Transfection of the genes for interleukin-12 into the K1735 melanoma and the EMT6 mammary sarcoma murine cell lines reveals distinct mechanisms of antitumor activity

Interleukin 12 (IL-12) is a pleiotropic cytokine with multiple effects on the immune system. The antitumor effects of locally produced IL-12 were examined in 2 tumor model systems. IL-12 expressing EMT6 mammary sarcomas (EMT6/IL-12) grew temporarily and then regressed resulting in mice that were immune to a further challenge of EMT6 cells. Interestingly, the IL-12 expressing K1735 melanomas (K1735/IL-12) maintained a lag phase of nonmeasurable growth for several weeks, followed by tumor outgrowth that was associated with a loss of IL-12 production. Tumor-infiltrating lymphocytes (TILs) isolated from EMT6/IL-12 tumors effectively lysed EMT6 target cells, whereas K1735/IL-12 TILs lacked lytic activity. Both IL-12 expressing tumors, however, grew progressively in nude mice indicating an important role for T cells in each case. Recombinant murine interferon gamma (rmIFN-gamma) inhibited the growth of EMT6 cells, but not K1735 cells in vitro, and strongly induced the expression of the antiangiogenic chemokine interferon-inducible protein 10 (IP-10) by both cell lines. Of interest, only the EMT6 cell line was able to secrete the proangiogenic molecule, vascular endothelial growth factor (VEGF), in response to low oxygen conditions. Fluorescent staining of the vascular endothelium at the tumor injection site provided images depicting early stages of angiogenesis prior to K1735/IL-12 tumor outgrowth. These results indicate that locally produced IL-12 likely mediates the rejection of EMT6 tumors through tumor cell lysis by host immune cells, whereas its antiangiogenic potential may be counterbalanced by the strong induction of VEGF by hypoxic tumor cells. In contrast, IL-12 does not induce protective immunity to K1735 tumors. However, an antiangiogenic mechanism may be responsible for controlling tumor growth.     

Quantitative and qualitative in vivo angiogenesis assay

We describe the development and optimization of an in vivo angiogenesis assay utilizing gelfoam sponges impregnated with 0.4% agarose and different proangiogenic factors, such as basic fibroblast growth factor (bFGF), vascular epidermal growth factor (VEGF), tumor growth factor-alpha (TGF-alpha), and endothelial growth factor (EGF). The sponges are implanted into the subcutis of mice and harvested after different times. The gelfoam sponges are fixed, sectioned, and stained with fluorescent antibodies against CD31. The median number of CD31+ cells is determined in 10 different 0.159-mm2 fields. Proangiogenic molecules induced significant migration and proliferation of endothelial cells. To demonstrate the utility of this assay for evaluation of an antiangiogenic agent, mice were implanted with gelfoam sponges containing different proangiogenic factors and treated orally with water or PTK 787, a novel tyrosine kinase inhibitor with specific activity against the VEGF-R. PTK 787 significantly inhibited angiogenesis in sponges containing agarose + VEGF but not other proangiogenic molecules. The data show that the implanted gelfoam sponges provide a reliable quantitative assay to study in vivo angiogenesis.     

Growth stimulation of COX-2-negative pancreatic cancer by a selective COX-2 inhibitor

Cyclooxygenase 2 (COX-2) inhibitors are promising antiangiogenic agents in several preclinical models. The aim of the present study was to evaluate the effect of selective COX-2 inhibitors on vascular endothelial growth factor (VEGF) production in vitro and angiogenesis and growth of pancreatic cancer in vivo, focusing on putative differences between COX-2-negative and COX-2-positive tumors. VEGF production and angiogenesis in vitro were determined by ELISA and endothelial cell migration assay. To determine whether the effect of COX-2 inhibitors was mediated by peroxisome proliferator-activated receptor gamma (PPAR-gamma), we used a dominant-negative PPAR-gamma and a pharmacologic inhibitor. In vitro findings were validated in a pancreatic cancer animal model. Microvessel density was assessed by CD31 immunostaining. Intratumoral prostaglandin and VEGF levels were measured by mass spectroscopy and ELISA. Selective COX-2 inhibitors had a concentration-dependent effect on VEGF production in vitro. Higher concentrations increased VEGF levels and stimulated angiogenesis by activating PPAR-gamma. In vivo, nimesulide increased VEGF production by cancer cells in COX-2-positive and COX-2-negative pancreatic tumors. In COX-2-negative pancreatic cancer, this effect was associated with an increase in angiogenesis and growth. In COX-2-positive pancreatic cancer, the nimesulide-induced increase of VEGF production by the cancer cells was offset by a decrease in VEGF production by the nonmalignant cell types leading to reduced tumor angiogenesis and growth. Selective COX-2 inhibitors had opposite effects on growth and angiogenesis in pancreatic cancer depending on COX-2 expression. These findings imply that assessing the COX-2 profile of the pancreatic tumor is mandatory before initiating therapy with a selective COX-2 inhibitor.     

Recombinant CD44-HABD is a novel and potent direct angiogenesis inhibitor enforcing endothelial cell-specific growth inhibition independently of hyaluronic acid binding

CD44 is the main cellular receptor for hyaluronic acid (HA). We previously found that overexpression of CD44 inhibited tumor growth of mouse fibrosarcoma cells in mice. Here, we show that soluble recombinant CD44 HA-binding domain (CD44-HABD) acts directly onto endothelial cells by inhibiting endothelial cell proliferation in a cell-specific manner. Consequently, soluble recombinant CD44-HABD also blocked angiogenesis in vivo in chick and mouse, and thereby inhibited tumor growth of various origins at very low doses (0.25 mg/kg x day). The antiangiogenic effect of CD44 is independent of its HA-binding capacity, since mutants deficient in HA binding still maintain their antiangiogenic and antiproliferative properties. Recombinant CD44-HABD represents a novel class of angiogenesis inhibitors based on a cell-surface receptor.     

Vascular endothelial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in human melanoma xenografts

Angiogenesis is a significant prognostic factor in melanoma, but the angiogenic factors controlling the neovascularization are not well defined. The purpose of this study was to investigate whether the angiogenesis and metastasis of melanoma are promoted by vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF). Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal angiogenesis assay. Lung colonization and spontaneous lung metastasis were determined after i.v. and intradermal inoculation of tumor cells, respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization, and spontaneous metastasis were assessed in mice treated with neutralizing antibody. The melanoma lines expressed multiple angiogenic factors, and each line showed a unique expression pattern. Multiple angiogenic factors promoted angiogenesis in the most angiogenic melanoma lines, whereas angiogenesis in the least angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may promote metastasis in melanoma, because inhibition of one of them could not be compensated for by the others. Our observations suggest that efficient antiangiogenic treatment of melanoma may require identification and blocking of common functional features of several angiogenic factors.     

Dopamine increases the efficacy of anticancer drugs in breast and colon cancer preclinical models.

PURPOSE: Because neurotransmitter dopamine inhibits vascular permeability factor/vascular endothelial growth factor (VEGF)-induced angiogenesis and as anti-VEGF agents act synergistically with anticancer drugs, we therefore investigated whether dopamine can increase the efficacies of these drugs. EXPERIMENTAL DESIGN: The effect of dopamine was investigated in human breast cancer-(MCF-7) and colon (HT29) cancer-bearing mice. Experimental groups received either dopamine or doxorubicin or dopamine plus doxorubicin in MCF-7 tumor-bearing mice, and either dopamine or 5-fluorouracil or dopamine plus 5-fluorouracil in HT29-bearing mice. Thereafter, tumor growth, angiogenesis, tumor cell apoptosis, life span, and the effect of dopamine on the growth and survival of tumor cells in vitro were determined. Finally, the effects of dopamine on tumor vascular permeability; on VEGF receptor-2, mitogen-activated protein kinase, and focal adhesion kinase phosphorylation; and also on the proliferation and migration of tumor endothelial cells were investigated. RESULTS: Dopamine, in combination with anticancer drugs, significantly inhibited tumor growth and increased the life span when compared with treatment with dopamine or anticancer drugs alone. Dopamine had no direct effects on the growth and survival of tumor cells. The antiangiogenic action of dopamine was mediated by inhibiting proliferation and migration of tumor endothelial cells through suppression of VEGF receptor-2, mitogen-activated protein kinase, and focal adhesion kinase phosphorylation. CONCLUSION: Our study shows that dopamine significantly enhances the efficacies of commonly used anticancer drugs and also indicates that an inexpensive drug like dopamine, which is being extensively used in the clinics, might have a role as an antiangiogenic agent for the treatment of breast and colon cancer.     

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.     

Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGF-producing ovarian cancer

Interleukin-10 (IL-10) is an immunosuppressive cytokine produced by T lymphocytes and drawing attention as an inhibitor of tumor angiogenesis. In this study, we investigated antiangiogenic and tumor suppressive effects of IL-10 in ovarian cancer cells. mIL-10-expressing plasmid was transferred into two ovarian cancer cell lines, SHIN-3 [vascular endothelial growth factor (VEGF) producing] and KOC-2S (non-VEGF producing). After selection, mIL-10-expressing cells were obtained as SHIN-3/mIL-10 and KOC-2S/mIL-10. No significant differences were observed in in vitro growth properties between mIL-10-expressing cells and control (luciferase expressing) cells in either KOC-2S or SHIN-3. The angiogenic activities of mIL-10-expressing cells were measured by dorsal air sac assay, which detected the number of newly formed blood vessels within a chamber in vivo. In addition, tumor formation was evaluated by s.c. tumor transplantation, and survival was monitored after i.p. injection of ovarian cancer cells into BALB/c nude mice. Both in vivo angiogenic activity and tumor growth were significantly inhibited in SHIN-3/mIL-10 cells compared with the control. Moreover, peritoneal dissemination was inhibited, and the survival period was significantly prolonged (mean survival days > 90 versus 36). In contrast, in the case of KOC-2S cells, no significant differences were observed in any of the parameters tested. These results indicate that IL-10 has suppressive effects on angiogenesis, tumor growth, and peritoneal dissemination of VEGF-producing ovarian cancer cells. Although the mechanisms of the antiangiogenic effect of IL-10 are still unclear, the potential usefulness of IL-10-mediated gene therapy of ovarian cancer was suggested.     

Antitumor effects of ZD6474 on head and neck squamous cell carcinoma

Angiogenesis is required for tumor growth and metastasis and, therefore, represents a target for cancer treatment. While many factors have been implicated in promoting angiogenesis, vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. ZD6474 is a potent VEGF receptor-2 (VEGFR-2) tyrosine kinase inhibitor which also has activity against the epidermal growth factor receptor (EGFR) tyrosine kinase. The purpose of this study was to investigate the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to ZD6474, and to evaluate its antitumor efficacy on HNSCC xenografts. This is the first demonstration of antitumor effects of ZD6474 on HNSCC. In vitro ZD6474 displayed antiproliferative effects on HNSCC cells and inhibition of VEGFR-2 and EGFR pathways. In vivo ZD6474 displayed antitumor activity, induced apoptosis and antiangiogenic activity on nude mice bearing an established xenograft of YCU-H891 cells. These results suggest that ZD6474 has the potential to inhibit two key pathways in tumor growth via inhibition of VEGF-dependent tumor angiogenesis and via inhibition of EGFR-dependent tumor cell proliferation.     

Angiogenesis and radiation response modulation after vascular endothelial growth factor receptor-2 (VEGFR2) blockade

The formation of new blood vessels (angiogenesis) represents a critical factor in the malignant growth of solid tumors and metastases. Vascular endothelial cell growth factor (VEGF) and its receptor VEGFR2 represent central molecular targets for antiangiogenic intervention, because of their integral involvement in endothelial cell proliferation and migration. In the current study, we investigated in vitro and in vivo effects of receptor blockade on various aspects of the angiogenic process using monoclonal antibodies against VEGFR2 (cp1C11, which is human specific, and DC101, which is mouse specific). Molecular blockade of VEGFR2 inhibited several critical steps involved in angiogenesis. VEGFR2 blockade in endothelial cells attenuated cellular proliferation, reduced cellular migration, and disrupted cellular differentiation and resultant formation of capillary-like networks. Further, VEGFR2 blockade significantly reduced the growth response of human squamous cell carcinoma xenografts in athymic mice. The growth-inhibitory effect of VEGFR2 blockade in tumor xenografts seems to reflect antiangiogenic influence as demonstrated by vascular growth inhibition in an in vivo angiogenesis assay incorporating tumor-bearing Matrigel plugs. Further, administration of VEGFR2-blocking antibodies in endothelial cell cultures, and in mouse xenograft models, increased their response to ionizing radiation, indicating an interactive cytotoxic effect of VEGFR2 blockade with radiation. These data suggest that molecular inhibition of VEGFR2 alone, and in combination with radiation, can enhance tumor response through molecular targeting of tumor vasculature.     

Enhanced antiangiogenic therapy of squamous cell carcinoma by combined endostatin and epidermal growth factor receptor-antisense therapy.

PURPOSE: We tested the combined effects of antiangiogenic endostatin and epidermal growth factor receptor (EGFR) antisense gene therapy on squamous cell carcinoma (SCC). EXPERIMENTAL DESIGN and Results: The 1483 cell line of human head and neck SCC (HNSCC) and SCC-VII/SF murine SCC cells was used to establish tumors in nude mice and immunocompetent C3H mice, respectively. Tumor-bearing mice were treated with endostatin (20 mg/kg/day, s.c.), liposomal EGFR-antisense expression plasmid (25 microg/mouse, three times/week, intratumoral), a combination of both agents, or liposomal EGFR-sense plasmid as a control. Endostatin or EGFR-antisense alone significantly, yet partially, inhibited the growth of 1483 and SCC-VII/SF tumors, and a combination of both treatments completely blocked tumor growth. Immunohistochemistry analysis demonstrated that a complete suppression of tumor angiogenesis was achieved by the combination treatment. Down-regulation of vascular endothelial growth factor was shown in EGFR-antisense-treated tumors. These results suggest that the EGFR-antisense treatment, in addition to its inhibitory activity on tumor cell proliferation, might have a synergistic effect with endostatin on SCC-induced angiogenesis. In vitro studies demonstrated that EGFR inhibition by antisense oligonucleotides or EGFR-specific tyrosine kinase inhibitor down-regulated the production of VEGF in HNSCC cells. Additional experiments demonstrated that these EGFR inhibition approaches also directly suppressed the growth of endothelial cells. CONCLUSION: A combination of endostatin and EGFR targeting strategies profoundly inhibited the angiogenesis and growth of SCC in vivo. EGFR-antisense therapy might have multiple inhibitory effects against both tumor cells and endothelial cells, leading to enhanced antitumor efficacy. Such a combination strategy might represent a novel and promising approach for HNSCC therapy.     

Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: implications for use as a surrogate marker of antiangiogenic activity.

PURPOSE: Circulating endothelial cells (CEC) comprise at least two distinct populations: bone marrow-derived circulating endothelial progenitors (CEP) and mature CECs derived from existing vasculature. We hypothesized that antiangiogenic agents may have differential effects on CEPs and mature CECs and that these changes may serve as a marker of biological activity. EXPERIMENTAL DESIGN: The effect of angiogenesis inhibitors on CECs was evaluated by flow cytometry after vascular endothelial growth factor (VEGF)-induced mobilization and in mice bearing Lewis lung carcinoma (LLC). Tumor angiogenesis was evaluated in parallel by immunohistochemistry. RESULTS: In nontumor-bearing mice, VEGF administration increased both mature CECs and CEPs. This increase was inhibited by the VEGF receptor 2 inhibitor ZD6474 as well as the VEGF inhibitor-soluble Flt-1. ZD6474 had no significant effect on CECs in the absence of exogenous VEGF stimulation. In contrast, LLC-bearing mice had an increase in mature CECs but not CEPs after 3 days of treatment with ZD6474. The increase in mature CECs was dose-dependent, accompanied by a decrease in tumor microvessel density, and preceded reduction in tumor volume. Treatment of LLC-bearing mice with the vascular targeting agent ZD6126 also increased mature CECs. CONCLUSIONS: VEGF inhibitors can have differential effects on mature CECs and CEPs, and agents inhibiting tumor angiogenesis may cause a concomitant increase in mature CECs. This increase occurs in tumor-bearing but not in nontumor-bearing mice, suggesting that tumor endothelium is a potential source of mature CECs. Therefore, assessing both mature CECs and CEPs may provide insights into the mechanism of antiangiogenic agents and serve as an early surrogate marker of biological activity.     

Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor

The melanoma differentiation-associated gene 7 (mda-7), also called interleukin (IL)-24, suppresses the growth of some cancers in vitro and in vivo as a result of the ectopic expression of its protein. However, the function of the secreted form of the protein in cancer has not been previously studied. The purpose of this study was to determine the antiangiogenic function of a secreted form of the MDA-7/IL-24 protein (sMDA-7/IL-24). In vitro, sMDA-7/IL-24 inhibited both endothelial cell differentiation and migration of endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor. The sMDA-7/IL-24-mediated inhibitory effect was 10-50 times more potent than endostatin, IFN-gamma, and IFN-inducible protein 10 in vitro. Furthermore, the inhibitory effect was not mediated by IFN or IFN-inducible protein 10. IL-22 receptor mediated the antiangiogenic activity of sMDA-7/IL-24. Administration of a blocking antibody to IL-22 receptor in conjunction with sMDA-7/IL-24 led to abrogation of inhibition of endothelial differentiation. sMDA-7/IL-24 inhibited vascular endothelial growth factor-induced angiogenesis as evidenced by reduced vascularization and hemoglobin content in in vivo Matrigel plug assays. In vivo, the growth of human lung tumor cells was significantly inhibited, and vascularization was reduced when the cells were mixed with 293 cells stably expressing sMDA-7/IL-24. Systemic administration of sMDA-7/IL-24 inhibited lung tumor growth in a mouse xenograft model. Associated with tumor growth inhibition was decreased tumor microvessel density and hemoglobin content, indicating the presence of antiangiogenic activity. These data demonstrate that sMDA-7/IL-24 is a novel and potent antiangiogenic effector and support the development of MDA-7/IL-24-based therapeutics.     

Interleukin-8 differentially regulates migration of tumor-associated and normal human brain endothelial cells

Interleukin-8 (IL-8) is a chemokine involved in angiogenesis, a process vital to tumor growth. Previously, we showed that endothelial cells derived from human tumor tissue have different functional and phenotypic properties compared with normal endothelial cells. This study analyzes the role of IL-8 in regulating angiogenesis of tumor-associated brain endothelial cells (TuBEC). Results show that TuBECs have a higher baseline migration rate compared with normal brain endothelial cells (BEC). TuBECs are unaffected when stimulated with IL-8 whereas BECs are activated. This lack of response of TuBECs to IL-8 is due to the constitutive production of IL-8. Endogenously produced IL-8 activates TuBECs in an autocrine manner as shown by IL-8 receptor inhibition. Blocking either CXCR1 or CXCR2 partially reduces TuBEC migration, whereas blocking both receptors further reduces migration. Treatment with antibody against vascular endothelial growth factor (VEGF) shows that production of IL-8 by TuBECs is dependent on VEGF. Transforming growth factor-beta1 (TGF-beta1), shown to down-regulate IL-8 production in BECs, does not inhibit IL-8 production in TuBECs. In summary, these studies show that TuBECs constitutively secrete IL-8 and autocrine activation by IL-8 is the result of VEGF stimulation. Furthermore, TuBECs do not respond to the feedback inhibition normally induced by TGF-beta1. These data emphasize the functional uniqueness of TuBECs. Understanding the functions and regulatory processes of tumor-associated endothelial cells is critical for developing appropriate antiangiogenic therapies.     

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.     

Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular function, and promotes tumor growth in vivo

The vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. However, clinical trials targeting the VEGF pathway are often ineffective, suggesting that other factors/pathways are also important in tumor angiogenesis. We have previously shown that the Notch ligand Delta-like 4 (DLL4) is up-regulated in tumor vasculature. Here, we show that DLL4, when expressed in tumor cells, functions as a negative regulator of tumor angiogenesis by reducing the number of blood vessels in all five types of xenografts, but acts as a positive driver for tumor growth in two of them (human glioblastoma and prostate cancer). The growth of in vivo models was not related to the effects on growth in vitro. DLL4 expressed in the tumor cells activated Notch signaling in host stromal/endothelial cells, increased blood vessel size, and improved vascular function within tumors. The promotion of tumor growth was, to some extent, due to a reduction of tumor hypoxia and apoptosis. DLL4-expressing tumor cells responded to anti-VEGF therapy with bevacizumab. A soluble form of DLL4 (D4ECD-Fc) blocked tumor growth in both bevacizumab-sensitive and bevacizumab-resistant tumors by disrupting vascular function despite increased tumor vessel density. In addition, we show that DLL4 is up-regulated in tumor cells and tumor endothelial cells of human glioblastoma. Our findings provide a rational basis for the development of novel antiangiogenic strategies via blockade of DLL4/Notch signaling and suggest that combined approaches for interrupting both DLL4 and VEGF pathways may improve antiangiogenic therapy.     

Recombinant adenovirus expressing wild-type p53 is antiagiogenic--implication for lung cancer gene therapy

Angiogenesis is required for the growth and progression of malignancies. Recent studies have demonstrated that genetic alterations may accompany acquisition of the angiogenic phenotype. Here we demonstrate that the recombinant adenovirus-mediated transfer of the wild-type p53 gene into a mutant p53-expressing human non-small cell lung cancer cell line markedly inhibited the expression of an angiogenic factor, vascular endothelial growth factor (VEGF), and increased the expression of a novel antiangiogenic factor, brain-specific angiogenesis inhibitor 1 (BAI 1), resulting in reduced neovascularization in vivo. These results may explain in part the mechanism of the bystander effect induced by wild-type p53 gene transfer of adjacent tumor cells.     

The recombinant kringle domain of urokinase plasminogen activator inhibits in vivo malignant glioma growth

In a previous report, the recombinant kringle domain (UK1) of the urokinase type plasminogen activator (uPA) showed antiangiogenic activity. Here, we investigated in vivo antitumor effects of the UK1 of human uPA employing a brain tumor model. The systemic administration of UK1 purified from pichia expression (10 and 50 mg/kg/day intraperitoneally for 25 days) led to suppress the growth of a U87 human glioma xenograft, implanted into the brains of male BALB/cSlc nude mice, by 35% and 80%, respectively. In the immunohistochemical analysis, the tumors treated with UK1 showed decreased vascularity and expression of angiogenesis-related factors including vascular endothelial growth factor (VEGF), angiogenin, alpha-smooth muscle actin, von Willebrand's factor, and CD31 (PECAM-1 [Platelet endothelial cell adhesion molecule-1]), and increased apoptosis. UKl inhibited the in vitro proliferation and tube formation of VEGF-stimulated endothelial cells but not the proliferation of glioma cells. These results suggest that UK1 inhibits the malignant glioma growth by suppression of angiogenesis.     

The antiangiogenic property of docetaxel is synergistic with a recombinant humanized monoclonal antibody against vascular endothelial growth factor or 2-methoxyestradiol but antagonized by endothelial growth factors

Numerous chemotherapeutic agents have been shown to have an inhibitory effect on endothelial cell proliferation and migration, and tubule formation. In this study, we examined the antiangiogenic activity of docetaxel. Docetaxel inhibited endothelial cell proliferation and tubule formation in vitro in a dose-dependent fashion. Docetaxel treatment also inhibited angiogenesis in an in vivo Matrigel plug assay. The endothelial stimulating factors, vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor are able to protect endothelial cells from the antiangiogenic properties of docetaxel. This protective effect can be overcome by a recombinant humanized monoclonal antibody directed against VEGF in both in vitro and in vivo models. Similarly, combination of docetaxel with the antiangiogenic agent 2-methoxyestradiol also overcomes the protective effect of VEGF in both in vitro and in vivo models. These data suggest that microenvironmental factors (e.g., local release of VEGF and basic fibroblast growth factor) could play a role in decreasing the antiangiogenic effects of docetaxel, whereas agents such as 2- methoxyestradiol and recombinant humanized monoclonal antibody directed against VEGF may reverse this protective effect.