ; Chiroptera ; virology ; Genetic Variation ; Host Specificity ; Phylogeny

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.
Adenoviridae ; genetics ; Animals ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cetuximab ; Drug Synergism ; Erlotinib Hydrochloride ; Female ; Genetic Therapy ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Nude ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; physiology ; Transfection ; Tumor Burden ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity.

METHODSStable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo.

RESULTSThe cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05).

CONCLUSIONSThe results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.

Adenoviridae ; physiology ; Animals ; Carcinoembryonic Antigen ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; metabolism ; pathology ; therapy ; Female ; Gene Silencing ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oncolytic Virotherapy ; Oncolytic Viruses ; physiology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tumor Burden

BACKGROUNDOur previous studies have demonstrated potent oncolysis efficacy of the E1A, E1B double-restricted replication-competent oncolytic adenovirus AxdAdB-3 for treatment of bladder cancer. Here, we reported the feasibility and efficacy of AxdAdB-3 alone, or in combination with gemcitabine for treating renal cell carcinoma.

METHODSCytopathic effects of AxdAdB-3 were evaluated in human renal cell carcinoma cell lines TOS-1, TOS-2, TOS-3, TOS-3LN, SMKT-R3, SMKT-R4 and ACHN, and in normal human renal proximal tubule epithelial cells (RPTEC). AxdAdB-3 induced down-regulation of the cell cycle was determined by flow cytometry. Combination therapies of AxdAdB-3 with gemcitabine were evaluated in vitro and in vivo on subcutaneous TOS-3LN tumors in a severe combined immunodeficiency disease (SCID) mouse model.

RESULTSAxdAdB-3 was potently cytopathic against the tested most renal cell carcinoma cell lines including TOS-2, TOS-3, TOS-3LN, SMKT-R3 and SMKT-R4, while normal human RPTEC were not destroyed. AxdAdB-3 effectively induced cell cycle S-phase entry. Combined therapy of AxdAdB-3 with gemcitabine demonstrated stronger antitumor effects in vitro and in vivo compared with either AxdAdB-3 or gemcitabine alone.

CONCLUSIONAxdAdB-3 alone, or in combination with gemcitabine may be a promising strategy against renal cell carcinoma.

Adenoviridae ; genetics ; metabolism ; physiology ; Adenovirus E1A Proteins ; genetics ; Adenovirus E1B Proteins ; genetics ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Carcinoma, Renal Cell ; drug therapy ; therapy ; Cell Cycle ; drug effects ; genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; Deoxycytidine ; analogs & derivatives ; pharmacology ; therapeutic use ; Flow Cytometry ; Humans ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Mice, SCID ; Oncolytic Virotherapy ; Receptors, Virus ; genetics ; metabolism ; Xenograft Model Antitumor Assays

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.
Adenoviridae ; genetics ; metabolism ; physiology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genes, p53 ; Genetic Therapy ; Genetic Vectors ; Humans ; Neoplasms ; metabolism ; pathology ; virology ; Oncolytic Viruses ; genetics ; metabolism ; physiology ; Quality Control ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Virus Replication

BACKGROUND AND OBJECTIVEAdenovirus vectors were widely used in gene therapy for tumors. We used adenovirus vector to transfer small interfering RNA (siRNA) against vascular epithelium growth factor A (VEGF-A) molecules to mouse lung adenoma LA795 cells and used low dose of chemotherapeutic drugs to further elevate the infection efficiency of adenovirus vector in and therapeutic effect of RNAi on tumor cells.

METHODSLA795 cells were infected by Ad/EGFP and treated with different dosages of gemcitabin, epirubicin, cisplatin, or 5-fluorouracil (5-FU). Cells were observed under fluorescence microscope continuously using green fluorescent protein (GFP) as the reporter gene. The percentage of GFP-positive cells and fluorescent intensity were tested by flow cytometry to determine optimum concentrations of drugs. Ad/siVEGF-A containing VEGF-A siRNA was constructed. Real-time PCR and ELISA were applied to measure the expression level of VEGF-A after LA795 cells were infected by Ad/siVEGF-A and treated with 5-FU. The combination of Ad/siVEGF-A and 5-FU was also applied in treating subcutaneous tumor in mice.

RESULTSLow dose of 5-FU elevated the Ad/EGFP infection in LA795 cells significantly, and also enhanced the effect of Ad/siVEGF-A in down-regulating VEGF-A mRNA and protein levels in tumor cells. When used in tumor in vivo, the combination strategy repressed tumor growth effectively.

CONCLUSIONLow dose of 5-FU can enhance the capability of adenovirus infecting tumor cells and promote the efficiency of gene therapy by adenovirus.

Adenocarcinoma ; metabolism ; pathology ; Adenoviridae ; genetics ; Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; Fluorouracil ; administration & dosage ; pharmacology ; Genetic Therapy ; Genetic Vectors ; Lung Neoplasms ; metabolism ; pathology ; Mice ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics ; physiology