Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

Introduction  

The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.     

Regulatory effects of PTEN gene transfection on vascular endothelial growth factor (VEGF) in leukemia cells北大核心

Objective: To investigate the effects of wild type tumor-suppressing gene PTEN (phosphatase and tensin hemology deleted on chromosome ten gene) on vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1) in leukemia cells and elucidate the mechanism. Methods: The recombinant adenovirus containing wild type PTEN and green fluorescent protein (GFP) (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transfected into K562 leukemia cells. PTEN, VEGF, and VEGFR1 mRNA expression levels were detected using quantitative PCR and the protein expression levels were detected using Western blotting. Transwell invasion test was used to examine the invasion ability of the leukemia cells. Human umbilical vein endothelial cells ECV304 were also transfected with or without PTEN gene. Then the proliferation and apoptosis were measured using MTT assay and flow cytometry (FCM), respectively. Chick chorioallantoic membrane (CAM) test was used to determine the effect of PTEN gene transfection on angiogenesis. Results: The expressions of VEGF and VEGFR1 were significantly inhibited after transfection of Ad-PTEN-GFP compared with the control group transfected with empty Ad-GFP. The inhibitory effects were in a dose-dependent manner. The invasion capability of K562 cells was markedly weakened after transfection of Ad-PTEN-GFP. PTEN gene transfection inhibited the proliferation and induced apoptosis of vascular endothelial ECV304 cells. The cells were arrested at S phase. CAM test showed that PTEN gene inhibited the angiogenesis in vivo. Conclusion: Tumor suppressor gene PTEN inhibited the growth of endothelial cells and the invasion of leukemia cells. The action mechanism may be related with down-regulation VEGF expression and angiogenesis of leukemia cells.     

Effect of vascular endothelial growth factor on remodeling of C6 glioma tissue in vivo

Pathological characteristics and biological behaviors of tumor are considered to result from pathological tissue remodeling regulated by the interaction of factors in the tumor microecosystem (TMES). Vascular endothelial growth factor (VEGF) is one of the factors that probably play an important role in the process of tissue remodeling. This study was a comprehensive investigation of the effects of VEGF on remodeling of glioma tissue in vivo. C6 cells with expression vectors containing sense (C6/VEGF+) or antisense (C6/VEGF−) VEGF₁₆₄ complementary DNA (cDNA) or an empty vector (C6/vec) were implanted into athymic mice, which served as an in vivo model with different levels of VEGF expression. VEGF expression, water content, and morphological characteristics of these tumor tissues were assayed. Expression of VEGF and water content in C6/VEGF− glioma (C6/VEGF−G) tissues were lower than in C6, C6/VEGF+, and C6/vec glioma (C6G, C6/VEGF+G, C6/vecG) (P < 0.01, P < 0.05); water content correlated with VEGF expression (r = 0.791, P = 0.000). In C6/VEGF−G, tumor cells were not tightly adhered to vascular walls, and the basal lamina surrounded by collagen fibers was monolayer and not continuous. However, in C6G, C6/VEGF+G, and C6/vecG, tumor cells were very close to the vascular walls, with some extending their processes to the wall. Generally, loose basal laminae surrounded by small amounts of collagen fibers were multilayer, integrated, and continuous. Vesicular vacuolar organelle (VVO) structures were observed in plasma of vascular endothelial cells (VECs), and significant correlation was found between VEGF expression level and VVO density (Spearman’s r = 0.642, P = 0.007). No correlation was found between VEGF expression and fenestrae formation in VECs (Pearson’s correlation r = −0.053, P = 0.846), and fenestrae in neither VECs nor intercellular clefts correlated with water content of tumor tissue (Pearson’s correlation r = 0.018, P = 0.947). These results demonstrate that VEGF can aggravate edema in tumor tissues by increasing VVOs and plays critical roles in the stickiness of tumor cells to vessel wall and in the integrity and continuity of the basal lamina of vessels. Our data indicate a possible mechanism of remodeling of glioma tissue and suggest that blocking VEGF might contribute to a therapeutic strategy for glioma.     

Lewis Y regulates signaling molecules of the transforming growth factor β pathway in ovarian carcinoma-derived RMG-I cells

LeY (Lewis Y) is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of LeY is frequently observed in epithelial-derived cancers and is correlated to pathological staging and prognosis. To study the role of LeY on cancer cells, a stably LeY-overexpressing cell line, RMG-I-H, was developed previously by transfection of the α1,2-fucosyltransferase gene, a key enzyme that catalyzes the synthesis of LeY, into ovarian carcinoma-derived RMG-I cells. Our studies have shown that LeY is involved in the changes in biological behavior of RMG-I-H cells. However, the mechanism is still largely unknown. In this study, we determined the structural relationship and co-localization between LeY and TβRI/TβRII, respectively, and the potential cellular signaling mechanism was also investigated. We found that both TβRI and TβRII contain the LeY structure, and the level of LeY in TβRI and TβRII in RMG-I-H cells was significantly increased. Overexpression of LeY up-regulates the phosphorylation of ERK, Akt and down-regulates the phosphorylation of Smad2/3. In addition, the phosphorylation intensity was attenuated significantly by LeY monoantibody. These findings suggest that LeY is involved in the changes in biological behavior through TGF?β receptors via Smad, ERK/MAPK and PI3K/Akt signaling pathways. We suggest that LeY may be an important composition of growth factor receptors and could be an attractive candidate for cancer diagnosis and treatment.     

Current concepts in the treatment of Ewing’s sarcoma

Current concepts in the treatment of patients with Ewing’s sarcoma are presented focusing on the role of chemotherapy, radiation therapy and surgical resection. Particular attention is given to current methods of limb salvage. Problem areas, including the pelvis, proximal femur and spine, are discussed.     

Effect of α-difluoromethyl-ornithine on the expression and function of the epidermal growth factor receptor in human breast epithelial cells in culture

We have previously shown that ornithine decarboxylase (ODC) overexpression enhances the transforming effects of HER-2neu and epidermal growth factor (EGF) in normal MCF-10A human breast epithelial cells. Our data suggest that such potentiation may be mediated by activation of the mitogen-activated protein kinase (MAPK) pathway and, possibly, STAT signalling. To further explore the interaction between the polyamine pathway and EGF/HER-2neu signalling in this system, we inhibited endogenous ODC activity with -difluoromethylornithine (DFMO) and assessed the effects of this blockade on the expression of EGF receptors (EGFR) and HER-2neu as well as activation of downstream EGF target genes. We found that DFMO administration to MCF-10A cells increased EGF-R mRNA and protein levels in a dose-response fashion, while HER-2neu expression was not affected. The effect of DFMO was mediated through polyamine depletion since it could be reversed by exogenous putrescine administration. Our results also indicated that the increase in EGFR induced by DFMO was not a non-specific consequence of inhibition of cell proliferation. The upregulated EGFRs were functional since they could be phosphorylated by EGF and they were able to promote phosphorylation of downstream signalling molecules including ERK, STAT-3, and STAT-5. We propose that physiologic levels of ODC activity may be critical for regulation of a yet undefined signalling pathway, whose blockade by DFMO leads to a compensatory increase in functional EGFR.     

Clinical usefulness of serum and plasma vascular endothelial growth factor in cancer patients: which is the optimal specimen?

Vascular endothelial growth factor (VEGF) is secreted by various human cancer cells and plays a key role in cancer angiogenesis and metastasis. Recently, evidence of VEGF storage in blood cells including platelets has been reported. The serum VEGF levels were reported to increase during clotting as a result of its release from platelets, and plasma sample instead of serum was recommended for measuring the circulating VEGF more accurately. However, platelets have been implicated in tumor metastasis since circulating tumor cells forming aggregates with platelets were observed. The purpose of this study was to clarify which is an optimal specimen to measure VEGF in cancer patients, serum or plasma. We measured serum and plasma VEGF levels and platelet counts in 173 cancer patients and 42 healthy people, and found that serum VEGF levels were significantly higher than matched plasma VEGF and the VEGF difference (serum VEGF - plasma VEGF) correlated with platelet counts (r=0.624, p<0.05) in both cancer patients and healthy controls. We selected cancer patients with normal platelet counts (130-400x103/microl, Plt-normal cancer group). Interestingly, serum VEGF levels were higher in Plt-normal cancer group than in healthy controls. The theoretical platelet-derived VEGF in serum, calculated based on actual blood platelet counts (pg per 106 platelets), was also significantly higher in Plt-normal cancer group than in normal controls. It is, therefore, suggested that, although the serum VEGF levels are affected by blood platelets, platelet-derived VEGF also reflect biology of cancer cells, and that serum would be the more useful specimen for measurement of circulating VEGF in cancer patients for prognosis.     

Clinical significance of vascular endothelial growth factor‐C (VEGF‐C) in breast cancer

Vascular endothelial growth factorC (VEGFC) is a specific ligand which induces lymphangiogenesis. We examined the expression of VEGFC protein to determine its role in the progression of breast cancer. Immunohistochemical analysis revealed that VEGFC was overexpressed in 39 of 98 breast cancer specimens (39.8%) but not in adjacent normal mammary glands. The expression of VEGFC showed a significant correlation with lymphatic vessel invasion (p=0.0004). It is noteworthy that the 5year disease free survival rate of the VEGFC positive group was significantly poorer than that of negative group (p=0.0356). We suggest that as expression of VEGFC is not implicated in lymphatic spread, it may prove to be a promising marker to predict the recurrence of breast cancer.     

Are basic fibroblast growth factor and vascular endothelial growth factor prognostic indicators in pediatric patients with malignant solid tumors?

Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors. Among angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) appear to be useful markers in adults with cancer. The aim of this pilot study was to determine the levels of VEGF in serum and bFGF in serum and urine of children with solid tumor at diagnosis (as measured by ELISA), and to investigate whether these parameters provide prognostic information. Forty consecutive patients with different types of cancer were prospectively included in this study. Median values of all studied angiogenic factors were higher in patients than in controls (n = 40), and the differences were statistically significant for bFGF in serum and urine: 10 versus 3 pg/ml (P = 0.0004) and 6406 versus 0 pg/g of creatinine (P < 0.0001), respectively. Among patients, median serum values of bFGF and VEGF were higher in children with metastatic disease (n = 14) than in those with localized disease (n = 26). The difference was statistically significant for serum bFGF: 17.5 versus 6 pg/ml (P = 0.02). Serum angiogenic factor levels correlated with outcome. The estimated event-free survival at 3 years was 79% for patients with normal bFGF values (n = 13) versus 42% (n = 26; P = 0.02) for those with high levels, and 71% in case of normal VEGF values (n = 20) versus 38% (n = 19; P = 0.04) for those with high levels. No benefit of normal urinary bFGF values was observed. Our results provide a rationale for exploring the clinical interest of bFGF and VEGF measurements in body fluids of a larger group of children with cancer.     

Interleukin 8 and vascular endothelial growth factor -- prognostic factors in human gastric carcinomas?

Gastric carcinoma cells express potent angiogenic factors including vascular endothelial growth factor (VEGF). We previously reported that interleukin-8 (IL-8) acts as an angiogenic factor for human gastric carcinomas. More recently, we found that IL-8 upregulates matrix metalloproteinase-9 (MMP-9) expression and increases invasive activity of gastric carcinoma cells. The purpose of this study was to determine whether the expression of IL-8 and VEGF correlates with clinicopathological parameters in human gastric carcinomas. IL-8 and VEGF expression levels were measured by an enzyme-linked immunosorbent assay (ELISA) in 56 gastric carcinomas and the surrounding normal mucosa. Macroscopic and histopathological tumour findings, presence of metastasis and prognosis were obtained from the patient records and endoscopic, surgical and pathological reports. IL-8 protein levels were higher in most neoplasms than in the corresponding normal mucosal tissue. In contrast, VEGF expression in the tumours was similar to that in normal mucosa. The IL-8 level in the neoplasms correlated significantly with the depth of invasion, venous invasion and lymphatic invasion. VEGF expression in the tumours correlated well with the depth of invasion and lymph node metastasis. No correlation between IL-8 and VEGF expression in the tumours was observed. The survival rates of patients with tumours displaying high IL-8 and VEGF expression levels were significantly lower (P<0.05) than those of patients with tumours displaying low IL-8 and VEGF expression. The results suggest that IL-8 and VEGF may be independent and important prognostic factors in human gastric carcinomas.     

Inhibition of hypoxia inducible factor-1α (HIF-1α) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas

SummaryIntroduction Hypoxia inducible factor-1α (HIF-1α) regulates vascular endothelial growth factor (VEGF), the presumed principal mediator of angiogenesis in malignant gliomas, under normal physiologic conditions. We examined the effect of HIF-1α on VEGF secretion, tumor growth, and angiogenesis in malignant gliomas.Methods We examined 175 human gliomas for expression of HIF-1α and its downstream-regulated proteins. HIF-1α expression and VEGF secretion in glioma cell lines under normoxia and hypoxia were examined using␣ELISA and Western blot. Malignant glioma cell lines were transfected with dominant-negative HIF-1α (DN-HIF-1α) expression vector or siRNA constructs against the HIF-1α gene. Growth studies were conducted on cells with the highest VEGF/HIF-1α inhibition isolated from stable transfected cell lines. MIB-1-labeling index and microvascular density (MVD) measurements were performed on the in vivo tumors.Results HIF-1 expression correlates with malignant glioma phenotype and was not confined to perinecrotic, pseudopalisading cells. VEGF and HIF-1 expression was high in glioma cell lines even under normoxia, and increased after exposure to hypoxia or growth factor stimulation. Cells transfected with DN-HIF-1α or HIF-1α siRNA demonstrated decreased HIF-1α and VEGF secretion. In vivo but not in vitro growth decreased in response to VEGF and HIF-1 inhibition. HIF-1 siRNA studies showed decreased VEGF secretion and in vitro and in vivo growth of glioma cell lines. MVD was unchanged but MIB-1 proliferation index decreased for both types of HIF-1 inhibition.Conclusions VEGF and HIF-1α are elevated in malignant gliomas. HIF-1α inhibition results in VEGF secretion inhibition. HIF-1α expression affects glioma tumor growth, suggesting clinical applications for malignant glioma treatment.     

Intraperitoneal vascular endothelial growth factor burden in peritoneal surface malignancies treated with curative intent: The first step before intraperitoneal anti-vascular endothelial growth factor treatment?

IntroductionVascular endothelial growth factor (VEGF) is one of the most important angiogenic factors in solid tumours and plays an important role in ascites development in peritoneal surface malignancies (PSM). The main goal of this study was to determine the evolution and factors influencing intraperitoneal (IP) VEGF burden during cytoreductive surgery (CRS) with curative intent.Patients and methodsNinety-seven consecutive patients with PSM were treated with CRS at a single centre with curative intent. Patient data were collected prospectively between February 2012 and October 2012. An enzyme-linked immunosorbent assay technique was used to assess VEGF levels in intravenous (IV) systemic blood samples before incision and after abdominal closure, and in IP samples during abdominal cavity exploration, after completion of CRS, after hyperthermic IP chemotherapy, and at 1 and 24 h after abdominal closure.ResultsThe IP VEGF burden increased significantly after CRS, and then decreased progressively (p < 0.005). In multivariate analysis, neoadjuvant IV bevacizumab significantly decreased the preoperative IP VEGF burden, tumour load according to Peritoneal Cancer Index value increased significantly the preoperative IP VEGF burden and a low preoperative IP VEGF burden was associated with significantly increased postoperative complications. Neoadjuvant IV bevacizumab is the only factor that influences the preoperative IV VEGF concentration.ConclusionFor patients with PSM who were treated with curative intent, the IP VEGF burden increased after CRS, and was reduced prior to surgery by the administration of neoadjuvant IV bevacizumab.