Thymidine phosphorylase activity required for tumor angiogenesis and growth

Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We examined the involvement of TP activity in tumor growth and angiogenesis. KB cells were transfected with wild-type or mutant (L148R) PD-ECGF cDNA, and two sublines with high TP activity, KB/wt4 and KB/wt6, and one subline with no TP activity, KB/L148R, were cloned, respectively. The doubling times of these subclones in vitro were similar to that of KB cells. However, the growth of KB/wt4 and KB/wt6 cells was significantly faster when xenografted into nude mice than that of control cells with no TP activity. The tumors with high TP activity (KB/wt4 and KB/wt6) had significantly more microvessels than those with no TP activity (KB/-, KB/CV and KB/L148R) (P<0.01). These results, taken together with previous reports, suggest that the TP enzyme activity itself is involved in angiogenesis and growth of the KB tumors.     

Twist is required for thrombin-induced tumor angiogenesis and growth

Twist, a master regulator of embryonic morphogenesis, induces functions that are also required for tumor invasion and metastasis. Because thrombin contributes to the malignant phenotype by up-regulating tumor metastasis, we examined its effect on Twist in five different tumor cell lines and two different endothelial cell lines. Thrombin up-regulated Twist mRNA and protein in all seven cell lines. Down-regulation of Twist in B16F10 tumor cell lines led to a approximately 3-fold decrease in tumor growth on a chorioallantoic membrane assay and approximately 2-fold decrease in syngeneic mice. Angiogenesis was decreased approximately 45% and 36%, respectively. The effect of Twist on angiogenesis was further examined and compared with the effect of thrombin. In studies using a Twist-inducible plasmid, several identical vascular growth factors and receptors were up-regulated approximately 2- to 3-fold in tumor cells as well as human umbilical vascular endothelial cells by both Twist as well as thrombin (vascular endothelial growth factor, KDR, Ang-2, matrix metalloproteinase 1, GRO-alpha, and CD31). Thrombin-induced endothelial cell chemotaxis and Matrigel endothelial cell tubule formation were similarly regulated by Twist. Thus, thrombin up-regulates Twist, which is required for thrombin-induced angiogenesis as measured by endothelial cell migration, Matrigel tubule formation, and tumor angiogenesis.     

The effects of aging on tumor growth and angiogenesis are tumor-cell dependent

It is generally accepted that histologically similar tumors grow more slowly, with less angiogenesis, in aged mice relative to young mice. We subcutaneously implanted TRAMP-C2 tumor cells, a prostate cancer cell line not previously examined in aging, into syngeneic C57/Bl6 young (4 month) and aged (20 month) mice and compared tumor growth and angiogenesis. Unexpectedly, the prostate tumors grew as fast in aged as in young mice. Angiogenesis in TRAMP-C2 tumors was robust, with no differences between the young and aged mice in the number of vessels, distribution of vessel sizes or features of vessel maturation. Aged mice had lower levels of serum testosterone than the young mice. VEGF levels were similar in the tumors and sera of the young and aged mice. Comparison with B16/F10 melanoma, a cancer cell line that is representative of previous studies in aged mice, showed that B16/F10 tumors grew minimally in the aged mice. In contrast to the B16/F10, TRAMP-C2 tumors had an extracellular matrix with significantly higher levels of MMP2 and MMP9 expression and activity. These unique results demonstrate that tumor progression can be as robust in aged tissues as young tissues. The ability of aged mice to grow large, vascularized prostate tumors is associated with high levels of MMP2/9 activity that may produce a permissive environment for tumor growth and angiogenesis. These data demonstrate that tumor-cell specific features determine the effect of aging on tumor growth and angiogenesis.     

Aquaporins in tumor growth and angiogenesis

The aquaporins (AQPs) are a family of transmembrane water channel proteins widely distributed and play a major role in transcellular and transepithelial water movement. Moreover, recent evidence indicates that AQPs may be involved in cell migration and angiogenesis. This review article summarizes literature data concerning the involvement of AQPs in tumor growth, angiogenesis and metastatic process and suggest a potential therapeutic approach by antagonizing their biological activity.     

Epidermal growth factor receptor in tumor angiogenesis

This article focuses on the preclinical evidence for activation of the epidermal growth factor receptor (EGFR) in promoting angiogenesis and the efficacy of anti-EGFR agents in inhibiting 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.     

Extracellular matrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis

Summary Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a soilid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM-resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM producedin vitro and in basement membranes of the cornea and blood vesselsin vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM-immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.     

Alpha1-antitrypsin inhibits angiogenesis and tumor growth

Disturbances of the ratio between angiogenic inducers and inhibitors in tumor microenvironment are the driving force behind angiogenic switch critical for tumor progression. Angiogenic inhibitors may vary depending on organismal age and the tissue of origin. We showed that alpha(1)-antitrypsin (AAT), a serine protease inhibitor (serpin) is an inhibitor of angiogenesis, which induced apoptosis and inhibited chemotaxis of endothelial cells. S- and Z-type mutations that cause abnormal folding and defective serpin activity abrogated AAT antiangiogenic activity. Removal of the C-terminal reactive site loop had no effect on its angiostatic activity. Both native AAT and AAT truncated on C-terminus (AATDelta) inhibited neovascularization in the rat cornea and delayed the growth of subcutaneous tumors in mice. Treatment with native AAT and truncated AATDelta, but not control vehicle reduced tumor microvessel density, while increasing apoptosis within tumor endothelium. Comparative analysis of the human tumors and normal tissues of origin showed correlation between reduced local alpha(1)-antitrypsin expression and more aggressive tumor growth.     

血管生成与肿瘤的生长及转移

新生血管既为肿瘤生长提供营养和氧气,也是肿瘤侵袭和转移的主要途径.宿主不能控制肿瘤血管生成,人为抑制血管生成能显著抑制肿瘤生长.血管生成抑制剂具有高效、低毒和不易产生耐药性的特点,抗血管生成疗法将成为不同于常规的新的肿瘤治疗策略.     

The role of angiogenesis in tumor growth.

Experimental and clinical evidence is here assembled in support of the concept that the development of a solid tumor progresses from a prevascular phase to a vascular phase. The prevascular tumor does not induce angiogenesis, is limited in size, and rarely metastasizes. The vascularized tumor induces host microvessels to undergo angiogenesis, has the potential to rapidly expand its cell population, and has a propensity to metastasize. Thus, angiogenesis is necessary but not sufficient for tumor growth and metastasis. Neovascularization of a tumor requires that a critical number of its cells have switched to the angiogenic phenotype. The mechanisms by which tumor cells become angiogenic, subjects of current study, are reviewed here. At least two general categories are recognized: (i) angiogenic activity arises from the tumor cell itself in the form of the release of angiogenic molecules such as basic fibroblast growth factor; (ii) angiogenic activity arises from host cells recruited by the tumor (e.g. macrophages), or is mobilized from the extracellular matrix, or requires concomitant loss of physiological inhibition of endothelial cell proliferation. Accumulating evidence indicates that for most tumors, the switch to the angiogenic phenotype depends upon the outcome of a balance between angiogenic stimulators and angiogenic inhibitors, both of which may be produced by tumor cells and perhaps by certain host cells.     

Second hand smoke stimulates tumor angiogenesis and growth

Exposure to second hand smoke (SHS) is believed to cause lung cancer. Pathological angiogenesis is a requisite for tumor growth. Lewis lung cancer cells were injected subcutaneously into mice, which were then exposed to sidestream smoke (SHS) or clean room air and administered vehicle, cerivastatin, or mecamylamine. SHS significantly increased tumor size, weight, capillary density, VEGF and MCP-1 levels, and circulating endothelial progenitor cells (EPC). Cerivastatin (an inhibitor of HMG-coA reductase) or mecamylamine (an inhibitor of nicotinic acetylcholine receptors) suppressed the effect of SHS to increase tumor size and capillary density. Cerivastatin reduced MCP-1 levels, whereas mecamylamine reduced VEGF levels and EPC. These studies reveal that SHS promotes tumor angiogenesis and growth. These effects of SHS are associated with increases in plasma VEGF and MCP-1 levels, and EPC, mediated in part by isoprenylation and nicotinic acetylcholine receptors.     

Fibroblast growth factor overexpressing breast carcinoma cells as models of angiogenesis and metastasis

Summary Progression of breast cancer from an estrogen-dependent, slowly growing tumor amenable to tamoxifen treatment to an aggressive, metastatic, estrogen-independent phenotype has been mimicked by the transfection of MCF-7 breast carcinoma cells with fibroblast growth factors 1 or 4. FGF-transfected cells are aggressively tumorigenic in ovariectomized or tamoxifen-treated nude mice, conditions under which the parental cells would not produce tumors. When detection of metastasis was enhanced bylacZ transfection, the FGF-transfected MCF-7 cells were reliably metastatic to lymph nodes and frequently metastatic to lungs, in further contrast to parental cells. An antiangiogenic drug, AGM-1470, given to mice bearing tumors produced by FGF-transfected MCF-7 cells, produced a decrease in tumor size. The decreased tumor size was not as marked as that produced by treatment with pentosan polysulfate, an agent which would abrogate all autocrine or paracrine effects of the transfected FGF. Thus, increased angiogenesis may be a component of the phenotypic change produced by the FGF transfection, but other autocrine or paracrine effects may also be important.Since a clonal FGF-4 andlacZ doubly-transfected cell line, MKL-4, progressively lost expression of the transfectedlacZ gene in individual cells, we performed successive rounds of fluorescence-activated cell sorting to select high-expressing cells. High-expressing cell populations thus obtained rapidly lost expression of ß-gal activity in continued culture. High ß-gal expressing clonal cell lines of MKL-4 cells established by either one or two rounds of low-density cloning also lostlacZ expression with continued culture. Southern analysis of DNA fromlacZ transfected cell lines showed the transfected sequences to be present and grossly intact in both high and low expressing populations. However, Northern analysis revealed that high-expressing populations of MKL-4 cells contained the mostlacZ mRNA, implying that in the unstable MKL-4 cell line, individual cells are down-regulating mRNA levels oflacZ. StablelacZ expression has been obtained in other FGF-transfected and parental MCF-7 cell lines using the same expression vector. Thus, the MKL-4 cell line is down-regulating mRNA encoding the transfected gene through a mechanism not dependent on the CMV promotor utilized in the expression vector. This evidence suggests thatlacZ expression is not a benign modification in certain cells.     

Recombinant adenovirus encoding vasohibin prevents tumor angiogenesis and inhibits tumor growth

Numerous lines of evidence have shown that angiogenesis plays a pivotal role in the development of tumors. Therefore anti‐angiogenesis therapy represents a potentially promising approach to cancer therapy. Recently, a new inhibitor called vasohibin was discovered to operate as an intrinsic and highly specific feedback inhibitor in the process of angiogenesis. However, to date, reports on the antitumor and anti‐angiogenic properties of vasohibin have been very limited. To explore the potential of vasohibin as an anti‐angiogenesis therapeutic, we constructed a recombinant adenovirus encoding vasohibin. Our data showed that the recombinant adenovirus encoding vasohibin could prevent tumor angiogenesis and tumor growth. Notably, angiogenesis in the tumors was prevented without any apparent side‐effects. Therefore, the findings suggested that the recombinant adenovirus encoding vasohibin might be valuable as a potential strategy for antitumor angiogenesis therapy in the clinic. (Cancer Sci 2009)     

MT1-MMP is required for efficient tumor dissemination in experimental metastatic disease

Membrane-type I matrix metalloproteinase (MT1-MMP) is associated with multiple forms of cancer including mammary cancer. To directly evaluate the significance of MT1-MMP expression in tumor progression and metastasis using a genetically induced cancer model, we crossed MT1-MMP-deficient mice to MMTV-polyoma virus middle T-antigen (PyMT) mice. Expression of PyMT in the MT1-MMP-deficient background consistently resulted in hyperplasia of the mammary gland as seen in wild-type PyMT littermates. Following orthotopic transplantation of PyMT+ glands into the cleared mammary fat pad of syngeneic recipient mice, MT1-MMP-deficient tumors were palpable earlier than wild-type tumors. Moreover, MT1-MMP-deficient tumors grew to the experimental end point size quicker than control tumors, but demonstrated markedly reduced ability to metastasize to the lungs of recipient mice. Accordingly, MT1-MMP-deficient mice displayed an overall reduction in metastasis count of 50%. MT1-MMP was expressed solely in the stroma of PyMT-induced tumors and those metastatic nodules that formed in the lungs were devoid of MT1-MMP expression. Stromal fibroblasts isolated from MT1-MMP-deficient tumors did not degrade type I collagen suggesting that efficient dissemination of tumor cells is dependent on stromal cell remodeling of the tumor environment. The data demonstrate directly that MT1-MMP-mediated proteolysis by stromal cells is important in the metastatic process.     

Glucocorticoids suppress tumor angiogenesis and growth of prostate cancer

Hormone-refractory prostate cancer ( HRPC) sometimes is responsive to treatment with glucocorticoids, such as prednisolone, hydrocortisone and dexamethasone, but the underlying mechanisms are not well established. In a recent paper (Clin Cancer Res, 2006, 12:3003-3009), Yano et al. Hypothesized and confirmed that the therapeutic effect of glucocorticoids on HRPC is attributed to inhibition of angiogenesis. A prostate cancer cell line DU145 that expresses glucocorticoid receptor was used to study the effect of dexamethasone (Dex) on the expres-     

Reduced tumor growth and angiogenesis in endoglin-haploinsufficient mice.

Endoglin is a transforming growth factor-beta(1) (TGF-beta(1)) accessory receptor which is highly expressed in tumor vessels. To study the role of endoglin in tumor growth and angiogenesis we induced a highly vascularized tumor in mice heterozygous for endoglin (Eng+/-) and in their control littermates (Eng+/+) by injecting 10(6) Lewis lung carcinoma (3LL) cells subcutaneously. Nine days after injection, the tumor was removed and weighed. Capillary density (CD31 immunohistochemistry), hemoglobin content and vascular cell adhesion molecule-1 (VCAM-1) expression were used to assess tumor vascularization. Tumor perfusion rate was measured by laser-Doppler technique. Expression of the hypoxia-inducible factor (HIF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were determined by Western blot analysis. The aerobic metabolism and oxygen dependency were inferred from the measurement of ATP in tumoral tissue. Tumor weight, capillary density, hemoglobin and VCAM-1 were reduced by about 30% in Eng+/- compared to Eng+/+ littermates. The protein levels of eNOS and phosphorylated eNOS were significantly reduced in Eng+/- compared to Eng+/+ mice. HIF expression was slightly reduced whereas VEGF level was slightly increased in Eng+/- compared to Eng+/+. Tumor tissue levels of ATP and ADP were similar in both types of mice. These data demonstrate that endoglin plays a major role in tumor neoangiogenesis.     

Neuropilin-1与肿瘤血管新生和生长转移

神经纤毛蛋白-1（Neuropilin-1）最早是作为轴突导向分子collapsin／semaphorin的受体被发现，在神经发育过程中引导轴突选择正确途径以成功到达靶区，与此同时又可作为血管内皮生长因子受体-2（VEGFR-2）的共受体表达于血管内皮细胞，在血管新生中发挥作用。近来的研究表明NRP-1可通过依赖于VEGF和独立于VEGF的方式参与调节肿瘤血管新生，在肿瘤生长转移中发挥作用。     

Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor tyrosine kinase c-Met have emerged as key determinants of brain tumor growth and angiogenesis. SF/HGF and c-Met are expressed in brain tumors, the expression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of SF/HGF and/or c-Met in brain tumor cells enhances their tumorigenicity, tumor growth, and tumor-associated angiogenesis. Conversely, inhibition of SF/HGF and c-Met in experimental tumor xenografts leads to inhibition of tumor growth and tumor angiogenesis. SF/HGF is expressed and secreted mainly by tumor cells and acts on c-Met receptors that are expressed in tumor cells and vascular endothelial cells. Activation of c-Met leads to induction of proliferation, migration, and invasion and to inhibition of apoptosis in tumor cells as well as in tumor vascular endothelial cells. Activation of tumor endothelial c-Met also induces extracellular matrix degradation, tubule formation, and angiogenesis in vivo. SF/HGF induces brain tumor angiogenesis directly through only partly known mechanisms and indirectly by regulating other angiogenic pathways such as VEGF. Different approaches to inhibiting SF/HGF and c-Met have been recently developed. These include receptor antagonism with SF/HGF fragments such as NK4, SF/HGF, and c-Met expression inhibition with U1snRNA/ribozymes; competitive ligand binding with soluble Met receptors; neutralizing antibodies to SF/HGF; and small molecular tyrosine kinase inhibitors. Use of these inhibitors in experimental tumor models leads to inhibition of tumor growth and angiogenesis. In this review, we summarize current knowledge of how the SF/HGF:c-Met pathway contributes to brain tumor malignancy with a focus on glioma angiogenesis.     

Centrosomal PKCbetaII and pericentrin are critical for human prostate cancer growth and angiogenesis

Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth, but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCbetaII was activated during increased angiogenesis and that PKCbetaII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCbetaII-selective inhibitor, betaIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCbetaII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCbetaII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be up-regulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is up-regulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCbetaII inhibitor such as betaIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.