The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin

In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.     

Roxithromycin and clarithromycin, 14-membered ring macrolides, potentiate the antitumor activity of cytotoxic agents against mouse B16 melanoma cells

We previously reported antiangiogenic activity of roxithromycin and clarithromycin, 14-membered ring macrolide antibiotics. In the present study, we examined the antitumor effects of roxithromycin and clarithromycin, alone and in combination with several cytotoxic drugs, on mouse B16BL6 melanoma cells in vivo and in vitro. Both roxithromycin and clarithromycin potentiated the inhibition of tumor growth induced by cyclophosphamide, cis-diamminedichloroplatinum(II), Adriamycin and vindesine in vivo. However, neither roxithromycin nor clarithromycin, altered the cytotoxicity of 4-hydroperoxycyclophosphamide, cis-diamminedichloroplatinum(II), Adriamycin or vindesine in an in vitro cell proliferation assay. These results suggest that the antiangiogenic activity of roxithromycin and clarithromycin may provide beneficial effects in combination with cytotoxic therapies against solid tumors.     

Combination of thalidomide and cisplatin in an head and neck squamous cell carcinomas model results in an enhanced antiangiogenic activity in vitro and in vivo

Thalidomide is an immunomodulatory, antiangiogenic drug. Although there is evidence that it might be more effective in combination with chemotherapy the exact mechanism of action is unclear. Therefore, we investigated its effect in combination with metronomically applied cisplatin in a xenotransplant mouse model characteristic for advanced head and neck squamous cell carcinomas, its possible synergistic action in vitro, and which tumor-derived factors might be targeted by thalidomide. Although thalidomide alone was ineffective, a combined treatment with low-dose cisplatin inhibited significant tumor growth, proliferation and angiogenesis in vivo as well as migration and tube formation of endothelial cells in vitro. Noteworthy, the latter effect was enhanced after coapplication of cisplatin in nontoxic doses. An inhibitory effect on tumor cell migration was also observed suggesting a direct antitumor effect. Although thalidomide alone did not influence cell proliferation, it augmented antiproliferative response after cisplatin application emphasizing the idea of a potentiated effect when both drugs are combined. Furthermore, we could show that antiangiogenic effects of thalidomide are related to tumor-cell derived factors including vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and Il-8 some known and with, granulocyte colony stimulating growth factor and granulocyte macrophage colony stimulating growth factor, some new target molecules of thalidomide. Altogether, our findings reveal new insights into thalidomide-mediated antitumor and antiangiogenic effects and its interaction with cytostatic drugs.     

Introduction of midkine gene into human bladder cancer cells enhances their malignant phenotype but increases their sensitivity to antiangiogenic therapy.

PURPOSE: Midkine (MK) is a member of a family of heparin-binding growth factors, which was reported to have an important role in angiogenesis. Although MK was reported to be associated with bladder cancer progression, the functional significance of MK expression in bladder cancer progression has not been elucidated. The objectives of this study were to determine whether overexpression of MK in bladder cancer cells enhances their malignant potential and to evaluate the inhibitory effect of the antiangiogenic agent TNP-470 on the growth of MK-overexpressing bladder cancer cells in vivo. EXPERIMENTAL DESIGN: We introduced the MK gene into human bladder cancer UM-UC-3 cells that do not secrete a detectable level of MK protein and generated the MK-overexpressing cell line UM-UC-3/MK. The biological activity of secreted MK was evaluated using a human umbilical vein endothelial cell proliferation assay. To investigate the in vivo effects of MK overexpression on tumor growth, each cell line was injected s.c. and orthotopically into nude mice. To evaluate the therapeutic effects of the antiangiogenic agent, mice were given TNP-470 after s.c. injection of each cell line. The microvessel density of tumors was quantitated by immunohistochemistry of CD31. RESULTS: The heparin affinity-purified conditioned media of UM-UC-3/MK cells significantly enhanced human umbilical vein endothelial cell proliferation. MK expression had no effect on in vitro growth but conferred a growth advantage on both s.c. and orthotopic tumors in vivo. Furthermore, enhanced tumor growth was closely associated with increased microvessel density. Significant inhibition of tumor growth by TNP-470 treatment was observed only in UM-UC-3/MK tumors and not in control tumors. CONCLUSIONS: We demonstrated that overexpression of the MK gene causes an increase in the angiogenic activity of cells through vascular endothelial cell growth, resulting in enhanced malignant potential of human bladder cancer cells. Moreover, the present findings suggest that TNP-470 could be used as a novel therapeutic adjunct to conventional agents for patients with advanced bladder cancer overexpressing MK.     

Antiangiogenic therapy with mammalian target of rapamycin inhibitor RAD001 (Everolimus) increases radiosensitivity in solid cancer.

PURPOSE: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth. EXPERIMENTAL DESIGN: Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 x 2 or 5 x 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy. RESULTS: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83%), L3.6pl (-8%), and CT-26 (-82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 x 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm(3) versus 1144 mm(3); CT-26: 210 mm(3) versus 636 mm(3); P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy. CONCLUSION: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.     

Antiangiogenic and antitumor activity of IDN 5390, a new taxane derivative.

PURPOSE: We previously reported that paclitaxel, a microtubule-stabilizing drug, inhibited angiogenesis, mainly by inhibiting endothelial cell motility (D. Belotti et al., Clin. Cancer Res., 2: 1843-1849, 1996). The aim of this study was to select a taxane with little cytotoxicity but with antimotility and hence antiangiogenic activity. EXPERIMENTAL DESIGN: Different taxanes, seco derivatives, and 14-beta-hydroxy-10-deacetyl baccatin III derivatives were tested for their effects on the proliferation and motility of human umbilical vein endothelial cells. The antiangiogenic and antineoplastic activities of the compound selected from this screening were further investigated in experimental models in vitro and in vivo. RESULTS: From the screening of different taxanes, we selected IDN 5390, a seco derivative that showed potent antimotility activity and less cytotoxicity than paclitaxel. In comparable experimental conditions, IDN 5390 inhibited endothelial cell migration without affecting proliferation. This compound dose-dependently inhibited the capacity of human umbilical vein endothelial cells plated on Matrigel to organize into a network of cords. In vivo, IDN 5390 significantly inhibited fibroblast growth factor-2-induced angiogenesis in Matrigel implants. Daily treatment with IDN 5390 in mice bearing established lung micrometastases from the B16BL6 murine melanoma caused a reduction in the size of metastases. Finally, IDN 5390 slowed the s.c. growth of the paclitaxel-resistant human ovarian carcinoma, 1A9/PTX22, xenografted in nude mice. CONCLUSIONS: The seco derivative IDN 5390 might represent the prototype of a new class of taxane derivatives with antiangiogenic properties.     

Augmentation of radiation response with the vascular targeting agent ZD6126

PURPOSE: To examine the antivascular and antitumor activity of the vascular targeting agent ZD6126 in combination with radiation in lung and head-and-neck (H and N) cancer models. The overall hypothesis was that simultaneous targeting of tumor cells (radiation) and tumor vasculature (ZD6126) might enhance tumor cell killing. METHODS AND MATERIALS: A series of in vitro studies using human umbilical vein endothelial cells (HUVEC) and in vivo studies in athymic mice bearing human lung (H226) and H and N (squamous cell carcinoma [SCC]1, SCC6) tumor xenografts treated with ZD6126 and/or radiation were performed. RESULTS: ZD6126 inhibited the capillary-like network formation in HUVEC. Treatment of HUVEC with ZD6126 resulted in cell cycle arrest in G2/M, with decrease of cells in S phase and proliferation inhibition in a dose-dependent manner. ZD6126 augmented the cell-killing effect of radiation and radiation-induced apoptosis in HUVEC. The combination of ZD6126 and radiation further decreased tumor vascularization in an in vivo Matrigel angiogenesis assay. In tumor xenografts, ZD6126 enhanced the antitumor activity of radiation, resulting in tumor growth delay. CONCLUSIONS: These preclinical studies suggest that ZD6126 can augment the radiation response of proliferating endothelial H and N and lung cancer cells. These results complement recent reports suggesting the potential value of combining radiation with vascular targeting/antiangiogenic agents.     

Cotargeting tumor and tumor endothelium effectively inhibits the growth of human prostate cancer in adenovirus-mediated antiangiogenesis and oncolysis combination therapy

Tumor-endothelial interaction contributes to local prostate tumor growth and distant metastasis. In this communication, we designed a novel approach to target both cancer cells and their "crosstalk" with surrounding microvascular endothelium in an experimental hormone refractory human prostate cancer model. We evaluated the in vitro and in vivo synergistic and/or additive effects of a combination of conditional oncolytic adenovirus plus an adenoviral-mediated antiangiogenic therapy. In the in vitro study, we demonstrated that human umbilical vein endothelial cells (HUVEC) and human C4-2 androgen-independent (AI) prostate cancer cells, when infected with an antiangiogenic adenoviral (Ad)-Flk1-Fc vector secreting a soluble form of Flk1, showed dramatically inhibited proliferation, migration and tubular formation of HUVEC endothelial cells. C4-2 cells showed maximal growth inhibition when coinfected with Ad-Flk1-Fc and Ad-hOC-E1, a conditional replication-competent Ad vector with viral replication driven by a human osteocalcin (hOC) promoter targeting both prostate cancer epithelial and stromal cells. Using a three-dimensional (3D) coculture model, we found that targeting C4-2 cells with Ad-hOC-E1 markedly decreased tubular formation in HUVEC, as visualized by confocal microscopy. In a subcutaneous C4-2 tumor xenograft model, tumor volume was decreased by 40-60% in animals treated with Ad-Flk1-Fc or Ad-hOC-E1 plus vitamin D3 alone and by 90% in a combined treatment group, compared to untreated animals in an 8-week treatment period. Moreover, three of 10 (30%) pre-established tumors completely regressed when animals received combination therapy. Cotargeting tumor and tumor endothelium could be a promising gene therapy strategy for the treatment of both localized and metastatic human prostate cancer.     

Rapamycin-induced endothelial cell death and tumor vessel thrombosis potentiate cytotoxic therapy against pancreatic cancer.

PURPOSE: Despite current chemotherapies, pancreatic cancer remains an uncontrollable, rapidly progressive disease. Here, we tested an approach combining a recently described antiangiogenic drug, rapamycin, with standard gemcitabine cytotoxic therapy on human pancreatic tumor growth. EXPERIMENTAL DESIGN: Tumor growth was assessed in rapamycin and gemcitabine-treated nude mice orthotopically injected with metastatic L3.6pl human pancreatic cancer cells. H&E staining was performed on tumors, along with Ki67 staining for cell proliferation and immunohistochemical terminal deoxynucleotidyl transferase-mediated nick end labeling and CD31 analysis. Rapamycin-treated tumor vessels were also directly examined in dorsal skin-fold chambers for blood flow after thrombosis induction. Cell death in human umbilical vein endothelial cells was assessed by flow cytometry after annexin-V staining. RESULTS: Rapamycin therapy alone inhibited tumor growth and metastasis more than gemcitabine, with remarkable long-term tumor control when the drugs were combined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage and thrombosis with rapamycin treatment. Indeed, dorsal skin-fold chamber analysis of rapamycin-treated tumors showed an increased susceptibility of tumor-specific vessels to thrombosis. Furthermore, terminal deoxynucleotidyl transferase-mediated nick end labeling/CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in vitro. In contrast, gemcitabine was not antiangiogenic and, despite its known cytotoxicity, did not reduce proliferation in orthotopic tumors; nevertheless, rapamycin did reduce tumor proliferation. CONCLUSIONS: Our data suggest a novel mechanism whereby rapamycin targets pancreatic tumor endothelium for destruction and thrombosis. We propose that rapamycin-based vascular targeting not only reduces tumor vascularization, it decreases the number of proliferating tumor cells to be destroyed by gemcitabine, thus introducing a new, clinically feasible strategy against pancreatic cancer.     

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.     

Petasiphenol which inhibits DNA polymerase lambda activity is an inhibitor of in vitro angiogenesis

Petasiphenol, a polyphenolic compound from a Japanese vegetable (Petasites japonicus) which is a DNA polymerase lambda selective inhibitor, was demonstrated as a potent antiangiogenic agent in this study. Petasiphenol showed suppressive effects on in vitro angiogenesis assays, human umbilical vein endothelial cell (HUVEC) proliferation, tube formation on reconstituted basement membrane and chemotaxis in a dose-dependent manner (10-100 microM). However, treatment of HUVEC with petasiphenol did not affect 72 kDa matrix metalloprotease activity at these concentrations. Petasiphenol at higher 50 microM also suppressed microvessel outgrowth in ex vivo angiogenesis assay using a rat aortic ring. Taken together, petasiphenol could act as a potent antiangiogenic compound for preventing tumor and be useful to design therapeutic agents.     

Selenium-induced inhibition of angiogenesis in mammary cancer at chemopreventive levels of intake

The trace element nutrient selenium (Se) has been shown to possess cancer-preventive activity in both animal models and humans, but the mechanisms by which this occurs remain to be elucidated. Because angiogenesis is obligatory for the genesis and growth of solid cancers, we investigated, in the study presented here, the hypothesis that Se may exert its cancer-preventive activity, at least in part, by inhibiting cancer-associated angiogenesis. The effects of chemopreventive levels of Se on the intra-tumoral microvessel density and the expression of vascular endothelial growth factor in 1-methyl-1-nitrosourea-induced rat mammary carcinomas and on the proliferation and survival and matrix metalloproteinase activity of human umbilical vein endothelial cells in vitro were examined. Increased Se intake as Se-enriched garlic, sodium selenite, or Se-methylselenocysteine led to a significant reduction of intra-tumoral microvessel density in mammary carcinomas, irrespective of the manner by which Se was provided: continuous exposure (7-wk feeding) with a chemoprevention protocol or acute bolus exposure (3 d) after carcinomas had established. Compared with the untreated controls, significantly lower levels of vascular endothelial growth factor expression were observed in a sizeable proportion of the Se-treated carcinomas. In contrast to the mammary carcinomas, the microvessel density of the uninvolved mammary glands was not altered by Se treatment. In cell culture, direct exposure of human umbilical vein endothelial cells to Se induced cell death predominantly through apoptosis, decreased the gelatinolytic activities of matrix metalloproteinase-2, or both. These results indicate a potential for Se metabolites to inhibit key attributes (proliferation, survival, and matrix degradation) of endothelial cells critical for angiogenic sprouting. Therefore, inhibition of angiogenesis associated with cancer may be a novel mechanism for the anticancer activity of Se in vivo, and multiple mechanisms are probably involved in mediating the anti-angiogenic activity.     

Combination strategy targeting the hypoxia inducible factor-1 alpha with mammalian target of rapamycin and histone deacetylase inhibitors.

PURPOSE: The hypoxia-inducible factor-1 alpha (HIF-alpha) is a key regulator of tumor angiogenesis. Mammalian target of rapamycin (mTOR) and histone deacetylase (HDAC) inhibitors suppress tumor-induced angiogenesis by reducing tumor HIF-1 alpha protein expression. Thus, we hypothesized that combination treatment of rapamycin and the HDAC inhibitor LBH589 has greater antiangiogenic and antitumor activity compared with single agents. EXPERIMENTAL DESIGN: To evaluate the effect of LBH589 and rapamycin on HIF-1 alpha in human prostate PC3, renal C2 carcinoma cell lines, and endothelial cells (human umbilical vein endothelial cells), we did Western blot analysis. To determine the antitumor activity of LBH589 and rapamycin, cell proliferation assays and xenograft experiments were conducted. RESULTS: Western blotting showed that combination treatment of human umbilical vein endothelial cells, C2 and PC3, significantly reduced HIF-1 alpha protein expression compared with single agents. Treatment with rapamycin resulted in inhibition of the downstream signals of the mTOR pathway and increased phosphorylation of Akt in C2 cells, whereas the constitutively activated Akt in PC3 cells was not modulated. LBH589 decreased both constitutively expressed and rapamycin-induced phosphorylated Akt levels in PC3 and C2 cell lines. In clonogenic assays, the combination treatment had a greater inhibitory effect in PC3 cells (93 +/- 1.4%) compared with single agents (66 +/- 9% rapamycin and 43 +/- 4% LBH589). Combination of rapamycin and LBH589 significantly inhibited PC3 and C2 in vivo tumor growth and angiogenesis as measured by tumor weight and microvessel density. CONCLUSIONS: Combination treatment of mTOR and HDAC inhibitors represents a rational therapeutic strategy targeting HIF-1 alpha that warrants clinical testing.     

Synergistic antitumor effect of antiangiogenic factor genes on colon 26 produced by low-voltage electroporation

Antiangiogenic factors are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vitro and tumor growth in mice. We have demonstrated the synergistic antitumor effect of antiangiogenic genes (mouse angiostatin: pBLAST-mAngio; and mouse endostatin: p-BLAST42-mEndo XV) delivered to tumors by low-voltage electroporation in mouse colon 26 models. A synergistic antitumor effect was strongly suggested by in vivo tumor growth kinetics, as well as in survival studies with the mice. RT-PCR confirmed that the fragments of each gene were transferred by low-voltage electroporation in the tumor. Decreased microvessel density measurements in tumors also confirmed the efficacy of the synergistic antitumor effect of both genes. Significant growth inhibition was observed in mice treated with a 1:1 proportion of angiostatin and endostatin genes, and the order of the both genes transferred (first the endostatin gene, followed 1 week later by the angiostatin gene) had a profound inhibitory effect on tumor growth. These data suggest that in vivo delivery of antiangiogenic genes with low-voltage electroporation could be a possible therapeutic strategy for established solid tumors when both genes were applied in combination.     

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis

BACKGROUND: The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. METHODS: The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. RESULTS: GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. CONCLUSIONS: In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.     

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.     

Antiangiogenic and antimetastatic effects of toremifene citrate

In this study, we demonstrated that toremifene citrate (TOR) inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVEC) in vitro. Moreover, TOR suppressed angiogenesis in rabbit cornea and lung metastasis of human fibrosarcoma HT-1080 cells in nude mice. The antiangiogenic activity in vitro was apparent at the concentration of 5 microM which is clinically achievable by oral administration of 120 mg/kg of TOR. These results suggest that clinical treatment with 120 mg/day of TOR might be expected to exhibit antiangiogenesis and antimetastasis effects, in addition to inhibition of estrogen-dependent tumor cell growth.     

AKT/NF-kappaB inhibitor xanthohumol targets cell growth and angiogenesis in hematologic malignancies

BACKGROUND: Leukemias are dependent on Akt/NF-kappaB activation and angiogenesis. METHODS: The antiangiogenic Akt/NF-kappaB inhibitor xanthohumol (XN) has in vitro activity against acute and chronic myelogenous leukemia cell lines (AML, CML) and fresh samples from patients were investigated. RESULTS: Inhibition of cell proliferation is associated with induction of apoptosis and reduced VEGF secretion. Decreased cell invasion, metalloprotease production, and adhesion to endothelial cells observed in the presence of XN could prevent in vivo life-threatening complications of leukostasis and tissue infiltration. CONCLUSIONS: As endothelial cells and hematopoietic cells are mutually correlated in their development and growth, targeting both tumor cells and endothelial cells with agents possessing cytotoxic and antiangiogenic activities may lead to synergistic antitumor effects interrupting a reciprocal stimulatory loop between leukemia and endothelial cells.