Melanoma cells secrete follistatin, an antagonist of activin-mediated growth inhibition

Using a proteomic approach to screen for new growth factors released by melanoma cells, we identified follistatin as a major heparin-binding factor in medium conditioned by the Bowes melanoma cell line. Since follistatin is primarily studied in relation to its neutralization of activin, a member of the transforming growth factor-beta family of ligands, the expression and function of this receptor system was investigated in a panel of melanoma cell lines and melanocytes. All cell lines expressed activin receptors and showed phosphorylation of Smad signal transduction molecules upon treatment with activin. Secretion of follistatin, either native or after retroviral transduction, efficiently prevented Smad activation or activation of an activin-responsive luciferase reporter construct. In melanocytes, activin treatment led to growth inhibition and induction of apoptosis. These effects were counteracted by cotreatment with follistatin. In summary, we characterized the activin-activin receptor system in melanocytes and melanoma cell lines and found that secretion of follistatin by melanoma cells may represent an effective way to circumvent activin's negative regulatory effects.     

Antiproliferative effects of interferon-alphaCon1 on ovarian clear cell adenocarcinoma in vitro and in vivo.

PURPOSE: We examined the antiproliferative effect of IFN-alphaCon1 and its mechanism on ovarian clear cell adenocarcinoma in vitro and in vivo. EXPERIMENTAL DESIGN: (a) The effects of IFN-alphaCon1 on growth, morphology, cell cycle, and type I IFN-alpha receptor (IFNAR-2) expression were examined on two ovarian clear cell adenocarcinoma cell lines (KOC-5C and KOC-7C) in vitro. (b) KOC-5C or KOC-7C cells were transplanted into nude mice, and changes in tumor volume, tumor weight, apoptosis, necrosis, and microvessel density were investigated. The expression of angiogenesis factors was examined in the serum and the developed tumors. RESULTS: Both cell lines expressed IFNAR-2 mRNA, but its protein was detected only in KOC-7C. In KOC-7C cells, antiproliferative effects were observed in a time- and dose-dependent manner and cell division was blocked at the S phase. The KOC-7C tumors showed decreases in tumor volume and weight; a decreasing tendency in basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and interleukin (IL)-8 protein expression in the tumor; a significant decrease in bFGF and IL-8 protein expression in the serum, and of microvessel density; and significant increase in apoptosis and necrosis in the tumor. In the KOC-5C tumors, these in vitro and in vivo changes were not apparent, and the antiproliferative effects of IFN-alphaCon1 were not obvious. CONCLUSIONS: IFN-alphaCon1 suppresses tumor proliferation by inducing apoptosis, blocking the cell cycle, and inhibiting tumor angiogenesis. Our findings show that the clinical efficacy of IFN-alphaCon1 can be predicted by examining IFNAR-2 expression on tumor cells, and the efficacy of IFN-alphaCon1 treatment can be evaluated by measuring serum bFGF and IL-8 levels.     

Squalamine and cisplatin block angiogenesis and growth of human ovarian cancer cells with or without HER-2 gene overexpression

Angiogenesis is important for growth and progression of ovarian cancers. Squalamine is a natural antiangiogenic sterol, and its potential role in treatment of ovarian cancers with or without standard cisplatin chemotherapy was assessed. Since HER-2 gene overexpression is associated with cisplatin resistance in vitro and promotion of tumor angiogenesis in vivo, the response of ovarian cancer cells with or without HER-2 gene overexpression to squalamine and cisplatin was evaluated both in tumor xenograft models and in tissue culture. Ovarian cancer cells with or without HER-2 overexpression were grown as subcutaneous xenografts in nude mice. Animals were treated by intraperitoneal injection with control vehicle, cisplatin, squalamine or cisplatin combined with squalamine. At the end of the experiment, tumors were assessed for tumor growth inhibition and for changes in microvessel density and apoptosis. Additional in vitro studies evaluated effects of squalamine on tumor and endothelial cell growth and on signaling pathways in human endothelial cells. Profound growth inhibition was elicited by squalamine alone and by combined treatment with squalamine and cisplatin for both parental and HER-2-overexpressing ovarian tumor xenografts. Immunohistochemical evaluation of tumors revealed decreased microvessel density and increased apoptosis. Although HER-2-overexpressing tumors had more angiogenic and less apoptotic activity than parental cancers, growth of both tumor types was similarly suppressed by treatment with squalamine combined with cisplatin. In in vitro studies, we found that squalamine does not directly affect proliferation of ovarian cells. However, squalamine significantly blocked VEGF-induced activation of MAP kinase and cell proliferation in human vascular endothelial cells. The results suggest that squalamine is anti-angiogenic for ovarian cancer xenografts and appears to enhance cytotoxic effects of cisplatin chemotherapy independent of HER-2 tumor status.     

Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo

Survivin is highly expressed in most cancers, including glioblastoma, and it plays a significant role in inhibiting apoptosis and promoting tumor growth. Treatment of cancer cells with N-(4-hydroxyphenyl) retinamide (4-HPR) induces apoptosis through destabilization of mitochondrial membrane and activation of caspase-mediated apoptotic pathways. We studied the efficacy of a combination of survivin knockdown and 4-HPR treatment to induce apoptosis and inhibit invasion, angiogenesis, and growth of human glioblastomas in vitro and in vivo. Using a plasmid encoding survivin shRNA, we downregulated survivin in glioblastoma U251MG and U118MG cells and simultaneously treated with 1 µM 4-HPR for 48 hours. Cells following treatments were subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and invasion assays. In vivo angiogenesis and tumor regression studies were performed in nude mice. TUNEL assay demonstrated apoptosis in more than 80% of cells after survivin knockdown and 4-HPR treatment. Matrigel invasion assays demonstrated marked decreases in tumor cell invasion. In vivo angiogenesis studies depicted a remarkable inhibition of neovascularization due to the knockdown of survivin and 4-HPR treatment. Imaging of intracerebral tumorigenesis and longitudinal studies on subcutaneous solid tumor formation showed dramatic decreases in tumorigenesis and solid tumor progression, respectively, after treatment with the combination. Studies to elucidate the molecular mechanisms of the inhibition of angiogenesis and tumor regression demonstrated marked decreases in proliferating cell nuclear antigen, metalloproteinase-9, vascular endothelial growth factor, basic fibroblast growth factor, and CD31 in solid tumors. Our data demonstrated that survivin knockdown and concurrent 4-HPR treatment could be a novel therapeutic strategy for controlling growth of human glioblastomas.     

Inhibition of tumor growth and angiogenesis by soluble EphB4

EphB receptors and their ephrinB ligands play a key role in the formation of a regular vascular system. Recent studies have also shown the involvement of Eph/ephrin interactions in malignant tumor progression and angiogenesis. We have generated soluble monomeric EphB4 (sEphB4)-expressing A375 melanoma cells to study the effect of dominant negatively acting sEphB4 on tumor growth and angiogenesis. Soluble EphB4-expressing A375 tumors grown subcutaneously in nude mice show dramatically reduced tumor growth compared to control tumors. The proliferative capacity of sEphB4-expressing cells in monolayer culture is not altered. Yet, sEphB4-expressing A375 cells cannot establish proper cell-cell contacts in three-dimensional spheroids. However, sEphB4 transfectants have reduced proliferation and apoptosis rates when grown in three-dimensional culture in vitro or in subcutaneous tumors in vivo. Analysis of the vascular phenotype of the tumors revealed a reduction of intratumoral microvessel density in sEphB4-expressing tumors. Corresponding to these mouse experiments, a matched pair analysis of EphB4 and ephrinB2 expression in human colon carcinomas revealed significantly upregulated levels of EphB4 expression compared to adjacent normal tissue. Taken together, the data identify dual effects of sEphB4 on the tumor and the vascular compartment that collectively inhibit tumor growth.     

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.     

转化生长因子β促肿瘤侵袭转移的作用机制

转化生长因子β(TGF-β)在肿瘤形成、进展过程中具有双重作用.在肿瘤发生的起始阶段,TGF-β能抑制肿瘤细胞的增殖和诱导肿瘤细胞的凋亡.在肿瘤进展期,肿瘤细胞义能通过自分泌和旁分泌过量TGF-β促进自身的侵袭和转移.TGF-β促进肿瘤侵袭转移的机制主要有以下几个方面:①诱导上皮细胞-间允质转化;②增强肿瘤细胞的侵袭能力;③诱导肿瘤周围基质环境的改变;④促进肿瘤血管形成;⑤抑制宿主免疫监视系统杀伤肿瘤细胞.     

Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages

S1P has been proposed to contribute to cancer progression by regulating tumor proliferation, invasion, and angiogenesis. We developed a biospecific monoclonal antibody to S1P to investigate its role in tumorigenesis. The anti-S1P mAb substantially reduced tumor progression and in some cases eliminated measurable tumors in murine xenograft and allograft models. Tumor growth inhibition was attributed to antiangiogenic and antitumorigenic effects of the antibody. The anti-S1P mAb blocked EC migration and resulting capillary formation, inhibited blood vessel formation induced by VEGF and bFGF, and arrested tumor-associated angiogenesis. The anti-S1P mAb also neutralized S1P-induced proliferation, release of proangiogenic cytokines, and the ability of S1P to protect tumor cells from apoptosis in several tumor cell lines, validating S1P as a target for therapy.     

Follistatin overexpression in rodent liver tumors: a possible mechanism to overcome activin growth control

The activin-follistatin system is a potent growth regulatory system of liver tissue homeostasis. Activin A inhibits hepatocellular DNA synthesis and induces cell death. Follistatin binds activin and sequesters it from the signaling pathway. Consistently, follistatin has been reported to act as an inducer of DNA synthesis in the liver. Using RNase protection analysis, we studied the expression of follistatin in rat and mouse liver tumors as a possible mechanism to overcome activin growth control. Approximately 40% of the tumors (nine of 24 each), most of them hepatocellular carcinomas, displayed increased levels of follistatin mRNA when compared to tumor-surrounding liver tissue. The degree of overexpression was highly variable but independent of the carcinogen treatment that animals had received. It was also independent from the histological stage of malignancy and further found in rat liver adenomas. Follistatin expression was also observed in cell lines derived from human hepatocellular carcinomas. Overexpression of follistatin may represent a unique strategy of hepatic tumors to overcome the inhibitory action of a growth factor, activin, by decreasing its local bioavailability.     

Antiangiogenic treatment with the three thrombospondin-1 type 1 repeats recombinant protein in an orthotopic human pancreatic cancer model.

PURPOSE: This study investigates the antiangiogenesis and antitumor efficacy of a recombinant protein composed of the three type 1 repeats (3TSR) of thrombospondin-1 in an orthotopic human pancreatic cancer model and provides useful preclinical data for pancreatic cancer treatment. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (AsPC-1) were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR (3 mg per kg per day) or PBS for 3 weeks. Subsequently, the effects of 3TSR on tumor growth, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. The in vitro effects of 3TSR on human pancreatic cancer cells were also studied. RESULTS: 3TSR treatment significantly reduced angiogenesis and tumor growth of orthotopic pancreatic cancer. 3TSR-treated mice had a 69% reduction in tumor volume (316.6 +/- 79.3 versus 1,012.2 +/- 364.5 mm(3); P = 0.0001), and a significant increase in tumor necrotic area. After 3TSR treatment, both the vessel number and average microvessel size were significantly decreased, and microvessel density was decreased from 8.0% to 3.7% (P < 0.0001). The apoptotic rate of tumoral endothelial cells in 3TSR-treated tumors increased to 14.7% comparing to 4.2% in control tumors (P < 0.0001). 3TSR showed no direct effects on pancreatic cancer cell proliferation or apoptosis either in vivo or in vitro. CONCLUSION: 3TSR, a domain of a natural occurring angiogenesis inhibitor, showed potent therapeutic effect in pancreatic cancer by inhibiting tumor angiogenesis and may prove to be a promising agent for clinical pancreatic cancer treatment.     

Follistatin as potential therapeutic target in prostate cancer

Follistatin is a single-chain glycosylated protein whose primary function consists in binding and neutralizing some members of the transforming growth factor-β superfamily such as activin and bone morphogenic proteins. Emerging evidence indicates that this molecule may also play a role in the malignant progression of several human tumors including prostate cancer. In particular, recent findings suggest that, in this tumor, follistatin may also contribute to the formation of bone metastasis through multiple mechanisms, some of which are not related to its specific activin or bone morphogenic proteins’ inhibitory activity. This review provides insight into the most recent advances in understanding the role of follistatin in the prostate cancer progression and discusses the clinical and therapeutic implications related to these findings.     

Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis

Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T-cell immunity in progression. Comparing Gal-1-deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immunodeficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immunomodulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.     

Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors.

Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.     

Inhibition of angiogenesis by the antiepidermal growth factor receptor antibody ImClone C225 in androgen-independent prostate cancer growing orthotopically in nude mice.

In human androgen-independent prostate cancer (PCa), epidermal growth factor receptor (EGFR) regulates angiogenesis, tumor growth, and progression. In this study, we evaluated whether the blockade of EGFR by the anti-EGFR antibody ImClone C225 (IMC-C225) inhibited tumor growth and metastasis by inhibiting angiogenesis, and whether paclitaxel enhanced the results of therapy in androgen-independent PCa. PC-3M-LN4 PCa cells were implanted orthotopically in athymic nude mice and treated with i.p. IMC-C225 (1 mg twice a week) and/or paclitaxel (200 microg once a week). In vitro treatment of PC-3M-LN4 with IMC-C225 inhibited EGFR autophosphorylation without any significant antiproliferative effect. In contrast, in vivo therapy with IMC-C225 alone (P < 0.05) or in combination with paclitaxel (P < 0.005) significantly inhibited PCa growth and metastasis. Serum levels of interleukin (IL) 8 were lower after therapy, and IL-8 mRNA expression was down-regulated within the tumors after therapy. The down-regulation of IL-8 correlated with reduced microvessel density. IMC-C225 reduced tumor cell proliferation, enhanced p27(kip1) expression, and induced tumor and endothelial cell apoptosis. These studies indicate that IMC-C225 has significant antitumor effect in this murine model, mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. The simultaneous administration of paclitaxel enhanced this effect.     

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.     

Tumor cell and endothelial cell therapy of oral cancer by dual tyrosine kinase receptor blockade

Expression of the epidermal growth factor (EGF) and activation of its receptor (EGFR), a tyrosine kinase, are associated with progressive growth of head and neck cancer. Expression of the vascular endothelial growth factor (VEGF) is associated with angiogenesis and progressive growth of tumor. The tyrosine kinase inhibitor NVP-AEE788 (AEE788) blocks the EGF and VEGF signaling pathways. We examined the effects of AEE788 administered alone, or with paclitaxel (Taxol), on the progression of human head and neck cancer implanted orthotopically into nude mice. Cells of two different human oral cancer lines, JMAR and MDA1986, were injected into the tongues of nude mice. Mice with established tumors were randomized to receive three times per week oral AEE788, once weekly injected paclitaxel, AEE788 plus paclitaxel, or placebo. Oral tumors were resected at necropsy. Kinase activity, cell proliferation, apoptosis, and mean vessel density were determined by immunohistochemical immunofluorescent staining. AEE788 inhibited cell growth, induced apoptosis, and reduced the phosphorylation of EGFR, VEGFR-2, AKT, and mitogen-activated protein kinase in both cell lines. Mice treated with AEE788 and AEE788 plus paclitaxel had decreased microvessel density, decreased proliferative index, and increased apoptosis. Hence, AEE788 inhibited tumor vascularization and growth and prolonged survival. Inhibition of EGFR and VEGFR phosphorylation by AEE788 effectively inhibits cellular proliferation of squamous cell carcinoma of the head and neck, induces apoptosis of tumor endothelial cells and tumor cells, and is well tolerated in mice. These data recommend the consideration of patients with head and neck cancer for inclusion in clinical trials of AEE788.     

Antitumor treatment efficacy by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma model

Using an orthotopic intracerebral model from our established HM55-BGIV-101 tumor line, we investigated the antitumor effect on the angiogenesis and growth of human glioblastoma after treatment with monoclonal antibody DC101 against the vascular endothelial growth factor receptor-2 and monoclonal antibody C225 against the epidermal growth factor receptor. Nude mice bearing intracerebral glioblastoma xenografts were treated intraperitoneally with DC101 and C225 either alone or in combination. Histopathological analysis of solid tumor volume, satellite tumor number, microvessel density, tumor cell proliferation, and apoptosis was performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7 and 64%, respectively; tumor cell proliferative activity was reduced by 53.2% and the apoptotic index (AI) was increased by 66.7%; satellite tumor number was enhanced by 84.4%. C225 alone reduced satellite tumor number by 43.3%, but had no effect on solid tumor volume, microvessel density, tumor cell proliferation, and apoptosis. C225 combined with DC101 not only reduced solid tumor volume, microvessel density, tumor cell proliferative activity, and increased AI, but also reduced satellite tumor number. Inhibition of angiogenesis achieved by DC101 can cause increased tumor cell invasiveness. In our studies this increased tumor cell invasiveness was inhibited simultaneously by C225, which provides a theoretical basis for treatment of glioblastoma by the method of combining drugs with different pharmacological activity.     

Synergistic inhibition of angiogenesis and glioma cell-induced angiogenesis by the combination of temozolomide and enediyne antibiotic lidamycin

Present work mainly evaluated the inhibitory effects of lidamycin (LDM), an enediyne antibiotic, on angiogenesis or glioma-induced angiogenesis in vitro and in vivo, especially its synergistic anti-angiogenesis with temozolomide (TMZ). LDM alone efficiently inhibited proliferations and induced apoptosis of rat brain microvessel endothelial cells (rBMEC). LDM also interrupted the tube formation of rat brain microvessel endothelial cells (rBMEC) and rat aortic ring spreading. The blockade of rBMEC invasion and C6 cell-induced rBMEC migration by LDM was associated with decrease of VEGF secretion in a co-culture system. TMZ dramatically potentiated the effects of LDM on anti-proliferation, apoptosis induction, and synergistically inhibited angiogenesis events. As determined by western blot and ELISA, the interaction of tumor cells and the rBMEC was markedly interrupted by LDM plus TMZ with synergistic regulations of VEGF induced angiogenesis signal pathway, tumor cell invasion/migration, and apoptosis signal pathway. Immunofluorohistochemistry of CD31 and VEGF showed that LDM plus TMZ resulted in synergistic decrease of microvessel density (MVD) and VEGF expression in human glioma U87 cell subcutaneous xenograft. This study indicates that the high efficacy of LDM and the synergistic effects of LDM plus TMZ against glioma are mediated, at least in part, by the potentiated anti-angiogenesis.     

Fibulins 3 and 5 antagonize tumor angiogenesis in vivo

Lethal tumor growth and progression cannot occur without angiogenesis, which facilitates cancer cell proliferation, survival, and dissemination. Fibulins (FBLN) 5 and 3 are widely expressed extracellular matrix proteins that regulate cell proliferation in a context-specific manner. Reduced FBLN-5 expression has been associated with cancer formation and progression in humans, whereas its constitutive expression antagonizes endothelial cell angiogenic sprouting in vitro. Thus, FBLN-5 may suppress tumorigenesis by preventing tumor angiogenesis. FBLN-3 is homologous to FBLN-5 and expressed in endothelial cells, yet its role in tumorigenesis and angiogenesis is unknown. We find FBLN-3 expression to be altered in some human tumors and that its constitutive expression in endothelial cells inhibited their proliferation, invasion, and angiogenic sprouting, as well as their response to vascular endothelial growth factor as measured by p38 mitogen-activated protein kinase activation. In endothelial cells, both FBLNs (a) reduced angiogenic sprouting stimulated by basic fibroblast growth factor (bFGF); (b) inhibited matrix metalloproteinase expression and activity; and (c) stimulated tissue inhibitor of metalloproteinase expression. More importantly, both FBLNs prevented angiogenesis and vessel infiltration into bFGF-supplemented Matrigel plugs implanted in genetically normal mice, as well as decreased the growth and blood vessel density in tumors produced by MCA102 fibrosarcoma cells implanted s.c. into syngeneic mice. Our findings establish FBLN-3 and FBLN-5 as novel angiostatic agents capable of reducing tumor angiogenesis and, consequently, tumor growth in vivo and suggest that these angiostatic activities may one day be exploited to combat tumor angiogenesis and metastasis in cancer patients.     

Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various antiangiogenic effects including endothelial cell apoptosis.

We determined whether the IFN-beta gene could suppress angiogenesis, tumor growth, and metastasis of human bladder transitional cell carcinoma. The highly tumorigenic and metastatic 253J B-V(R) human bladder transitional cell carcinoma (TCC) cell line (resistant to the antiproliferative effects of IFN-beta) was infected in vitro with adenoviral beta-galactosidase (Ad-LacZ), murine adenoviral IFN-beta (Ad-mIFN-beta), or human adenoviral IFN-beta (Ad-hIFN-beta) and implanted into the bladders of athymic nude mice. Ad-mIFN-beta and Ad-hIFN-beta were used because of the species specificity of IFN-beta. The transient production of mIFN-beta and hIFN-beta from the infected 253JB-V(R) tumor cells significantly inhibited tumorigenicity and spontaneous lymph node metastasis. Subsequently, the 253J B-V(R) cells were implanted into the subcutis of athymic nude mice, and established tumors were treated by direct intratumoral injection with Ad-mIFN-beta, Ad-hIFN-beta, Ad-LacZ, or PBS. By in situ hybridization (ISH) and immunohistochemical analysis (IHC), expression of hIFN-beta and mIFN-beta mRNA and protein within the tumors was demonstrated after Ad-hIFN-beta and Ad-mIFN-beta gene therapy, respectively. The therapy also induced necrosis in both the Ad-mIFN-beta- and Ad-hIFN-beta-treated tumors. IHC revealed decreased tumor cell proliferation and the sequestration of activated macrophages within the tumors after Ad-mIFN-beta therapy. In addition, the expression of the proangiogenic factors bFGF, and MMP-9 protein (by IHC) was significantly down-regulated by Ad-hIFN-beta gene therapy. Double-immunofluorescent IHC for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and CD-31 demonstrated tumor and endothelial cell apoptosis in those tumors treated with Ad-hIFN-beta gene therapy. Tumor-induced angiogenesis, as determined by the microvessel density, was decreased in tumors treated with both Ad-mIFN-beta and Ad-hIFN-beta. These data suggest that the inhibition of tumorigenicity and the metastasis of the 253J B-V(R) cells after infection with Ad-IFN-beta is caused by the inhibition of angiogenesis and the activation of host effector cells.