angiogenin and Lung-Neoplasms

Squamous cell carcinoma of the lung is one of the most aggressive cancers, and its aggressiveness is in part due to its intrinsic high rate of metastasis. Moreover, the process of epithelial-mesenchymal transition (EMT) appears to be involved in these neoplastic processes. Furthermore, EMT-type cells share many biological characteristics with the function of angiogenin (ANG) in squamous cell lung carcinoma. We conducted immunohistochemical analysis to detect the expression of ANG, E-cadherin, vimentin, N-cadherin, β-catenin and TGF-β1 in 60 cases of squamous cell lung carcinoma tissues. Western blot analysis was adopted to detect the protein expression levels of ANG and EMT markers. The effects of ANG on proliferation, migration and invasion of squamous cell lung carcinoma cells was analyzed by Cell Counting Kit-8, scratch assay and Transwell invasion chamber in order to reveal the role of ANG in the process of EMT in squamous cell lung carcinoma. The results revealed that ANG was aberrantly expressed in the squamous cell lung carcinoma specimens and was closely correlated with the differentiation of the cell lines. The expression of ANG was also significantly associated with metastasis and the stage of the squamous cell lung carcinoma cases. In addition, we validated that ANG influenced the expression of vimentin, E-cadherin, N-cadherin, β-catenin and TGF-β1 in SK-MES-1 cells. Most importantly, overexpression of ANG enhanced the migration and invasion of SK-MES-1 cells, while knockdown resulted in opposite effects. In the present study, we found that ANG plays an important role in EMT in squamous cell lung carcinoma and may be a valuable therapeutic target for squamous cell lung carcinoma.

Topics: beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Neoplasm Invasiveness; Ribonuclease, Pancreatic; Transforming Growth Factor beta1; Vimentin

Angiogenin (ANG) is a multifunctional secreted protein that belongs to the pancreatic ribonuclease A super family, which has been conceived to play a more important role in cell survival, growth and proliferation than the mediation of angiogenesis. Accumulating evidences suggest that the expression and activity of ANG increased significantly in a variety of human cancers. Recent studies showed that ANG activates cell signaling pathway through the putative receptor on endothelial cells. However, the underlying mechanisms remain largely unknown. AKT/mTOR signaling pathway participates in cell growth, cell-cycle progression and cell apoptosis. The purpose of our study was to determine whether ANG implicated in growth and metastasis of bladder cancer cells through regulating AKT/mTOR signaling pathway. In this study, we constructed ANG siRNA plasmids that transfected into human bladder cancer T24 cells. We demonstrated that knockdown of ANG could inhibit cell proliferation, regulate cell cycle and induce apoptosis. We also found that down-regulation of ANG remarkably reduced the phosphorylation of signaling targets AKT, GSK-3β and mTOR. Furthermore, down-regulation of ANG increased expression of ribonuclease inhibitor, which is a cytoplasmic acidic protein with many functions. Finally, ANG siRNA led to the suppression for tumorigenesis and metastasis in vivo. Taken together, these findings highlight for the first time that ANG could play a pivotal role in the development of bladder cancer through regulating AKT/mTOR signaling pathway. The targeting of ANG and associated factors could provide a novel strategy to inhibit human bladder cancer.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Shape; Down-Regulation; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Ribonuclease, Pancreatic; Signal Transduction; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Angiogenin (ANG), a member of RNase A superfamily, is the only angiogenic factor that possesses ribonucleolytic activity. Recent studies showed that the expression of ANG was elevated in various types of cancers. Accumulating evidence indicates that ANG plays an essential role in cancer progression by stimulating both cancer cell proliferation and tumor angiogenesis. Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats (LRRs), which are present in a large family of proteins that are distinguished by their display of vast surface areas to foster protein-protein interactions. RI might be involved in unknown biological effects except inhibiting RNase A activity. The experiment demonstrated that RI also could suppress activity of angiogenin (ANG) through closely combining with it in vitro. PI3K/AKT/mTOR signaling pathway exerts a key role in cell growth, survival, proliferation, apoptosis and angiogenesis. We recently reported that up-regulating RI inhibited the growth and induced apoptosis of murine melanoma cells through repression of angiogenin and PI3K/AKT signaling pathway. However, ANG receptors have not yet been identified to date, its related signal transduction pathways are not fully clear and underlying interacting mechanisms between RI and ANG remain largely unknown. Therefore, we hypothesize that RI might combine with intracellular ANG to block its nuclear translocation and regulate PI3K/AKT/mTOR signaling pathway to inhibit biological functions of ANG. Here, we reported for the first time that ANG could interact with RI endogenously and exogenously by using co-immunoprecipitation (Co-IP) and GST pull-down. Furthermore, we observed the colocalization of ANG and RI in cells with immunofluorescence staining under laser confocal microscope. Moreover, through fluorescence resonance energy transfer (FRET) assay, we further confirmed that these two proteins have a physical interaction in living cells. Subsequently, we demonstrated that up-regulating ANG including ANG His37Ala mutant obviously decreased RI expression and activated phosphorylation of key downstream target molecules of PI3K/AKT/mTOR signaling pathway. Finally, up-regulating ANG led to the promotion of tumor angiogenesis, tumorigenesis and metastasis in vivo. Taken together, our data provided a novel mechanism of ANG in regulating PI3K/AKT/mTOR signaling pathway via RI, which suggested a new therapeutic target for cancer therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Fluorescence Resonance Energy Transfer; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mutant Proteins; Phosphatidylinositol 3-Kinases; Placental Hormones; Plasmids; Protein Binding; Proto-Oncogene Proteins c-akt; Ribonuclease, Pancreatic; Signal Transduction; TOR Serine-Threonine Kinases; Transfection; Up-Regulation; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Vascular endothelial growth factor (VEGF) is an important signaling protein and a predominant mediator of angiogenesis in tumor growth and metastasis. Therefore, antagonism of the VEGF pathway results in inhibition of abnormal angiogenesis, then suppression of tumor growth and metastasis. VEGF-Trap, a high-affinity soluble decoy receptor, is currently in phase II clinical trails, and has demonstrated more efficacy in different types of solid tumors by intravenous injection every two weeks. In our study, we used recombinant AAV2 as a delivery vehicle to achieve long-lasting expression of VEGF Trap protein in a mouse model for the first time. We report that AAV2-VEGF-Trap can be safely administered and sustained expression in vivo via a single intravenously administration, simultaneously suppressing primary tumor growth and preventing the pulmonary metastases of 4T1 tumors. Decreased microvessel density and increased tumor cell apoptosis were observed in the treatment group. AAV2-VEGF-Trap can obviously decrease not only the concentration of VEGF in sera, but also the concentration of other angiogenic factors, such as aFGF, bFGF, angiopoietin-1 and others. These studies suggest that AAV-mediated long-term expression of VEGF-Trap is a useful and safe tool to block tumor progression and inhibit spontaneous pulmonary metastases.

Topics: Animals; Apoptosis; Breast Neoplasms; Dependovirus; Disease Models, Animal; Female; Gene Transfer Techniques; Genetic Therapy; Injections, Intravenous; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Receptors, Vascular Endothelial Growth Factor; Recombinant Fusion Proteins; Ribonuclease, Pancreatic; Thymidine Phosphorylase; Vascular Endothelial Growth Factor A

Early recognition of lung cancer is a prerequisite for any strategy to improve lung cancer treatment outcome. Here we report a cross-sectional study intended as a proof of principle investigation using breath based detection (exhaled breath condensate, EBC) of angiogenic markers (VEGF, bFGF, angiogenin), TNF-alpha and IL-8 to discriminate 74 individuals, with confirmed presence or absence (X-ray, CT) of non-small lung cancer (NSCLC). Levels of angiogenic markers bFGF, angiogenin and VEGF in EBC significantly discriminated between 17 individuals with newly detected NSCLC versus stable and exacerbated chronic obstructive pulmonary disease (COPD) patients as well as healthy volunteers. Levels of IL-8 and TNF-alpha in EBC indicated acute inflammation, e.g. in acute exacerbated COPD (AECOPD) and were not indicative of lung cancer. In a different group of patients that were already treated with two cycles of chemotherapy and who responded with at least a 25% reduction in primary tumor diameter, levels of angiogenic markers were lower compared to patients with newly diagnosed NSCLC. We suggest that breath based detection of angiogenic markers may help in the early detection of lung cancer.

Topics: Aged; Biomarkers, Tumor; Breath Tests; Carcinoma, Non-Small-Cell Lung; Cross-Sectional Studies; Diagnosis, Differential; Disease Progression; Feasibility Studies; Female; Fibroblast Growth Factors; Humans; Lung Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Pulmonary Disease, Chronic Obstructive; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

Angiogenin, a basic heparin-binding protein, has been shown to play a key role in tumor growth and angiogenesis. It was found in the present study that 67 out of 100 lung adenocarcinomas exhibited angiogenin nuclear expression, and this nuclear expression correlated with vascular and pleural invasion as well as positive lymph node metastasis. To down-regulate angiogenin expression, we constructed an adenoviral-vector based short hairpin RNA system. ELISA, real-time qPCR and immunocytochemical staining demonstrated that adenoviral-vector based siRNA decreased angiogenin mRNA level and protein secretion, and inhibited angiogenin nuclear expression in A549 cells, resulting in marked inhibition on ribosomal RNA transcription, in vitro cell proliferation, soft agar colony formation, and xenograft tumor proliferation and angiogenesis. Experiments with neomycin further confirmed that angiogenin nuclear expression played an important role in tumor growth. Based on these data, we concluded that angiogenin nuclear expression played a dual role in the growth of lung adenocarcinoma with respect to cancer cell proliferation and angiogenesis.

Topics: Adenocarcinoma; Aged; Angiogenesis Inducing Agents; Cell Line, Tumor; Cell Proliferation; Disease Progression; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Ribosomes; RNA Interference; RNA, Messenger

To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 through adeno-associated virus (AAV)-mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation.. Recombinant AAV expressing H1-promoter-induced small-interference- RNA (siRNA) targeting ANG (AAV-shANG) was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and microvessel-density of the carcinoma were observed.. In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells (P < 0.01). Cell cycle analysis showed that the proliferation index (PI) of normal A549 cells, AAV-Null cells and AAVshANG cells were 0.32 +/- 0.29, 0.35 +/- 0.38 and 0.31 +/- 0.43, respectively. There was no statistic difference in the PIs among the 3 groups (P > 0.05). In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 +/- 1.5, 9.8 +/- 2.1 and 5.7 +/- 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were (7.7 +/- 3.1)%, (8.5 +/- 5.4)%, (17.1 +/- 8.6)%, respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were (84.8 +/- 9.7)%, (85.8 +/- 9.8)%, and (70.4 +/- 10.1)%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups.. AAV-mediated expression of siRNA could reduce the expression of ANG in cancer cells, significantly enough to inhibit cell proliferation, promote cell apoptosis and inhibit tumor growth.

Topics: Adenocarcinoma; Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dependovirus; Humans; Lung Neoplasms; Mice; Mice, Nude; Recombination, Genetic; Ribonuclease, Pancreatic; RNA Interference; Transfection

A sensitive method for rapid angiogenin (Ang) detection based on fluorescence resonance energy transfer (FRET) has been described. A dual-labeled probe based on high affinity aptamer for Ang was constructed. As donor and acceptor, 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TMR) were labeled at 5'- and 3'-termini of the aptamer probe, respectively. The dual-labeled probe showed obvious fluorescence changes due to the specific binding between aptamer and Ang. By monitoring the fluorescence intensity of donor and acceptor, quantitative Ang detection could be achieved. This assay is highly specific and sensitive, with a detection limit of 2.0 x 10(-10) mol L(-1) and a linear range of 5.0 x 10(-10) to 4.0 x 10(-8) mol L(-1) Ang. Ang in serum samples of health and lung cancer were also detected.

Topics: Angiogenesis Inducing Agents; Aptamers, Nucleotide; Base Sequence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Lung Neoplasms; Molecular Probe Techniques; Molecular Sequence Data; Neoplasm Proteins; Reproducibility of Results; Ribonuclease, Pancreatic; Sensitivity and Specificity; Time Factors

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a novel angiogenesis inhibitor. In the previous study, purified Pichia-derived TK1-2 has been shown to suppress in vivo growth of human lung and colon cancer cells. Here, we demonstrate that E. coli-derived non-glycosylated TK1-2 suppresses tumor growth more potently than Pichia-derived TK1-2 and prolongs the survival of tumor bearing mice. The recombinant TK1-2 prepared through E. coli expression, His-tag affinity chromatography and in vitro refolding was injected intraperitoneally once daily into nude mice 7 days after subcutaneous implantation with PC14 lung cancer cells (n=10). Measurement of tumor volumes indicated that low-dose TK1-2 treatment (10 mg/kg) suppressed tumor growth by approximately 85.2% (p<0.01), while high-dose TK1-2 treatment (50 mg/kg) even more potently inhibited tumor growth (>93.8%) (p<0.005). Treatment of TK1-2 also prolonged the survival of tumor-bearing mice in a dose-dependent fashion. In an independent HCT116 xenograft model, E. coli-derived TK1-2 was more effective in suppressing tumor growth than Pichia-derived TK1-2. Immunohistochemical analysis of tumor tissue also revealed that the expression of VEGF, SMA-alpha, TNF-alpha and angiogenin was less positive in the E. coli-derived TK1-2-treated group than in the Pichia-derived TK1-2-treated group. These results suggest that E. coli-derived refolded, non-glycosylated TK1-2 can be used more effectively as an anti-cancer agent.

Topics: Animals; Antineoplastic Agents; Colorectal Neoplasms; Dose-Response Relationship, Drug; Escherichia coli; HCT116 Cells; Humans; Kringles; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Recombinant Proteins; Ribonuclease, Pancreatic; Tissue Plasminogen Activator; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

Topics: Allantois; Animals; Apolipoproteins A; Cell Division; Cell Movement; Cells, Cultured; Chickens; Chorion; Colonic Neoplasms; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelium, Vascular; Escherichia coli; Female; Fibroblast Growth Factor 2; Gene Expression; Humans; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Kringles; Lung Neoplasms; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Peptide Fragments; Phosphorylation; Recombinant Proteins; Ribonuclease, Pancreatic; RNA, Messenger; Tumor Cells, Cultured; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis and vascular permeability. We hypothesized that malignant pleural effusions may contain high levels of VEGF protein as well as other cytokines implicated in these processes. Pleural effusions cytologically proven to be malignant were collected from 39 patients with various types of cancer, and VEGF, interleukin-8, and angiogenin levels in the effusions were determined by immunoassay. Negative controls were nonmalignant ascites and serum samples from healthy individuals. VEGF levels were significantly higher than those of control samples in pleural effusions secondary to breast, mesothelioma, and non-small cell lung cancer and when all malignant pleural effusion samples were pooled. Neither interleukin-8 nor angiogenin levels were elevated in malignant pleural effusions relative to the control samples. Vascular permeability, which was measured by using the Miles assay in nude mice, was increased proportionately with VEGF levels in the malignant pleural effusions; this increase in permeability induced by injection of recombinant VEGF or the malignant effusions was reduced by pretreating the mice with a VEGF receptor antibody.

Topics: Angiogenesis Inducing Agents; Animals; Breast Neoplasms; Capillary Permeability; Carcinoma, Non-Small-Cell Lung; Endothelial Growth Factors; Female; Humans; Interleukin-8; Lung Neoplasms; Lymphokines; Lymphoma; Male; Mesothelioma; Mice; Mice, Nude; Pleural Effusion, Malignant; Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Recombinant Proteins; Ribonuclease, Pancreatic; Sarcoma; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A noncytotoxic neutralizing monoclonal antibody (mAb), 26-2F, to human angiogenin (Ang), a potent inducer of neovascularization, has been reported to prevent or delay the establishment of HT-29 human tumor xenografts in athymic mice. In the present study the tumor model was modified to increase sensitivity to Ang antagonists to facilitate further investigations and comparisons of their capacity to inhibit tumor growth. An increase in the percentage of tumor-free mice from 10-25% to 65% is observed in this modified model after treatment with mAb 26-2F. An additional neutralizing mAb, 36u, that interacts with a different epitope on Ang similarly prevents the appearance of tumors, both alone and in combination with mAb 26-2F. In those tumors that develop in mice treated with these agents, the number of vascular elements is reduced. Actin, an Ang antagonist that unlike the mAbs binds both human and mouse Ang, also prevents the establishment of tumors while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse Ang. Ang antagonists also inhibit the appearance of tumors derived from two other Ang-secreting human tumor cell lines--i.e., A549 lung adenocarcinoma and HT-1080 fibrosarcoma. These results demonstrate that inhibition of the action of Ang is an effective therapeutic approach for the treatment of malignant disease.

Topics: Actins; Angiogenesis Inducing Agents; Animals; Antibodies, Monoclonal; Carcinoma; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Interactions; Fibrosarcoma; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasms, Experimental; Proteins; Ribonuclease, Pancreatic; Survival Analysis