Prostacyclin receptor in tumor endothelial cells promotes angiogenesis in an autocrine manner

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in re-endothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TEC-specific marker and might be a useful therapeutic target.     

Beta-elemene inhibits melanoma growth and metastasis via suppressing vascular endothelial growth factor-mediated angiogenesis

Purpose

It was to assess antiangiogenic effect of ??-elemene in?vitro and in?vivo, and it was involved in inhibiting melanoma growth and metastasis, as well as to elucidate its intrinsic mechanism.

Methods

Inhibitive effect of ??-elemene on B16F10 cells was performed by cell proliferation assay. Angiogenesis assays in vitro including rat aortic ring and chick embryo chorioallantoic membrane were used, as well as melanoma growth and metastasis assay in C57BL/6 mice. Vascular endothelial growth factor (VEGF) expression in?vitro and in?vivo was measured respectively by western blot analysis and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry analysis of CD34 and VEGF expression in primary melanoma was also presented.

Results

??-Elemene inhibited B16BF10 cell proliferation starting from 200???M, but VEGF from 20???M. Both 20 and 50???M ??-elemene in?vitro inhibited VEGF-induced sprouting vessel of rat aortic ring and microvessel formation of chick embryo chorioallantoic membrane. In?vivo, tumor size of primary melanoma in mice intraperitoneally treated with ??-elemene was significantly smaller than that of the control; CD34 expression of primary melanoma was also suppressed; and the metastatic melanoma colonies and content of melanin in lung were detected obviously decreased in mice of ??-elemene-treated groups. Furthermore, results of VEGF expressing in primary melanoma, serum and lung of mice also disclosed that VEGF was inhibited in?vivo.

Conclusions

??-Elemene inhibited melanoma growth and metastasis through suppressing VEGF-mediated angiogenesis. It is a natural potential antiangiogenic agent.     

Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor

Reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. However, the direct roles of endogenous ROS production still remain to be elucidated. In this study, we found that high levels of ROS were spontaneously produced by ovarian and prostate cancer cells. This elevated ROS production was inhibited by NADPH oxidase inhibitor diphenylene iodonium (DPI) and mitochondria electron chain inhibitor rotenone in the cells. To further analyze the source of ROS production, we found that ovarian cancer cells have much higher expression of NOX4 NADPH oxidase, and that specific inhibition of NADPH oxidase subunit p47(phox) diminished ROS production. To analyze the functional relevance of ROS production, we showed that ROS regulated hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in ovarian cancer cells. Elevated levels of endogenous ROS were required for inducing angiogenesis and tumor growth. NOX4 knockdown in ovarian cancer cells decreased the levels of VEGF and HIF-1 alpha and tumor angiogenesis. This study suggests a new mechanism of higher ROS production in ovarian cancer cells and provides strong evidence that endogenous ROS play an important role for cancer cells to induce angiogenesis and tumor growth. This information may be useful to understand the new mechanism of cancer cells in inducing tumorigenesis and to develop new therapeutic strategy by targeting ROS signaling in human cancer in the future.     

Hormones, autocrine and paracrine regulators of tumor growth in osteosarcoma

The data on 300 patients, aged 14-56, treated at the Center's Clinic have been evaluated. They include findings on androgen metabolism, basal level of secretion of sex steroid hormones in blood serum and their receptors in tumor, blood concentrations of sex steroids, binding globulin and pituitary hormones, arachidonic acid metabolism, expression of epidermal growth factor and its receptors and ligands, soluble Fas-antigen, vascular endothelial growth factor as well as calmodulin and cAMP levels in sarcoma. New trends of research in pathogenetic methods of treatment of osteosarcomas used in practice of oncology have been identified. Among them are androgen metabolism regulation, correction of the cyclooxygenase pathway of arachidonic acid metabolism, expression of epidermal growth factor and its ligands and neoangiogenesis in tumor.     

c-Myc interacts with hypoxia to induce angiogenesis in vivo by a vascular endothelial growth factor-dependent mechanism

The proto-oncogene c-myc is involved in the regulation of cell proliferation, differentiation, and apoptosis. In this study, we used an inducible transgenic mouse model in which c-Myc was targeted to the epidermis and, after activation, gave rise to hyperplastic and dysplastic skin lesions and to dermal angiogenesis, involving both vascular endothelial growth factor (VEGF) receptor-1 and VEGF receptor-2. After c-Myc activation, VEGF mRNA was expressed in postmitotic keratinocytes where it colocalized with transgene expression and areas of tissue hypoxia, suggesting a role of hypoxia in VEGF induction. In vitro, c-Myc activation alone was able to induce VEGF protein release and in conjunction with hypoxia, c-Myc activation further increased VEGF protein. Blocking VEGF signaling in vivo significantly reduced dermal angiogenesis, demonstrating the importance of VEGF as a mediating factor for the c-Myc-induced angiogenic phenotype.     

The oncogene phosphatidylinositol 3'-kinase catalytic subunit alpha promotes angiogenesis via vascular endothelial growth factor in ovarian carcinoma

The gene of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) has been implicated as an oncogene in ovarian cancer [L. Shayesteh et al., Nat. Genet., 21: 99-102, 1999]. In this study, we examined the expression of PIK3CA mRNA and its p110alpha protein product in human ovarian carcinoma and investigated its role in regulating angiogenesis via vascular endothelial growth factor (VEGF). PIK3CA mRNA was detected in 66.6% of stage I and 93.9% of advanced stage ovarian cancer specimens and in all 17 ovarian cancer cell lines. PIK3CA mRNA levels were significantly higher in invasive carcinomas compared with benign and low malignant potential neoplasms (P = 0.007), but no significant difference was seen between early and advanced stage carcinomas (P = 0.812). Strong expression of immunoreactive p110alpha was detected in tumor cells and/or stroma endothelium. PIK3CA expression in vivo positively correlated, both at the mRNA and the protein level, with the expression of VEGF as well as with the extent of microvascular development. Furthermore, PIK3CA mRNA overexpression positively correlated with increased proliferation and decreased apoptosis of tumor cells in vivo. In vitro, PIK3CA expression positively correlated with the expression of VEGF in ovarian cancer cells, whereas the phosphatidylinositol 3'-kinase inhibitor Ly294002 reduced both the constitutive and inducible expression of hypoxia-inducible factor-1alpha at the mRNA and protein levels and abrogated VEGF up-regulation by glucose starvation. Furthermore, Ly294002 suppressed cell proliferation and, at higher doses, induced marked apoptosis in ovarian cancer cells. Collectively, these data strongly indicate that PIK3CA supports ovarian cancer growth through multiple and independent pathways affecting cell proliferation, apoptosis and angiogenesis, and plays an important role in ovarian cancer progression.     

Overexpression of vascular endothelial growth factor by MCF-7 breast cancer cells promotes estrogen-independent tumor growth in vivo

Alteration of the phenotype of breast cancers from estrogen-dependent to estrogen-independent growth often leads to the failure of antiestrogenic tumor therapies. We report that overexpression of vascular endothelial growth factor (VEGF) by estrogen-dependent MCF-7 breast cancer cells could abolish estrogen-dependent tumor growth in ovariectomized mice. In the absence of estrogen, MCF-7 VEGF-expressing tumors with increased vessel density showed growth kinetics similar to, or even greater than, that of parental MCF-7 tumors with estrogen supplementation. Overexpression of VEGF by MCF-7 cells or treatment on parental MCF-7 cells with recombinant VEGF also stimulated cell proliferation in culture. Our data suggest that VEGF stimulation of MCF-7 tumor angiogenesis and growth is mediated by both autocrine and paracrine mechanisms.     

Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.

New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. Hence, the molecular basis of tumor angiogenesis has been of keen interest in the field of cancer research. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. The VEGF/VEGF-receptor axis is composed of multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities, cell-type expression, and function. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. More recently, an important role for VEGF has emerged in mobilization of endothelial progenitor cells from the bone marrow to distant sites of neovascularization. The well-established role of VEGF in promoting tumor angiogenesis and the pathogenesis of human cancers has led to the rational design and development of agents that selectively target this pathway. Studies with various anti-VEGF/VEGF-receptor therapies have shown that these agents can potently inhibit angiogenesis and tumor growth in preclinical models. Recently, an anti-VEGF antibody (bevacizumab), when used in combination with chemotherapy, was shown to significantly improve survival and response rates in patients with metastatic colorectal cancer and thus, validate VEGF pathway inhibitors as an important new treatment modality in cancer therapy.     

Energy intake and prostate tumor growth, angiogenesis, and vascular endothelial growth factor expression

BACKGROUND: A sedentary lifestyle coupled with excessive energy intake is speculated to be a factor associated with increased incidence of prostate cancer. We have investigated the effects of energy intake on prostate tumor growth in experimental animals. METHODS: Two transplantable prostate tumor models, i.e., the androgen-dependent Dunning R3327-H adenocarcinoma in rats and the androgen-sensitive LNCaP human carcinoma in severe combined immunodeficient mice, were studied. R3327-H tumor growth and relevant tumor biomarkers (proliferation index, apoptosis [programmed cell death], microvessel density, and vascular endothelial growth factor [VEGF] expression) were compared in ad libitum fed control rats, ad libitum fed castrated rats, and groups restricted in energy intake by 20% or 40%. A second set of experiments involving both tumor models examined tumor growth in ad libitum fed rats or in animals whose energy intake was restricted by 30% using three different methods, i.e., total diet restriction, carbohydrate restriction, or lipid restriction. All P values are two-sided. RESULTS: R3327-H tumors were smaller in energy-restricted or castrated rats than in control rats (P<.001). Tumors from energy-restricted rats exhibited changes in tumor architecture characterized by increased stroma and more homogeneous and smaller glands. In castrated rats, the tumor proliferation index was reduced (P<.0001), whereas apoptosis was increased in both energy-restricted (P<.001) and castrated (P<.001) rats. Tumor microvessel density and VEGF expression were reduced by energy restriction and castration (P<.003 versus control). Restriction of energy intake by reduction of carbohydrate intake, lipid intake, or total diet produced a similar inhibition of growth of R3327-H or LNCaP tumors. These effects were associated with reduced circulating insulin-like growth factor-I. CONCLUSIONS: Our observations are consistent with the hypothesis that energy restriction reduces prostate tumor growth by inhibiting tumor angiogenesis. Furthermore, dietary fat concentration does not influence prostate tumor growth when energy intake is reduced.     

Fetal stromal-dependent paracrine and intracrine vascular endothelial growth factor-a/vascular endothelial growth factor receptor-1 signaling promotes proliferation and motility of human primary myeloma cells

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.     

Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.     

Linomide blocks angiogenesis by breast carcinoma vascular endothelial growth factor transfectants.

The blocking of angiogenesis provides a novel therapeutic target to inhibit tumour spreading. In this study, we investigated the effect of linomide on angiogenesis induced in vivo by highly angiogenic breast carcinoma cells. The rabbit cornea was used to assess neovascular growth in the absence of a tumour mass. MCF-7 cells stably transfected with the cDNA encoding for vascular endothelial growth factor 121 (VEGF121) (V12 clone) were used to elicit a potent VEGF-dependent corneal angiogenesis. After tumour cell implant, albino rabbits received 100 mg kg(-1) day(-1) linomide for 5 consecutive days. Daily observation of neovascular progression indicated that linomide blocked angiogenesis. The antiangiogenic effect of linomide was apparent within 48 h from the beginning of the treatment and was both angiosuppressive and angiostatic. The block of neovascular growth lasted over 10 days from treatment suspension, and preformed vessels, which had regressed, remained dormant, suggesting the persistence of unfavourable conditions for capillary progression. Linomide (50-200 microg ml[-1]) was not cytotoxic in vitro on resting capillary endothelial cells but blocked endothelial cell replication induced by VEGF. Our data indicate that linomide can efficiently and persistently block VEGF-dependent angiogenesis in vivo in the absence of a growing tumour mass. These data suggest that linomide could be a chemopreventive drug in breast cancer patients and a valuable tool in clinical settings in which metastatic spreading occurs in the absence of a detectable tumour mass.     

An autocrine loop directed by the vascular endothelial growth factor promotes invasiveness of human melanoma cells

The vascular endothelial growth factor-A (VEGF-A) is a cytokine that promotes angiogenesis through the activation of two tyrosine kinase receptors, VEGFR-1 and VEGFR-2, on vascular endothelial cells. Moreover, several experimental evidences indicate that VEGF-A may also play a role in tumor progression by acting on neoplastic cells expressing VEGFRs. In this study we show that human melanoma cells that simultaneously produce VEGF-A and express VEGFRs exhibit a higher spontaneous ability to invade the extracellular matrix (ECM) than melanoma cells not expressing either VEGF-A or VEGFRs. Exposure of VEGFR expressing melanoma cells to exogenous VEGF-A further increases their ability to invade the ECM. Moreover, an inhibitor of VEGFR tyrosine kinase activity is able to abrogate VEGF-A-induced stimulation of ECM invasion. A cell clone (13443/N2) derived from a VEGF-A responsive melanoma cell line and expressing high levels of VEGFR-2 invades the ECM eight-fold more efficiently than a cell clone derived from the same cell line and expressing extremely low levels of the receptor. Exposure of 13443/N2 cells to VEGF-E, which selectively binds and activates VEGFR-2, increases their ability to invade the ECM. Finally, the expression of the VEGF-A mRNA antisense sequence in 13443/N2 cells markedly reduces the release of VEGF-A and ECM invasion. In conclusion, our data show for the first time that a VEGF-A-driven autocrine loop promotes human melanoma cell ability to invade the ECM, and strongly support the hypothesis that activation of VEGFR-2 plays a primary role in this process.     

Morphine stimulates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growth

Morphine is used to treat pain in several medical conditions including cancer. Here we show that morphine, in a concentration typical of that observed in patients' blood, stimulates human microvascular endothelial cell proliferation and angiogenesis in vitro and in vivo. It does so by activating mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation via Gi/Go-coupled G protein receptors and nitric oxide in these microvascular endothelial cells. Other contributing effects of morphine include activation of the survival signal PKB/Akt, inhibition of apoptosis, and promotion of cell cycle progression by increasing cyclin D1. Consistent with these effects, morphine in clinically relevant doses promotes tumor neovascularization in a human breast tumor xenograft model in mice leading to increased tumor progression. These results indicate that clinical use of morphine could potentially be harmful in patients with angiogenesis-dependent cancers.     

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.     

Nitroxyl inhibits breast tumor growth and angiogenesis

Nitroxyl (HNO) can inhibit the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Because of the importance of glycolysis in many malignant cells, we thus propose that HNO can adversely affect tumor growth. This hypothesis was tested using in vitro and in vivo models of breast cancer. We report here for the first time that HNO suppresses the proliferation of both estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines, in a dose dependent manner. Mice treated with HNO either injected into the tumor itself or via the intraperitoneal approach had smaller xenograft tumor size. In addition to significantly decreased blood vessel density in the HNO-treated tumors, we observed lower levels of circulating serum vascular endothelial growth factor (VEGF). Accordingly, there was a decrease in total HIF-1alpha (hypoxia-inducible factor) protein in HNO-treated tumor cells. Further studies showed inhibition of GAPDH activity in HNO-treated human breast cancer cell lines and in HNO-treated tumor tissue derived from xenografts. One explanation for the multiplicity of actions observed after HNO treatment could be the effect from the initial inhibition of GAPDH, providing a potential therapeutic avenue based upon blocking glycolysis resulting in decreased HIF-1alpha, thus leading to angiogenesis inhibition. Therefore, HNO appears to act via mechanism(s) different from those of existing breast cancer drugs, making it a potential candidate to overcome known and emerging drug resistance pathways.