angiogenin and Adenocarcinoma

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Cell Line, Tumor; Chick Embryo; Chorioallantoic Membrane; Collagen; Colorectal Neoplasms; Down-Regulation; Drug Combinations; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HEK293 Cells; HSP90 Heat-Shock Proteins; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Laminin; Mice, Nude; Platelet-Derived Growth Factor; Proteoglycans; Rectal Neoplasms; Ribonuclease, Pancreatic; RNA, Messenger; Specific Pathogen-Free Organisms; STAT3 Transcription Factor; Transforming Growth Factor beta1; Triazoles; Vascular Endothelial Growth Factor A; Vesicular Transport Proteins; Xenograft Model Antitumor Assays

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

Angiogenin (ANG), an efficient angiogenesis factor, is up-regulated in human colorectal carcinoma, pancreatic carcinoma, and other tumors. But the function of ANG in tumor angiogenesis is still unknown. This study was to explore the effect of ANG on angiogenesis of gastric carcinoma.. The expressions of ANG, CD34, and Ki-67 in 68 specimens of gastric carcinoma were detected by immunohistochemistry. Microvessel density (MVD) was figured out according to the expression of CD34. The proliferation index of carcinoma cells in gastric carcinoma tissue was figured out according to the expression of Ki-67.. Of the 68 specimens of gastric carcinoma, 19 showed strongly positive expression of ANG (group A), 3 showed negative expression of ANG (group B). MVD of group A was significantly higher than that of group B (308.4+/-25.6 vs. 196.0+/-31.3, P<0.01). Proliferation index of group A was significantly higher than that of group B (579.6+/-31.4 vs. 341.3+/-84.0, P<0.01).. Protein level of ANG positively correlates with MVD of gastric carcinoma tissue, and proliferation index of carcinoma cells.

Topics: Adenocarcinoma; Antigens, CD34; Cell Proliferation; Female; Humans; Ki-67 Antigen; Lymphatic Metastasis; Male; Microcirculation; Middle Aged; Neoplasm Staging; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Stomach Neoplasms

Angiogenin is a polypeptide involved in the formation and establishment of new blood vessels necessary for growth and metastasis of numerous malignant neoplasms, including prostatic adenocarcinoma. Antiangiogenin therapy inhibits the establishment, growth, and metastasis of prostatic adenocarcinoma in animal studies. In this study, we have investigated the expression of angiogenin in prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia, and adjacent benign prostatic epithelium in a large cohort of prostatectomy specimens.. We have studied the expression of angiogenin by immunohistochemistry in prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia, and adjacent benign prostatic tissue in 107 human total prostatectomy specimens.. The percentage of cells staining positively for angiogenin in benign prostatic glandular epithelium (mean = 17%) was significantly less than for high-grade prostatic intraepithelial neoplasia (mean = 58%, P < 0.001) and prostatic adenocarcinoma (mean = 60%, P < 0.001). Compared with adjacent benign prostatic epithelium, the staining intensity was significantly greater in high-grade prostatic intraepithelial neoplasia (P < 0.001) and prostatic adenocarcinoma (P < 0.001). Furthermore, staining intensity has significantly stronger in prostatic adenocarcinoma versus high-grade prostatic intraepithelial neoplasia (P = 0.0023). However, there was no correlation of angiogenin expression with various clinical and pathologic variables examined, including age at surgery, Gleason scores, pathologic stage, tumor extent, angiolymphatic invasion, extraprostatic extension, seminal vesical invasion, lymph node metastasis, surgical margin status, presence of prostatic intraepithelial neoplasia, and perineural invasion.. Angiogenin expression in prostatic tissue increases as prostatic epithelial cells evolve from a benign to an invasive phenotype. The increasing expression of prostatic adenocarcinoma in the progression from benign prostate to high-grade prostatic intraepithelial neoplasia and ultimately to prostatic adenocarcinoma are consistent with previous studies showing the influential role that angiogenin plays in the growth, invasion, and metastasis of prostatic adenocarcinoma and many other malignant tumors.

Topics: Adenocarcinoma; Adult; Aged; Enzyme-Linked Immunosorbent Assay; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Prostatectomy; Prostatic Hyperplasia; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Ribonuclease, Pancreatic; Treatment Outcome

We have previously demonstrated that the increased expression of angiogenin (ANG) in pancreatic cancer is related to cancer aggressiveness; however, the relationship between ANG expression and its clinical relevance in colorectal cancer has not been demonstrated. We therefore investigated the correlation between serum ANG (sANG) concentration and colorectal cancer progression or the changes in sANG concentrations before and after cancer resection. To determination sANG concentration by ELISA, sera were obtained from colorectal cancer patients (the cancer group) preoperatively (n = 34) and postoperatively (n = 25), from hernia patients (the nonneoplastic group) preoperatively (n = 9) and postoperatively (n = 4), and from 23 healthy volunteers. The amount of ANG in the colorectal cancer tissues (n = 19) was determined by the same method. Before surgery, the mean sANG concentration in the cancer group (411.8 +/- 106.3 ng/ml) was significantly higher than that in both the nonneoplastic group (344.0 +/- 60.7 ng/ml; P = 0.04) and in the healthy volunteers (321.7 +/- 59.7 ng/ml; P = 0.0001). The degree of elevation of sANG concentration in the cancer group was more significant in the more progressed subgroups as compared with that in the normal group (versus T(is) + T1 + T2 cancer, P = 0.01; versus T3 + T4 cancer, P = 0.002; versus stage 0 + I cancer, P = 0.02; versus >stage III cancer, P = 0.001; versus Dukes' A cancer, P = 0.02; versus Dukes' C cancer, P = 0.006). After cancer resection, the mean sANG concentrations in each subgroup decreased to the same levels as those of the normal group; the degrees of reduction were more significant in the more progressed subgroups. The tissue ANG amount correlated significantly with sANG concentration (P = 0.007). These results suggest that the increased concentration of sANG that is derived from colorectal cancer correlates with cancer progression.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Hernia, Inguinal; Humans; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Neovascularization, Pathologic; Postoperative Period; Proteins; Ribonuclease, Pancreatic

Human angiogenin is an excellent substrate for the adhesion of HT-29 human colon adenocarcinoma cells. These cells adhere more quickly to human angiogenin than to fibronectin, laminin, collagen I, and collagen IV. Anti-angiogenin antibodies and the angiogenesis inhibitors platelet factor-4 and placental ribonuclease inhibitor prevent adhesion of HT-29 cells to angiogenin. Calcium and magnesium ions are not required for adhesion and Arg-Gly-Asp-Ser has no effect, indicating that the interaction is integrin-independent. Instead, adhesion seems to involve a heparan/chondroitin sulfate proteoglycan. Treatment of the cells with heparinase or heparitinase decreases HT-29 cell adhesion onto angiogenin but not onto collagen I. Moreover, cell adhesion is decreased by the presence of heparin or chondroitin sulfates and by preincubation of the cells with inhibitors of proteoglycan synthesis or secretion. In addition, angiogenin binds tightly to heparin-Sepharose, requiring 0.78 M NaCl for elution. Angiogenin-affinity chromatography of a 35S-, 3H-labeled HT-29 cell fraction enriched in cell-surface proteoglycans yields a single, heparinase-sensitive component of apparent molecular mass > 200 kDa, as detected by autoradiography after SDS-polyacrylamide gel electrophoresis. These results suggest that angiogenin could be an effective substrate for tumor cell adhesion during metastasis and may provide a basis for the design of inhibitors of this process.

Topics: Adenocarcinoma; Calcium; Cell Adhesion; Cell Adhesion Molecules; Colonic Neoplasms; Glycosaminoglycans; Humans; In Vitro Techniques; Magnesium; Neovascularization, Pathologic; Oligopeptides; Proteins; Proteoglycans; Ribonuclease, Pancreatic; Tumor Cells, Cultured

Using digoxigenin-labelled, synthetic oligonucleotide probe cocktails of angiogenin and bFGF genes, the expression of the two genes was observed by in situ hybridization in ten colonic adenocarcinomas, seven gastric adenocarcinomas, and four hepatocellular carcinomas. The angiogenin gene was expressed in eight of the ten cases of colonic adenocarcinoma and in all of the four cases where dysplastic glands were found. Angiogenin expression was evident in four of the seven cases of gastric adenocarcinoma. bFGF expression was detected in only five of the seven gastric carcinoma cases. The mRNAs for angiogenin and bFGF were mainly cytoplasmic in distribution and were only occasionally seen in the nuclei of the positive cells. Neither the angiogenin gene mRNA nor the bFGF mRNA was expressed in the four cases of hepatocellular carcinoma. It is postulated that the angiogenin gene may play an important role in angiogenesis in colonic adenocarcinomas; in gastric cancers, both angiogenin and bFGF were involved in this process. For hepatocellular carcinomas, neither angiogenin nor bFGF production appeared to be related to angiogenesis.

Topics: Adenocarcinoma; Angiogenesis Inducing Agents; Carcinoma, Hepatocellular; Colonic Neoplasms; Fibroblast Growth Factor 2; Gastrointestinal Neoplasms; Humans; In Situ Hybridization; Liver Neoplasms; Neoplasm Proteins; Neovascularization, Pathologic; Proteins; Ribonuclease, Pancreatic; RNA, Messenger; Stomach Neoplasms

Human angiogenin, a potent inducer of neovascularization, is secreted by HT-29 colon adenocarcinoma cells. microgram doses of a monoclonal antibody that neutralizes the in vitro and in vivo activities of angiogenin prevent or delay the appearance of s.c. HT-29 tumors in athymic mice in a statistically significant, dose-dependent manner. The antibody is not cytotoxic to tumor cells in vitro, which indicates that inhibition of tumor growth most likely occurs by neutralization of the activity of angiogenin in vivo and further implies a critical role for angiogenin in the early development of HT-29 tumors. The results suggest a therapeutically useful approach to the treatment of angiogenin-dependent malignancy.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Immunoglobulin G; Male; Mice; Mice, Nude; Proteins; Ribonuclease, Pancreatic; Specific Pathogen-Free Organisms; Tumor Cells, Cultured

The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology. With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine. However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate. Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme. Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa. The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.

Topics: Adenocarcinoma; Base Sequence; Cell Line; Colonic Neoplasms; Escherichia coli; Humans; Molecular Sequence Data; Neoplasm Proteins; Nucleic Acid Conformation; Polyribonucleotides; Ribonuclease, Pancreatic; RNA, Ribosomal; RNA, Ribosomal, 5S; Saccharomyces cerevisiae; Substrate Specificity

Topics: Adenocarcinoma; Angiogenesis Inducing Agents; Animals; Cell Line; Cricetinae; Female; Growth Substances; Humans; Mesocricetus; Neoplasm Proteins; Neovascularization, Pathologic; Ovarian Neoplasms; Ribonuclease, Pancreatic

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Amino Acid Sequence; Angiogenesis Inducing Agents; Animals; Cell Line; Colonic Neoplasms; Cyanogen Bromide; Growth Substances; Horses; Humans; Hydroxylamine; Hydroxylamines; Indicators and Reagents; Neoplasm Proteins; Peptide Fragments; Pyroglutamyl-Peptidase I; Ribonuclease, Pancreatic; Species Specificity; Trypsin

The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

Topics: Adenocarcinoma; Allantois; Amino Acid Sequence; Amino Acids; Angiogenesis Inducing Agents; Animals; Biological Assay; Cell Line; Chick Embryo; Chorion; Colonic Neoplasms; Culture Media; Growth Substances; Humans; Molecular Weight; Neoplasm Proteins; Ribonuclease, Pancreatic