Copper binding by tetrathiomolybdate attenuates angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase 1.

PURPOSE: A second-generation tetrathiomolybdate analogue (ATN-224; choline tetrathiomolybdate), which selectively binds copper with high affinity, is currently completing two phase I clinical trials in patients with advanced solid and advanced hematologic malignancies. However, there is very little information about the mechanism of action of ATN-224 at the molecular level. EXPERIMENTAL DESIGN: The effects of ATN-224 on endothelial and tumor cell growth were evaluated in cell culture experiments in vitro. The antiangiogenic activity of ATN-224 was investigated using the Matrigel plug model of angiogenesis. RESULTS: ATN-224 inhibits superoxide dismutase 1 (SOD1) in tumor and endothelial cells. The inhibition of SOD1 leads to inhibition of endothelial cell proliferation in vitro and attenuation of angiogenesis in vivo. The inhibition of SOD1 activity in endothelial cells is dose and time dependent and leads to an increase in the steady-state levels of superoxide anions, resulting in the inhibition of extracellular signal-regulated kinase phosphorylation without apparent induction of apoptosis. In contrast, the inhibition of SOD1 in tumor cells leads to the induction of apoptosis. The effects of ATN-224 on endothelial and tumor cells could be substantially reversed using Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, a catalytic small-molecule SOD mimetic. CONCLUSIONS: These data provide a distinct molecular target for the activity of ATN-224 and provide validation for SOD1 as a target for the inhibition of angiogenesis and tumor growth.     

RhoC is essential for angiogenesis induced by hepatocellular carcinoma cells via regulation of endothelial cell organization

The angiogenesis induced by tumor cells is essential for metastasis of hepatocellular carcinoma. Available information suggests that RhoC participates in angiogenesis through regulation of vascular endothelial growth factor expression in tumor cells. For its broad functions in cell migration and cytoskeletal organization, we hypothesized that RhoC regulating angiogenesis does not exclusively depend on regulation of vascular endothelial growth factor expression. To address this question, in the present study, we used a retroviral small interfering RNA approach to selectively knockdown the expression of RhoC in a neovascularization model in vivo and in vitro. Our present results indicate that RhoC is the downstream regulator of vascular endothelial growth factor in endothelial cells and is essential for angiogenesis induced by vascular endothelial growth factor, notwithstanding that RhoC regulates the expression of vascular endothelial growth factor in tumor cells. Furthermore, we show that knockdown of RhoC is associated with the inhibition of invasion and migration but not apoptosis of endothelial cells. Knockdown of RhoC results in inhibition of endothelial cell organization through restraining the reorganization of F‐actin filaments, which represses endothelial cell network and sprout formation. In conclusion, our results demonstrate that knockdown of RhoC inhibits angiogenesis induced by tumor cells not only through effecting the release of vascular endothelial growth factor, but also through inhibiting endothelial cell migration and organization, which implies that it blocks tumor metastasis by specifically inhibiting RhoC in endothelial cells. (Cancer Sci 2008; 99: 2012–2018)     

Angiopoietin-3 inhibits pulmonary metastasis by inhibiting tumor angiogenesis

Angiopoietins (Ang-1, Ang-2, and Ang-3) are the ligands of Tie-2 receptor tyrosine kinase. The essential roles of Ang-1 and Tie-2 in embryonic angiogenesis have been established, and studies have demonstrated the involvement of Ang-1 and Ang-2 in tumor angiogenesis. However, the role of Ang-3 in tumor angiogenesis and metastasis and the mechanism underlying its function are totally unknown. We have shown recently that Ang-3 is tethered on cell surface via heparan sulfate proteoglycans. In our current study, we have demonstrated that overexpression of Ang-3 inhibits pulmonary metastasis of Lewis lung carcinoma and TA3 mammary carcinoma (TA3) cells by inhibiting tumor angiogenesis and promoting apoptosis of the tumor cells. In addition, we have demonstrated that the binding of Ang-3 to the cell surface is required for the effective inhibition of Ang-3 on tumor metastasis and that Ang-3 inhibits endothelial cell proliferation and survival and blocks Ang-1- and vascular endothelial growth factor-induced activation of extracellular signal-regulated kinase 1/2 and Akt kinases, which likely underlie the Ang-3-mediated inhibition on tumor angiogenesis and metastasis.     

人canstatin基因治疗人食管癌裸鼠移植瘤的实验研究

背景与目的：Canstatin是一种新内源性血管生成抑制剂,以往的研究显示canstatin能有效的抑制肿瘤的生长,作用甚至强于endostatin。本研究将探讨人canstatin基因对人食管鳞状细胞癌移植瘤模型的抑制作用。方法：应用人食管癌细胞株KYSE150建立移植瘤模型。将裸鼠随机分成3组：腺病毒携带的canstatin基因组（Ad—GFP—canstatin）、腺病毒携带的绿色荧光蛋白组（Ad—GFP）和磷酸盐缓冲液组（PBS）。治疗期间测量皮下移植瘤的长径和短径;30d后处死裸鼠,取下肿瘤行常规病理切片,观察药物的毒性反应;检测肿瘤组织中caspase-3、内皮细胞生长因子受体1（fetal liver kinase-1,Flk-1）和血管内皮生长因子（vascular endothelial growth factor,VEGF）的表达和微血管密度（microvessel density,MVD）。结果：第二次注射病毒后3d,canstatin基因治疗组肿瘤体积显著小于其余两组（P〈0．05）,治疗第6天抑瘤率达到61％;HE染色显示：各组肿瘤组织中都有坏死．尤其在canstatin基因治疗组坏死更加明显;canstatin基因治疗组caspase-3表达高于空载体组和对照组（P〈0．05）;Flk-1的表达低于空载体组和对照组（P〈0．05）：VEGF的表达3组相比差异无统计学意义（P〉0．05）：canstatin基因治疗组MVD低于空载体组和对照组（P〈0．05）。结论：人canstatin基因对人食管癌移植瘤的生长具有抑制作用,作用机制可能是降低Flk-1的表达进而抑制肿瘤的血管生成,抑制肿瘤的生长。     

Silibinin strongly inhibits growth and survival of human endothelial cells via cell cycle arrest and downregulation of survivin, Akt and NF-kappaB: implications for angioprevention and antiangiogenic therapy

Recently, we observed that suppression of tumor xenograft growth by silibinin was associated with reduction in tumor vasculature and an increased apoptosis. Here, we provide evidence for molecular events associated with antiangiogenic efficacy of pharmacologically achievable doses of silibinin in endothelial cell culture system. Our data show that silibinin almost completely (P<0.001) inhibits growth of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC-dermal origin) together with induction of cell death in a dose- and time-dependent manner. Growth inhibition was associated with a strong induction of G1 arrest accompanied by an increase in Kip1/p27, Cip1/p21 and p53. Apoptosis induction (up to 14- to 17-fold in both cell lines, P<0.001) was an underlying mechanism in silibinin-induced death of endothelial cells. In the studies elucidating the molecular events involved in apoptosis, silibinin caused loss of mitochondrial membrane potential and an increase in cytochrome c release from mitochondria. An increase in Bax and a decrease in Mcl-1 proteins were also observed. Silibinin-induced apoptosis involved both caspase-dependent and -independent mechanisms. Silibinin also decreased survivin level and inhibited Akt and NF-kappaB signaling. Two different PI-3K inhibitors, wortmannin and LY294002, showed Akt-independent activation of NF-kappaB. Further, silibinin showed a concentration-dependent strong inhibition of capillary tube formation on matrigel, retraction and disintegration of preformed capillary network, inhibition of matrigel invasion and migration, and a decrease in matrix metalloproteinase-2 secretion by HUVEC. Together, these findings identify pleiotropic mechanisms for antiangiogenic efficacy of silibinin, and suggest its usefulness in angioprevention and antiangiogenic therapy.     

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.     

血管能抑素canstatin治疗肿瘤研究进展

血管能抑素canstatin是一种来源于Ⅳ型胶原α2链非胶原(NC1)区的血管生成和肿瘤生长内源性抑制因子.相比放疗、化疗等传统的肿瘤治疗方法,canstatin具有特异性高,不易产生抗药性等特点.临床研究表明,canstatin联合抑制肿瘤血管生成的其它疗法协同作用,在未来肿瘤的治疗中有着广阔的应用前景.     

The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors

Angiogenesis, or the formation of new blood vessels from preexisting vasculature, plays a major role in tumor growth and metastasis formation. Therefore, inhibiting tumor angiogenesis may be a promising therapeutic strategy. Paracrine stimuli from tumor cells are the main promoters of angiogenesis. They activate endothelial cells to proliferate and migrate, subsequently resulting in new tube formation and blood flow. This complex process involves numerous biological activities. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic factor. Originally identified for its ability to induce vascular permeability and stimulate endothelial cell growth, VEGF is now known to be a key requirement for tumor growth. Currently, three high-affinity tyrosine kinase receptors for VEGF have been identified, of which VEGF receptor (VEGFR)-Flk-1/KDR (VEGFR-2) is exclusively expressed in vascular endothelial cells. Because the VEGFR-2 system is a dominant signal-transduction pathway in regulating tumor angiogenesis, specific inhibitors of this pathway inhibit metastases, microvessel formation, and tumor-cell proliferation. Induction of apoptosis in tumor cells and endothelial cells has also been observed. The clinical importance of VEGF for tumor growth is supported by the fact that most tumors produce VEGF and that the inhibition of VEGF-induced angiogenesis significantly inhibits tumor growth in vivo. In this review, we discuss the biologic role of VEGF and the therapeutic options for inhibiting VEGF in cancer patients.     

Inhibition of androgen-responsive LNCaP prostate cancer cell tumor xenograft growth by dietary phenethyl isothiocyanate correlates with decreased angiogenesis and inhibition of cell attachment

Phenethyl isothiocyanate (PEITC) is a candidate anticancer compound found in certain cruciferous vegetables. In our tumor cell xenograft model, dietary administration of PEITC (100-150 mg/kg body weight/d) inhibited androgen-responsive LNCaP human prostate cancer cell tumor growth. We found that dietary treatment with PEITC significantly inhibited tumor platelet/endothelial cell adhesion molecule (PECAM-1/CD31) expression, a marker of angiogenesis. By contrast, we did not find the inhibitory effects of PEITC on tumor growth to be associated with alteration of specific markers for apoptosis, cell proliferation or androgen receptor-mediated pathways. Consistent with in?vivo results, PEITC exerted little effects on cell proliferation, cell cycle and androgen-dependent pathways. Interestingly, PEITC significantly attenuated LNCaP cell plating efficiency that correlated with inhibition of integrin family proteins integrin β1, α2 and α6 mRNA expression. Thus, PEITC may be a dietary factor that inhibits androgen-responsive prostate tumor growth indirectly by selectively targeting factors involved in the tumor microenvironment.     

Kringle 5 of human plasminogen suppresses hepatocellular carcinoma growth both in grafted and xenografted mice by anti-angiogenic activity

Plasminogen kringle 5 (K5), a proteolytic fragment of plasminogen, is an endogenous angiogenic inhibitor. We have previously shown that K5 inhibits ischemia-induced retinal neovascularization in a rat model. However, its anti-angiogenic potential and application in the treatment of neoplastic diseases have not been well investigated. Our present study was designed to test its effect on the neovascularization and growth of hepatocellular carcinoma, a typical hypervascular tumor. Recombinant human K5 was expressed in E. coli and purified by affinity chromatography. K5 inhibited proliferation and induced apoptosis of primary endothelial cells in dose-dependent manner, but no effect on pericytes from the same origin of endothelial cells, which suggested an endothelial cell-specific inhibition. Moreover, K5 had no effect on the proliferation and apoptosis of mouse HepA and human Bel7402 hepatoma cell lines even in the enhanced concentration range, which suggested K5 having no direct effect on tumor cells. Ventral injection of K5 significantly suppressed the tumor growth in graphed hepatocarcinoma mice model, which was established by injection of mouse HepA hepatoma cells. In xenografted hepatocarcinoma athymic mice model, which mimicked human tumors by injection of human Bel7402 hepatoma cells, K5 significantly suppressed the tumor growth. An average of 68% suppression of primary tumor growth was observed in the K5-treated mice compared with control group. K5 also inhibited intratumoral neovascularization in the two cancer models determined by micro vessel density (MVD) analysis. Injection of K5 significantly induced the cleavage of pro-caspase-3 in tumor tissues of grafted mouse model, which suggested K5 also induced apoptosis of tumor tissues and the decreased intratumoral microvascular density in K5 treated group may correlate with K5-induced endothelial cell apoptosis. These results suggest that tumor growth suppression of K5 depends on its anti-angiogenic activity and K5 could have therapeutic potential in hepatocellular carcinoma.     

Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases

BACKGROUND: Recent studies have suggested that vascular endothelial growth factor (VEGF), in addition to its proangiogenic properties, also functions as a survival factor for endothelial cells. The authors hypothesized that inhibition of VEGF activity by blockade of VEGF receptor-2 (R-2) function prevents angiogenesis and decreases tumor growth in colon carcinoma liver metastases. METHODS: Spleens of mice were injected with human colon carcinoma cells producing liver metastases. After 7 days of tumor growth, groups of mice received either antibody to VEGFR-2 (DC101) or phosphate-buffered saline (control). In a follow-up experiment, a similar treatment regimen was followed except that mice were sacrificed at 1-week intervals to assess the time course of endothelial cell and tumor cell apoptosis. RESULTS: After 21 days of therapy, the authors observed a significant decrease in vessel counts in liver metastases from human colon carcinoma in nude mice after therapy with VEGFR-2 antibody. Tumor cell apoptosis was increased significantly in the tumors of mice receiving DC101. Temporal studies with immunofluorescent double staining for the microvasculature and apoptotic cells revealed an increase in endothelial cell apoptosis that preceded an increase in tumor cell apoptosis. In vitro, treatment of human umbilical vein endothelial cells with antibody to VEGFR-2 produced a > 2.5-fold increase in endothelial cell apoptosis. CONCLUSIONS: Therapy targeting the VEGFR-2 inhibited tumor growth in a murine model of colon carcinoma liver metastasis. Surprisingly, this therapy did not only inhibit angiogenesis but also led to endothelial cell death. These findings suggest that VEGF, via VEGFR-2 signaling, functions as a survival factor for tumor endothelial cells in liver metastases from colon carcinoma.     

BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.     

Hypoxia-inducible factor: Achilles' heel of antiangiogenic cancer therapy (review).

Could a rational, hypothesis-driven and well-tolerated therapy drive tumor progression? This scenario can be foreseen for antiangiogenic therapy, despite it is one of the most elegant anticancer strategies. Antiangiogenic agents inhibit growth of endothelial cells resulting in tumor hypoxia and starvation which in turn inhibit tumor growth. On the other hand, it is known that hypoxia selects for a highly aggressive and metastatic cancer and is associated with unfavorable prognosis. This review attempts to reconcile these opposite notions and to revisit the thesis that antiangiogenic therapy is "resistant to resistance". The latter logical paradigm is based on the notion that endothelial cells cannot become drug resistant. Although endothelial cells may not acquire drug-resistance, cancer cells can acquire hypoxia-resistance which is also associated with the resistance to growth arrest and apoptosis as well as high metastatic potentials. Hypoxia-inducible factor (HIF-1) renders cells capable of surviving hypoxia and stimulating endothelial growth. Disruption of the HIF-1 pathway inhibits tumor growth, indicating HIF-1 as a potential anticancer target. Furthermore, inhibition of HIF-1 is a mechanism-based antiangiogenic strategy because it is the HIF-mediated response that drives tumor angiogenesis. Pharmacological approaches to HIF-1 inhibition are discussed.     

凝血酶敏感蛋白-1在肿瘤中的研究进展

凝血酶敏感蛋白-1(TSP-1)在多种肿瘤中表达,并通过诱导内皮细胞凋亡等机制抑制肿瘤血管生成,从而阻止肿瘤生长.其功能结构域(Type I序列)3TSR保留了抑制新生血管形成的功能,在临床肿瘤治疗中具有一定的实际应用意义.     

凝血酶敏感蛋白-1在肿瘤中的研究进展

凝血酶敏感蛋白-1(TSP-1)在多种肿瘤中表达,并通过诱导内皮细胞凋亡等机制抑制肿瘤血管生成,从而阻止肿瘤生长.其功能结构域(Type I序列)3TSR保留了抑制新生血管形成的功能,在临床肿瘤治疗中具有一定的实际应用意义.     

MAZ51, an indolinone that inhibits endothelial cell and tumor cell growth in vitro, suppresses tumor growth in vivo

We have recently described MAZ51, an indolinone that blocks the ligand-induced autophosphorylation of VEGFR-3, a receptor tyrosine kinase that plays a central role in the regulation of lymphangiogenesis. Here we show that MAZ51 is able to block the proliferation of VEGFR-3-expressing human endothelial cells and is less potently able to induce their apoptosis. MAZ51 also inhibits the proliferation and induces the apoptosis of a variety of non-VEGFR-3-expressing tumor cell lines. These data suggest that MAZ51 blocks the activity of tyrosine kinases in addition to VEGFR-3. In vivo, MAZ51 significantly inhibits the growth of rat mammary carcinomas. These data establish MAZ51 as a compound with antitumor properties that inhibits tumor growth directly and also indirectly by interfering with tumor-host interactions.     

Enhancement of radiation response with bevacizumab

ABSTRACT: BACKGROUND: Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis. Bevacizumab is a humanized monoclonal antibody that neutralizes VEGF. We examined the impact on radiation response by blocking VEGF signaling with bevacizumab. METHODS: Human umbilical vein endothelial cell (HUVEC) growth inhibition and apoptosis were examined by crystal violet assay and flow cytometry, respectively. In vitro HUVEC tube formation and in vivo Matrigel assays were performed to assess the anti-angiogenic effect. Finally, a series of experiments of growth inhibition on head and neck (H&N) SCC1 and lung H226 tumor xenograft models were conducted to evaluate the impact of bevacizumab on radiation response in concurrent as well as sequential therapy. RESULTS: The anti-angiogenic effect of bevacizumab appeared to derive not only from inhibition of endothelial cell growth (40%) but also by interfering with endothelial cell function including mobility, cell-to-cell interaction and the ability to form capillaries as reflected by tube formation. In cell culture, bevacizumab induced a 2 ~ 3 fold increase in endothelial cell apoptosis following radiation. In both SCC1 and H226 xenograft models, the concurrent administration of bevacizumab and radiation reduced tumor blood vessel formation and inhibited tumor growth compared to either modality alone. We observed a siginificant tumor reduction in mice receiving the combination of bevacizumab and radiation in comparison to mice treated with bevacizumab or radiation alone. We investigated the impact of bevacizumab and radiation treatment sequence on tumor response. In the SCC1 model, tumor response was strongest with radiation followed by bevacizumab with less sequence impact observed in the H226 model. CONCLUSIONS: Overall, these data demonstrate enhanced tumor response when bevacizumab is combined with radiation, supporting the emerging clinical investigations that are combining antiangiogenic therapies with radiation.     

Canstatin分泌增强体系的建立及其抗肺癌作用

目的:评估canstatin分泌增强体系在不同氧条件下的生物学效应,观察其对裸鼠肺癌模型的抗肿瘤作用。方法:基因重组技术构建canstatin分泌型载体,将其转染肿瘤细胞观察其在不同氧条件下的canstatin mRNA表达量,稳定转染的肺癌细胞与人脐静脉内皮细胞混合培养,观察混合培养对内皮细胞增殖、凋亡的效应。建立裸鼠肺癌移植瘤模型,将此体系转染荷瘤裸鼠,观察其对裸鼠移植瘤血管生成的作用,及肿瘤体积重量等指标,评估其抗肺癌效果。结果:成功构建canstatin缺氧增强分泌体系。缺氧条件下其转染的肺腺癌A549细胞canstatin mR-NA的表达显著增加。其稳定转染的A549细胞与人脐静脉内皮细胞HUVEC混合培养可使HUVEC生长增殖缓慢并大量凋亡,此体系转染的裸鼠移植瘤生长缓慢,瘤重、瘤体积及血管生成均显著低于对照组。(P<0.01)。结论:canstatin分泌增强体系在缺氧条件下能显著增加目的基因表达量及其生物学效应,并具有显著抑制肺癌生长和血管生成的作用。