Angiogenesis, formation of new microvessels providing oxygen and nutrient supply, is essential for tumor growth. It is dependent on the production of angiogenic growth factors by tumor cells. Angiopoietin 1 (Ang-1) and 2 (Ang-2) and their common receptor, Tie2, are thought to be critical regulators of tumor angiogenesis. We examined expression of Ang-1, Ang-2, and their common receptor Tie2 mRNAs and proteins in gastric cancers using in situ hybridization and immunohistochemistry. We also investigated the relationship between their expression and differentiation of cancer cells, lymph node metastasis, tumor size, depth of cancer cell invasion, TNM staging and microvessel density (MVD). The expression of Ang-1, Ang-2, and Tie2 mRNA in cancer cells significantly correlated with the MVD (p<0.001, <0.001 and =0.019, respectively). Ang-1 and Tie2 positivity correlated with advanced gastric cancers (p<0.05) and larger cancers had higher positive rates of Ang-1, Ang-2, and Tie2 mRNA expression (p<0.001, =0.010 and =0.039, respectively). Significant positive correlations were also found between mRNA expression of Tie2 and those of Ang-1 and Ang-2 (p<0.01 and <0.001, respectively). These findings indicate that the expression of Ang-1 and Ang-2 is important for tumor angiogenesis, and suggest a possible role of autocrine/paracrine function of angiopoietin/Tie2 system in gastric cancer progression.
Stomach Neoplasms/blood supply/*genetics/*metabolism/pathology ; Receptor, TIE-2/*genetics/*metabolism ; RNA, Neoplasm/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Neovascularization, Pathologic ; Middle Aged ; Male ; In Situ Hybridization ; Immunohistochemistry ; Humans ; Gene Expression ; Female ; Carcinoma, Signet Ring Cell/blood supply/genetics/metabolism/pathology ; Angiopoietin-2/*genetics/*metabolism ; Angiopoietin-1/*genetics/*metabolism ; Aged ; Adult ; Adenocarcinoma/blood supply/genetics/metabolism/pathology

OBJECTIVETo investigate the transcription level and protein expression of HIF-1alpha and VEGF in SW480 cell line and colorectal adenocarcinoma, and to determine whether HIF-1alpha plays a role in angiogenesis through its regulation of VEGF.

METHODSHIF-1alpha mRNA expression was analyzed by in situ hybridization. HIF-1alpha and VEGF protein expressions were determined by immunochemical streptavidin/peroxidase (SP) in SW480 cells and colorectal carcinoma tissue samples and Western blot, using proteins extracted from SW480 cells. Tumor tissue microvessel density (MVD) was determined by CD34 immunostaining of colorectal carcinomas.

RESULTSThe levels of HIF-1alpha mRNA changed significantly in response to different oxygen concentrations and an addition of genistein in SW480 cells. Immunocytochemistry revealed that the levels of HIF-1alpha, VEGF protein expression in SW480 cells were significantly higher under hypoxia than those in nomoxia (P < 0.01, P < 0.05 respectively). However, addition of genistein, an inhibitor of HIF-1alpha, suppressed such responses to hypoxia. Western blot analysis showed that SW480 cells exposed to hypoxia expressed a high level of HIF-1alpha protein, compared to a weak expression in nomoxia. The addition of genistein in hypoxia suppressed the over-expression of HIF-1alpha. The positive rates of HIF-1alpha mRNA by in situ hybridization in colorectal adenomas and adenocarcinomas were 38.9% (7/18) and 67.7% (42/62), respectively. The percentage of HIF-1alpha mRNA positive cells varied significantly from colorectal adenomas to adenocarcinomas at different Duke stages (P < 0.05), and HIF-1alpha mRNA was higher in adenocarcinomas than in adenomas (P < 0.01). The positive rates of HIF-1alpha and VEGF protein expression in adenocarcinomas were 43.5% (27/62) and 37.1% (23/62), respectively. The expression of VEGF elevated as the Duke tumor staging increased. The conformation rate of HIF-1alpha and VEGF was 74.2% (46/62). MVD was significantly higher in HIF-1alpha and/or VEGF positive tumors than those without (P < 0.01 and P < 0.05 respectively). Among the four groups, i.e. HIF-1alpha+/VEGF+, HIF-1alpha+/VEGF-, HIF-1alpha+/VEGF- and HIF-1alpha-/VEGF-, the difference of MVD was highly significant (P < 0.01). HIF-1alpha expression was correlated significantly with VEGF expression and microvessel density.

CONCLUSIONSThese findings suggest hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinoma. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinoma.

Adenocarcinoma ; blood supply ; metabolism ; pathology ; Colorectal Neoplasms ; blood supply ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Microcirculation ; pathology ; Neovascularization, Pathologic ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of angiopoietin (Ang)-1 and Ang-2 in colorectal tumors and its relations to microvessel density (MVD) in tumor tissue.

METHODSAng-1, Ang-2 and factor VIII-related antigen were stained immunohistochemically in 91 cases of primary colorectal adenocarcinoma, 20 cases of colorectal adenoma and 24 cases of normal colorectal mucosal tissue, and MVD was also assayed in above tissue specimens.

RESULT(1) A significantly higher Ang-1 (7.07+/-2.00) was observed in normal tissue compared with 1.75 +/-1.98 in the adenoma and 1.40 +/- 1.22 in the adenocarcinoma (P<0.01). (2) Ang-2 protein positive rate in adenocarcinoma was significantly higher than that in normal tissue and adenoma (P<0.01). The expression of Ang-2 in adenocarcinoma was closely associated with poor differentiation and vessel invasion. (3) There were significant correlations between Ang-1 and Ang-2 (r=-0.338, P<0.01), Ang-1 and MVD (r=-0.388, P<0.01), Ang-2 and MVD (r=0.594, P<0.01) in the 135 cases.

CONCLUSIONThe overexpression of Ang-2 may play an important role in angiogenesis of colorectal adenocarcinoma. It can be regarded as an index for malignancy and prognosis in colorectal adenocarcinoma.

Adenocarcinoma ; blood supply ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Angiopoietin-1 ; biosynthesis ; genetics ; Angiopoietin-2 ; biosynthesis ; genetics ; Biomarkers, Tumor ; Capillaries ; pathology ; Colorectal Neoplasms ; blood supply ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; von Willebrand Factor ; biosynthesis ; genetics

OBJECTIVE: To investigate the relationship between the expression of vascular endothelial growth factor C (VEGF-C) and angiogenesis and lymphangiogenesis in papillary thyroid carcinoma (PTC). METHODS: Seventy-two PTC cases were divided into 3 groups according to the level of invasion: papillary microcarcinoma group (PMC group), intrathyroid carcinoma group (IPC group), and extrathyroid carcinoma group (EPC group). They were again divided into 2 groups according to lymph node metastasis: lymph node metastasis group and lymph node no-metastasis group. The expressions of VEGF-C, CD105 and vascular endothelial growth factor receptor-3 (VEGFR-3) were detected by SP method of immunohistochemical staining. The expression of VEGF-C was analyzed quantitatively by image analysis system, and the PI of VEGF-C (VEGF-C-PI), the number of MVD (microvessel density), and LVD (lymphaticvessel density) were obtained. RESULTS: The VEGF-C-PI of lymph node metastasis group (23.15 +/- 3.75) was higher than that of lymph node non-metastasis group (14.54 +/- 2.93) (P <0.01). MVD was 35.25 +/- 2.06 in the PMC group, 41.75 +/- 5.46 in the IPC group, and 52.58 +/- 4.16 in the EPC group, which showed the elevatory tendency with the increase of invasion (P < 00.5). LVD was 6.00 +/- 0.81 in the PMC group, 13.80 +/- 1.81 in the IPC group, and 19.17 +/- 2.96 in the EPC group, which again showed the elevatory tendency with the increase of invasion (P <0.05). The LVD of lymph node metastasis group (19.56 +/- 2.45) was significantly higher than that of lymph node non-metastasis group (12.48 +/- 2.84) (P < 0.05). VEGF-C was positively correlated with MVD and LVD (r = 0.743, 0.90, P <0.01). CONCLUSION The expressions of VEGF-C and LVD are related to lymph node metastasis of PTC. MVD and LVD are related to the invasion of PTC. VEGF-C may play an important role in the angiogenesis and lymphangiogenesis.
Adenocarcinoma, Papillary ; blood supply ; metabolism ; pathology ; Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Lymphangiogenesis ; Male ; Middle Aged ; Neovascularization, Pathologic ; metabolism ; Thyroid Neoplasms ; blood supply ; metabolism ; pathology ; Vascular Endothelial Growth Factor C ; biosynthesis ; genetics

OBJECTIVETo investigate whether correlation exists between mRNA expression of IGF-II and hepatocyte growth factor (HGF) and tumor progression and prognosis in gastric cancer.

METHODSIn situ hybridization technique was used to examine mRNA expression of IGF-II and HGF, and immunohistochemical technique was used to examine protein expression of CD34 in 105 specimens of gastric carcinoma.

RESULTSIn situ hybridization revealed that the positive rates of IGF-II mRNA and HGFmRNA were 49.5% and 57.1%, respectively. In stage T3-T4 cases, positive mRNA expression rates of IGF-II and HGF, the frequencies of vessel invasion, lymph node metastasis and distant metastasis were significantly higher than those in stage T1-T2 cases. The mean microvascular density (MVD) in stage T3-T4 tumors, vessel invasion, lymph node metastasis and distant metastasis were significantly more frequent than those in stage T1-T2 tumors. The mean MVD in tumors with positive IGF-II and HGF expressions was significantly higher than that in tumors without IGF-II and HGF expression. There were positive correlations between MVD and expression of IGF-II and HGF. The mean survival time and 5-year survival rate in cases with positive IGF-II and HGF expression and MVD value > or = 39.5 were significantly shorter those that in cases with negative IGF-II and HGF expression and MVD value < 39.5.

CONCLUSIONIGF-II and HGF promote angiogenesis in gastric cancer, and take part in tumor invasion and metastasis. They can be used as prognostic markers of gastric cancer in clinical practice.

Adenocarcinoma, Papillary ; blood supply ; metabolism ; secondary ; Adult ; Aged ; Carcinoma, Ductal ; blood supply ; metabolism ; secondary ; Carcinoma, Signet Ring Cell ; blood supply ; metabolism ; secondary ; Disease Progression ; Female ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Insulin-Like Growth Factor II ; biosynthesis ; genetics ; Liver Neoplasms ; blood supply ; metabolism ; secondary ; Lymphatic Metastasis ; Male ; Microcirculation ; Middle Aged ; Neoplasm Invasiveness ; Neovascularization, Pathologic ; Peritoneal Neoplasms ; blood supply ; metabolism ; secondary ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; blood supply ; metabolism ; pathology ; Survival Rate

Adenocarcinoma ; blood supply ; metabolism ; mortality ; secondary ; Adult ; Aged ; Female ; Humans ; Lymphatic Metastasis ; Male ; Microcirculation ; Middle Aged ; Neovascularization, Pathologic ; metabolism ; pathology ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Receptors, Urokinase Plasminogen Activator ; Stomach Neoplasms ; blood supply ; metabolism ; mortality ; pathology ; Survival Rate ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effects of silencing heparanase (HPA) on growth, angiogenesis and metastasis of human gastric carcinoma transplanted in nude mice.

METHODSHuman gastric carcinoma SGC-7901 cells and those cells with silenced HPA (gastric carcinoma SGC-7901-HPA(-)) were separately transplanted subcutaneously in 6 nude mice. The time, size and speed of tumor growth were recorded. RT-PCR and Western-blot were used to detect the expression of HPA mRNA and protein in the subcutaneous tumors of the two groups. Immunohistochemical staining was used to detect microvessel density (MVD) in the subcutaneous tumors of the two groups. Cells of the subcutaneous transplanted tumors of the two groups were separately injected into the peritoneal cavity of nude mice, 6 mice each. The growth of metastatic tumors in nude mice was observed.

RESULTSHuman gastric carcinoma SGC-7901 cells and SGC-7901-HPA(-) cells were subcutaneously inoculated in nude mice, and tumors appeared at 4 days and 7 days after inoculation, respectively. The MVD was (20.69 ± 1.20)/HP and (11.35 ± 1.94)/HP, respectively (P < 0.05). The expressions of HPA mRNA and protein of the subcutaneously transplanted SGC-7901-HPA(-) tumor were decreased. Four voluminous metastatic tumors caused by SGC-7901 cells occurred in 3 mice in the liver, right kidney, omentum and intestine. Two smaller abdominal metastatic tumors of SGC-7901-HPA(-) cells were found in the liver and right kidney.

CONCLUSIONSilencing HPA can inhibit the tumor growth, angiogenesis and metastasis of human gastric cancer in nude mice. It suggests that HPA might become a new target for prevention and treatment of gastric cancer.

Adenocarcinoma ; blood supply ; enzymology ; genetics ; pathology ; secondary ; Animals ; Cell Line, Tumor ; Gene Silencing ; Glucuronidase ; biosynthesis ; genetics ; physiology ; Humans ; Kidney Neoplasms ; secondary ; Liver Neoplasms ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; pathology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; blood supply ; enzymology ; genetics ; pathology

OBJECTIVETo investigate the effect of small interfering RNA (siRNA) targeting human vascular endothelial growth factor (hVEGF) on A549 cell growth in nude mice and angiogenesis on chorioallantoic membrane (CAM) assay.

METHODSThree pairs of hVEGF siRNA-plasmid and non-silencing-plasmid were constructed, and transfected into A549 cells through lipofectamine 2000, respectively. The most effective pair of hVEGF siRNA-plasmid was selected by ELISA and real-time RT-PCR. A549 cells transfected with selected hVEGF siRNA- plasmid, A549 cells transfected with non-silencing-plasmid and A549 cells without transfection were inoculated into nude mice, respectively. Chick embryos were randomly divided into four groups and CAM was treated by different solutions for 48 h: culture media DMEM as negative control group,un-transfected A549 cell culture supernatants as positive control group, hVEGF siRNA A549 cell culture supernatants as hVEGF siRNA group and nonsilencing siRNA A549 cell culture supernatants as non-silencing siRNA group. The CAMs were harvested on d12 for microscopic assays.

RESULTCompared with control group, hVEGF siRNA-plasmid induced 48% reduction in hVEGF secretion by A549 cells accompanied by 70% reduction in hVEGF mRNA. Compared with non-silencing siRNA group, the mean tumor volume of murine xenograft was reduced by 58% in hVEGF siRNA group; time for xenografts growing to 50 mm(3)was delayed by 5.4 d. hVEGF contents in xenograft were reduced by 54%; but mean doubling time of tumors and the growth rate of tumors were not significantly reduced. In CAM assays, hVEGF content was zero in negative group, and in hVEGF siRNA group that was 40%-44% of non-silencing siRNA group or positive group; vessels branch points of CAM in hVEGF siRNA group or non-silencing siRNA group or positive group were increased by 45%-55% compared with negative group; total vessel length of CAM in hVEGF siRNA group was increased by 53% compared with negative group, while in non-silencing siRNA group or positive group that was increased by 97% or 99%. Compared with negative control group, the proliferation of microvessels was increased when cell culture supernatant with hVEGF was added in hVEGF siRNA group, significant proliferated vessels were observed in non-silencing siRNA group or positive group.

CONCLUSIONA plasmid-mediated hVEGF siRNA has been constructed and verified, which can effectively downregulate the expression of hVEGF in human A549 cells, resulting in the inhibition of angiogenesis. hVEGF siRNA can delay initial growth of A549 tumor xenograft but not reduce the growth rate.

Adenocarcinoma ; pathology ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; metabolism ; Female ; Humans ; Lung Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Physiologic ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics

The aim of the present study was to investigate the involvements of insulin-like growth factor-1 (IGF-1) and estrogen receptor α (ERα) in the inhibitory effect of wogonin on the breast adenocarcinoma growth. Moreover, the effect of wogonin on the angiogenesis of chick chorioallantoic membrane (CAM) was also investigated. MCF-7 cells (human breast adenocarcinoma cell line) were subjected to several drugs, including IGF-1, wogonin and ER inhibitor ICI182780, alone or in combination. MTT assay was used to detect breast cancer proliferation. Western blot was used to analyze ERα and p-Akt expression levels. CAM models prepared from 6-day chicken eggs were employed to evaluate angiogenesis inhibition. The results showed wogonin and ICI182780 both exhibited a potent ability to blunt IGF-1-stimulated MCF-7 cell growth. Either of wogonin and ICI182780 significantly inhibited ERα and p-Akt expressions in IGF-1-treated cells. The inhibitory effect of wogonin showed no difference from that of ICI182780 on IGF-1-stimulated expressions of ERα and p-Akt. Meanwhile, wogonin at different concentrations showed significant inhibitory effect on CAM angiogenesis. These results suggest the inhibitory effect of wogonin on breast adenocarcinoma growth via inhibiting IGF-1-mediated PI3K-Akt pathway and regulating ERα expression. Furthermore, wogonin has a strong anti-angiogenic effect on CAM model.
Adenocarcinoma ; metabolism ; pathology ; Angiogenesis Inhibitors ; pharmacology ; Animals ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Flavanones ; pharmacology ; Humans ; Insulin-Like Growth Factor I ; antagonists & inhibitors ; pharmacology ; Scutellaria ; chemistry