Tumor Endothelial Cell Proliferation Research Articles

Erythropoietin Receptor Expression Is a Potential Prognostic Factor in Human Lung Adenocarcinoma

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.

A phase II study of trebananib (AMG 386) and abiraterone in metastatic castration resistant prostate cancer.

TPS5102 Background: Preclinical studies support the use of an antiangiogenic approach in the treatment of prostate cancer. Trebananib is a novel peptide-Fc fusion protein that sequesters angiopoeitin 1 and angiopoeitin 2, thereby preventing their interaction with their common receptor Tie2, and inhibiting tumor endothelial cell proliferation and tumor growth. Trebananib is currently in Phase 3 trials for the treatment of ovarian carcinoma and has been shown to have clinical activity in multiple tumor types. Previous studies have demonstrated that in vivo alterations of testosterone levels regulate the expression of vascular endothelial growth factor, fibroblast growth factor, and angiopoietin associated factors. Dual inhibition of the androgen and angiogenic axis represents a novel strategy of combined targeted therapy for patients with metastatic castration-resistant prostate cancer (mCRPC). We hypothesize that the addition of trebananib to CYP17 inhibitor abiraterone and prednisone will increase the median progression free survival (PFS) in chemotherapy-naïve mCRPC. Methods: This phase 2 study will evaluate the treatment effect as measured by progression free survival in patients treated with trebananib plus abiraterone/prednisone relative to abiraterone/prednisone alone. 72 patients with progressive, mCRPC will be randomized 1:1 to either study arm. Trebananib is administered intravenously every week, on days 1, 8, 15 and 22 of each 28-day cycle. Abiraterone acetate is taken once daily with prednisone 5 mg twice daily. We have completed the initial run-in phase of trebananib at 15mg/kg and 30mg/kg. The randomized phase of the study will use the 30 mg/kg dose of trebananib with the standard dose (1000 mg) of abiraterone. The primary end point is radiographic PFS. Secondary end points include overall survival, changes in genetic biomarkers related to the androgen and angiogenesis signaling axis, molecular markers of angiogenesis, circulating tumor cells and androgen receptor signaling status in circulating tumor cells before and after treatment. This combination of angiogenesis inhibition and abiraterone has the potential to improve clinical outcomes in front-line therapy for mCRPC. Clinical trial information: NCT01553188.

Abstract 2797: AMG 511, a potent and selective class I PI3K inhibitor, demonstrates anti-tumor activity in multiple xenograft models

Abstract Background: Constitutive activation of the phosphoinositide 3-kinases (PI3K) signaling pathway is a frequent event in many cancers and results in increased cell growth and survival. Therefore, PI3K is an attractive target for oncology. This study evaluated the correlation between the pharmacokinetics (PK) and pharmacodynamics (PD) of AMG 511, a potent and selective class I PI3K inhibitor, and anti-tumor activity in preclinical models of human cancer which harbor mutations in the PI3K pathway. Method: To determine the PD, PK, and efficacy relationship of AMG 511, U87 MG (PTEN null), HCT-116 (PI3K & KRAS mutant), or BT474 (PI3K mutant, HER2 amplified) tumor bearing mice were treated with up to 10 mpk AMG 511 daily by oral gavage. For tumor PD time course and dose response studies, AKT phosphorylation (pAKT) was measured using an electrochemiluminescence detection assay after a single-dose of AMG 511 in U87 MG tumor bearing mice. To determine in vivo efficacy, mice bearing established U87 MG, HCT-116, or BT474 tumors were treated daily and measured 2x/week. The mechanism of action of AMG 511 was determined by examining effects on cell proliferation (BrdU incorporation) and apoptosis (caspase staining) using a combination of immunohistochemistry and flow cytometry analyses of ex-vivo U87 MG xenograft tumors following treatment with AMG 511. Results: Inhibition of pAKT in U87 MG tumor tissue was associated with increased plasma concentration of AMG 511 after a single dose (EC50=59 ng/mL). Treatment with 0.3, 1, and 3 mpk AMG 511 inhibited pAKT in tumor tissue by 39, 81, and 92 %, respectively. Treatment with 10 mpk AMG 511 significantly inhibited pAKT up to 16 hrs (p<0.01). A dose-dependent inhibition of xenograft growth was observed with AMG 511 versus vehicle control in multiple tumor xenograft models with PI3K pathway mutations (p<0.03). Flow cytometry analysis demonstrated that AMG 511 treatment resulted in a dose-dependent decrease of BrdU incorporation in tumor and CD31 positive endothelial cells at peak and trough plasma levels (p<0.05). Immunohistochemical analysis confirmed that AMG 511 treatment induced tumor cell apoptosis as measured by caspase staining in U87 MG tumor samples. Conclusion: AMG 511 is a potent pan-PI3K inhibitor, which has significant anti-tumor activity in human tumor xenograft models which harbor PI3K pathway mutations. Tumor growth inhibition is the result of an increase in AMG 511 exposure, a reduction in PI3K signaling, an inhibition of tumor cell and endothelial cell proliferation and an induction of tumor cell apoptosis. These data together provide evidence for the potential clinical investigation of AMG 511 in patients whose tumors harbor mutations in the PI3K pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2797. doi:1538-7445.AM2012-2797

Abstract LB-3: Evidence that dibenzofuran based anti-angiogenic, anti-tumor peptidomimetics target galectin-1

Abstract Galectins, named for their selective binding to ß-galactoside-containing glycans, play crucial roles in many physiological as well as pathological processes. Galectin-1 (gal-1) in particular, has been shown to be differentially elevated in tumor endothelial cells in several cancers including cancer of the breast, colon, prostate and ovaries. Gal-1 targeting 33mer peptide, Anginex, inhibits tumor endothelial cell proliferation via anoikis and attenuates tumor angiogenesis and tumor growth. Based on structure-activity relationships of anginex, partial-peptide mimetic were designed, using dibenzofuran (DBF) as a ß-turn mimetic to maintain the two key ß-strands and the bioactive amphipathic ß-sheet conformation of the peptide. The best DBF-based compound 6DBF7 (13mer) inhibited tumor angiogenesis and tumor growth in vivo better than parent anginex. Here we report on the optimization of the anti-angiogenic activity of 6DBF7 analogs in vitro, by substitution of natural and non-natural amino acids, and subsequently the antiangiogenic and anti-tumor effects of the most promising analogs in vivo. In addition, by using NMR spectroscopy and flow cytometry, we demonstrate that the lead 6DBF7 analogs interact with galectin-1 at a site near to its carbohydrate binding domain and by doing so can compete off galectin-1 from binding to ß-galactoside-containing glycans on the surface of cells in vitro. Overall, this research contributes to our ability to design pharmaceutical agents that target galectin-1 and thereby inhibit tumor growth and are potentially clinical candidates for use in the management of human cancers. This work was supported by NIH grant CA096090 to KHM Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-3. doi:1538-7445.AM2012-LB-3

Soluble CD44 Interacts with Intermediate Filament Protein Vimentin on Endothelial Cell Surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12–37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.

Abstract B9: Evidence that the small molecule antiangiogenic agent PTX008 targets galectin-1.

Abstract Inhibition of angiogenesis is a promising avenue for the treatment of cancer in the clinic. Although many or most angiostatic compounds being developed are inhibitors related to various components of growth factor pathways, these have had limited success in the clinic, and new angiostatic compounds that target different systems are sorely needed. Currently, there is growing evidence indicating the importance of galectin-1 in tumor angiogenesis. Galectins are proteins that generally bind β-galactoside-containing glycoconjugates and are involved in various pathological disorders like tumor endothelial cell adhesion, proliferation and migration. Recently, we designed a non-peptidic, calixarene-based galectin-1 antagonist, PTX008, as a relatively small, protein surface topomimetic of the antiangiogenic peptide anginex. Although we had demonstrated that PTX008 inhibits tumor angiogenesis and tumor growth in vivo, we have yet to confirm that it indeed targets galectin-1. Here, we do just that. By using NMR spectroscopy and flow cytometry, we demonstrate that PTX008 interacts with galectin-1 at a site near to its carbohydrate binding domain and by doing so can compete off galectin-1 from binding to β-galactoside-containing glycans on the surface of cells in vitro. Overall, this research contributes to our ability to design pharmaceutical agents that target galectin-1 and thereby inhibit tumor growth in animals and potentially in the clinic. This work was supported by a research grant from the NIH National Cancer Institute (CA-096090) to KHM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B9.

Sequential therapy with sunitinib and sorafenib in metastatic hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third most common cause of cancer-related death [1]. More than 75% of cases occur in the Asian-Pacific region, largely in association with chronic hepatitis B virus (HBV) infection, and 360,000 patients in Eastern Asia die from this disease each year [1]. Diagnosed at early stages, it is potentially curable by surgery and by locoregional procedures. However, disease that is diagnosed at an advanced stage or continues to progress after locoregional therapy has a dismal prognosis because of underlying liver cirrhosis and lack of effective alternative treatments. Sorafenib (Nexavar; Bayer, West Haven, CT, USA and Onyx, Emeryville, CA, USA) is an oral multitargeted tyrosine kinase inhibitor (TKI) and the first agent shown to improve overall survival (OS) in advanced HCC [2, 3]. Sorafenib inhibits angiogenesis by targeting receptors for VEGF (VEGFR2 and VEGFR3) and blocks tumor cell proliferation by inhibiting RAF/MEK/ERK signaling [4]. This combined inhibition of VEGF and PDGF pathways decreases tumor vascularization and endothelial cell proliferation and thus retards tumor progression. Sunitinib malate (Sutent; Pfizer, New York, NY, USA) is another oral, multitargeted TKI that acts on VEGFR-1, VEGFR-2, and VEGFR-3, PDGFR-α and PDGFR-β, and partially overlaps with sorafenib in its target inhibition profile [5]. Sunitinib is approved for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumors (GIST) [6]. Observational phase II studies suggest that sunitinib has antitumor activity in patients with advanced HCC, and a multicenter phase III clinical trial to compare its effectiveness with that of sorafenib in advanced HCC is in progress. Improving treatment outcomes for patients with advanced HCC requires development of alternative agents and regimens that are active, tolerable and safe. Further knowledge of drug mechanisms, and of biomarkers to predict tumor response and/or resistance are also needed. At present we have no standardized second-line regimen in HCC patients after progression on first line sorafenib. In metastatic RCC patients, sorafenib and sunitinib are currently used in sequence. Observational studies on the efficacy and cross-resistance of these drugs in RCC patients failed to define optimal strategy of sequential treatment [7– 13], however data for sequential treatment with these two TKIs in advanced HCC patients are lacking. We performed this retrospective study to evaluate the efficacy and safety of sequential administration of sunitinib following failure of sorafenib in advanced HCC patients.

Abstract 3493: Expression of VEGFR and FRGFR on endothelial cells isolated from hepatocellular carcinoma and adjacent liver tissue

Abstract Background Heterogenity in gene expression profiles in tumor endothelial cells (TEC) has been reported reported. Our previous studies showed TEC is also different from endothelial cells isolated from adjacent normal liver tissue (NEC) in term of response to chemotherapeutics and anti-angiogenesis drugs. Methods TEC and NEC were isolated by CD31 and CD105 antibody conjuaged magnetic beads from tumor and adjacent nontumor liver tissues from 5 patients who were diagnosed as hepatocellular carcinoma with a positive alpha fetoprotein (> 20ng/ml). The purity of ECs was proved by LDL internalization assay, tube formation, immunostaining and RT-PCR to exclude tumor cell contamination. VEGFR1, VEGFR2, FGFR-2, CD31 and CD105 expression on the isolated ECs were assayed by flow cytometry within 3-5 passages. Response of different types of ECs to sorafenib treatment was observed, including cell proliferation, VEGFRs and FGFR expression. Results The isolated ECs were proved by tumor formation and LDL internalization. More than 97% cells are LDL positive. AFP was not detectable on the isolated ECs in both flow cytometry assay and immunostaining assay. VEGFRs and FGFR expression were different on CD105+TEC and CD31+TEC among different patients, ranging from 18% to 67% for VEGFR1, 1% to 8% for VEGFR2, and 18% to 25% for FGFR-2 on CD105+TEC, and 18% to 90% for VEGFR1, 10% to 40% for VEGFR2, and 18% to 30% for FGFR-2 on CD31+TEC. Expression of VEGFRs and FGFR is higher on CD105+ECs than those on CD31+ECs. While between TEC and NEC, the expression of VEGFRs and FGFR is also heterogeneous. Sorafenib at 5 mM inhibted proliferation of TEC and NEC in a dose-dependent manner, while NEC is more sensitive to sorafenib treatment. Conclusions VEGFRs and FGFR expression were different among different patients; CD105+EC and CD31+EC are probably two types of ECs with different biological features. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2011-3493

Effect of recombinant human endostatin on antitumor efficacy of paclitaxel/gemcitabine/cisplatin (TGP) chemotherapy in the treatment of advanced urothelial carcinoma.

265^ Background: Rh-endostatin is a new recombinant human endostatin that can inhibit tumor endothelial cell proliferation, angiogenesis, and tumor growth in the pre-clinical studies. A phase III study revealed that rh-endostatin in combination with chemotherapy shows a synergic activity in advanced NSCLC patients. This study aims to evaluate the response, median progression-free survival (PFS), overall survival time (OS), and safety of the regimen, paclitaxel/gemcitabine/cisplatin (TGP) plus rh-endostatin, in patients with advanced urothelial transitional carcinoma. Methods: The phase II trial included 35 patients with histologically diagnozed advanced or metastatic urothelial carcinoma, ECOG performance status 0-1, and GFR>60ml/min. TGP chemotherapy plus rh-endostatin as a first line treatment included: paclitaxel (80 mg/m2, d1&8), gemcitabine (1000 mg/m2, d1&8), cisplatin (30 mg/m2, d1 to 3) and rh-endostatin (15mg, d1 to 14, every 21d). For every 2 cycles, patients were evaluated for progression or response. Patients who had achieved an objective response (complete or partial) or who had stable disease were further treated for two cycles. Results: The overall objective response rate was 62.8% (95% confidence interval [CI, 46.8% to 78.8%]), including 4 (11.4%) complete response (CR) and 18 (51.4%) partial responses (PRs). Six patients (17.1%) had stable disease (SD), and 7 (20.0%) had progression disease (PD). The disease control rate was 80.0%. The median progression-free survival and overall survival have not been reached. The most common adverse events included gastrointestinal reaction, alopecia, and myelosuppression. Grade 3/4 adverse events, including thrombocytopenia in 42.9% (15/35) of patients and leucopenia in 34.3% (12/35) of patients, were observed. Conclusions: The addition of rh-endostatin to TGP regimen for advanced urothelial carcinoma patients might have higher response rate. Rh-Endostatinin combination with TGP chemotherapy showed a synergic activityand a favorable toxic profile in advanced urothelial carcinoma patients. No significant financial relationships to disclose.

A randomized phase II study of recombinant human endostatin plus gemcitabine/cisplatin compared with gemcitabine/cisplatin alone as first-line therapy in advanced non-small-cell lung cancer

Studies indicate that recombinant human endostatin (rh-endostatin) can inhibit tumor endothelial cell proliferation, angiogenesis, and tumor growth. This study assessed the efficacy of the combination of standard gemcitabine plus cisplatin chemotherapy with rh-endostatin in patients with non-small-cell lung cancer (NSCLC). Chemotherapy-naive patients with stage IIIB to IV NSCLC were randomly (1:1) assigned to receive gemcitabine/cisplatin chemotherapy alone or with 7.5 mg/ m(2) of intravenously rh-endostatin on days 1 to 14 of each 3-week cycle. The primary end point was objective response rate (ORR). Baseline characteristics were similar between treatment arms. The best ORRs for rh-endostatin arm (n = 33) and chemotherapy-alone arm were 37.5% (95% CI: 21.3 to 47.2%) and 28.6% (95% CI: 19.8 to 37.6%), respectively. Median survival was 12.4 months in the rh-endostatin arm and 9.8 months in the chemotherapy-alone arm, and 1-year survival was 51.6% and 38.7%, respectively. Mild palpitions, diarrhea, and liver dysfunction were the most common rh-endostatin-related adverse events. Grade 3/4 hematological toxicities were all reported similar for patients in the two arms. The addition of rh-endostatin to gemcitabine plus cisplatin chemotherapy for first-line treatment of NSCLC improves objective response and may improve survival.

Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings.

Liposomal pravastatin inhibits tumor growth by targeting cancer-related inflammation

The chronic inflammatory environment of tumors is a target for novel antitumor therapeutic strategies. Besides cholesterol lowering effects, statins have been studied for their anti-inflammatory and immunomodulatory properties. These pleiotropic effects result mainly from the altered post-translational modification of GTP-binding proteins which regulate many intracellular pathways involved in cell growth and survival. Although pre-clinical studies suggest that statins may be effective anticancer agents required doses that are 100 to 500 fold higher than those needed to lower cholesterol levels. Furthermore, in view of their wide-ranging effects on cellular metabolism, target site-specific delivery is preferred. In this study, we investigated tumor-specific delivery of pravastatin using small long-circulating liposomes. In vitro studies on the effects of (liposomal) pravastatin on viability and proliferation of tumor cells, endothelial cells and macrophages revealed that the latter were the most sensitive cell type towards (liposomal) pravastatin treatment . In vivo, liposome-encapsulated pravastatin (5 mg/kg) inhibited murine B16F10-melanoma growth over 70% as compared to free pravastatin, which was ineffective. As expected, treatments did not influence serum cholesterol levels within the time frame of the study. At 48 h post-injection, 3 μg of pravastatin could still be recovered from the tumors of liposomal pravastatin treated mice, whereas pravastatin could not be detected in tumors of the free drug treated mice (i.e. < 20 ng). In contrast to the free drug, liposomal pravastatin treatment effectively inhibited the production of several pro-inflammatory/pro-angiogenic mediators involved in inflammation and angiogenesis, out of a range of a panel of 24 proteins studied. Furthermore, liposomal pravastatin treatment increased MHC class I protein expression in the tumor tissue whereas free drug showed no effect. Taken together, targeted delivery of statins can improve their tumor growth inhibiting activity by increasing local drug concentration and direct modulation of macrophage function. The antitumor activity seems to result primarily from a local inhibition of tumor inflammation and stimulation of antitumor immune response.

Abstract C105: Preclinical evaluation of galectin-1 targeting antiangiogenic, antitumor peptidomimetics

Abstract We have showed previously that the peptide 33mer anginex targets galectin-1 (gal-1) thereby inhibiting tumor endothelial cell proliferation via anoikis and attenuating tumor angiogenesis and tumor growth. Gal-1 binds β-galactoside groups on various cell surface receptors and is highly upregulated in tumor-activated endothelial cells. Differential stromal elevation of gal-1 over the tumor parenchyma has been reported in several cancers, including cancer of the breast, colon, prostate and ovaries. The aim of the present study was to develop gal-1 targeting anti-angiogenic peptidomimetics and anti-tumor clinical candidates for use in the management of human cancers. Based on structure-activity relationships of anginex, a partial-peptide mimetic was designed, using dibenzofuran (DBF) as a β-turn mimetic to maintain the two key β-strands and the bioactive amphipathic β-sheet conformation of the peptide. The best DBF-based compound 6DBF7 (13mer) inhibited tumor angiogenesis and tumor growth in vivo better than parent anginex. Subsequently, by substituting natural and non-natural amino acids, new DBF-based analogues were generated and tested in vitro. The analogs with improved anti-angiogenic activity and pharmacokinetic profile were then tested in tumor mouse models in vivo. The pre-clinical development of these novel anti-angiogenic and anti-cancer peptidomimetics will aid in the development of small-molecule mimetics and subsequently hold great promise for the management of human cancers in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C105.

TU‐C‐BRB‐07: Incorporation of Angiogenesis Into a Stochastic Imaging‐Based Tumor Vasculature Model

Aim Controlling interaction between tumor growth and vasculature development are the basis of anti‐angiogenic therapies. A model simulating tumor growth and angiogenesis was developed and the tumor oxygenation status was evaluated with respect to two parameters: tumor proliferation rate (TPR) and endothelial cell proliferation rate (EPR). Methods and Materials Our previous image‐based model of vasculature was expanded by incorporating angiogenic development. The tissue volume encapsulating the tumor and vasculature was simulated as a MATLAB matrix. Vessels were simulated to sprout from the existing vasculature towards the tumor driven by hypoxia‐induced growth factors. The relative magnitude of TPR and EPR determined the simulation time steps. Xenografted and spontaneous tumors were simulated and the tumor oxygenation status was evaluated with respect to TPR &amp; EPR. The model was verified against the experimental data obtained from CE‐CT and Cu‐ATSM PET imaging. Results The time‐dependant growth of vessels towards hypoxic regions in the tumor was demonstrated. TPR was found to have the most profound impact on development of hypoxia and heterogeneity of the oxygen concentration inside the tumor. Higher TPR led to faster development of hypoxia. Due to the limited vasculature available, xenografted tumors become hypoxic faster than spontaneous tumors for similar TPRs. When comparing simulations to the experiments, the overall development of vasculature and consequent hypoxia matched well with the imaging data. Conclusions A model capable of simulating temporal development of tumor and vasculature has been developed and tested on the imaging‐derived experimental data. The presented model has a potential to study the response of tumor and the vasculature to therapy, which can be a valuable input for optimizing anti‐angiogenic therapies.

Effects of cloned tumstatin-related and angiogenesis-inhibitory peptides on proliferation and apoptosis of endothelial cells

Tumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent. The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide (peptide 21), to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity: specifically inhibiting tumor angiogenesis like tumstatin. Peptide 21 was designed and synthesized using biological engineering technology. To determine its biological action, the human umbilical vein endothelial cell line ECV304, the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves. Transmission electron microscopy (TEM) and flow cytometry (FCM) were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively. In animal experiments, tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight, size and microvessel density (MVD). To initially investigate the role of peptide 21, the effect of peptide 21 on the expression of vascular endothelial growth factors (VEGFs) by tumor tissue was semi-quantitatively analyzed. The in vitro MTT test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner (P < 0.01); TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis (P < 0.01). Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly. The weight and size of the tumors after 21 days of treatment were smaller than those in the control group (P < 0.05), with a mean tumor inhibition rate of 67.86%; MVD of the tumor tissue in the peptide 21 group was significantly lower than in the control group (P < 0.05); the number of cells positive for VEGF in the peptide 21 group was significantly fewer than in the control group (P < 0.01). Similar to the activity of tumstatin in specifically inhibiting tumor angiogenesis, peptide 21 may specifically inhibit tumor endothelial cell proliferation and induce their apoptosis, thereby suppressing tumor angiogenesis and indirectly inhibit the growth, infiltration and metastasis of tumors. Peptide 21 may exert its effect through down-regulating the VEGF expression of tumor cells and vascular endothelial cells.

Pharmacokinetic-directed dosing of vandetanib and docetaxel in a mouse model of human squamous cell carcinoma.

Docetaxel, usually administered according to maximum tolerated dose (MTD), can inhibit endothelial cell proliferation at low nanomolar concentrations. Docetaxel may exert antiangiogenic effects if dosed so plasma levels are maintained at low nanomolar concentrations over a prolonged time. We evaluated metronomic and MTD-based dosing of docetaxel with and without vandetanib, a vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity, in a head and neck xenograft model. A murine physiologically based pharmacokinetic model was modified to predict docetaxel distribution following i.p. administration to design dosing regimens that target prespecified plasma concentrations, for antiendothelial effects (metronomic), or exposure, to mimic 30 mg/m2 (weekly/MTD) docetaxel in humans. Animals were treated for 28 days with 1 mg/kg/d (DTX1) or 6 mg/kg q4d (DTX6) docetaxel with or without vandetanib (15 mg/kg/d p.o.) in mice bearing UMSCC2 tumor xenografts. The DTX1 dosing scheme was adjusted to treatment for 10 days followed by 9 days off due to severe gastrointestinal toxicity. All treatment groups significantly reduced tumor volume, tumor proliferation (Ki-67), and tumor endothelial cell proliferation (Ki-67/von Willebrand factor) compared with control. Addition of vandetanib to docetaxel treatment significantly enhanced tumor growth inhibition over single-agent therapy. A positive correlation of tumor endothelial cell proliferation with tumor growth rates demonstrates vandetanib and docetaxel antiangiogenic effects. Due to the morbidity observed with DTX1 treatment, it is difficult to clearly ascertain if metronomic schedules will be effective for treatment. Docetaxel with vandetanib is effective in treating UMSCC2 xenografts at concentrations relevant to exposures in humans.

Liposomal glucocorticoids as tumor-targeted anti-angiogenic nanomedicine in B16 melanoma-bearing mice

This study evaluates whether the inhibitory effects of prednisolone phosphate (PLP) encapsulated in long-circulating liposomes (LCL-PLP) on tumor growth and tumor angiogenesis described previously can be generalized to other types of glucocorticoids (GC) encapsulated in LCL (LCL-GC). Four types of synthetic GC, i.e. budesonide disodium phosphate (BUP), dexamethasone disodium phosphate (DXP), methylprednisolone disodium phosphate (MPLP), and PLP, were selected based on the difference in their potency to activate the human glucocorticoid receptor. The effects of all LCL-GC on the production of angiogenic/inflammatory factors in vivo in the B16.F10 murine melanoma model as well as on the viability and proliferation of tumor cells and endothelial cells in vitro were investigated. Our results show that all four selected LCL-GC formulations inhibit tumor growth, albeit to different degrees. The differences in antitumor activity of LCL-GC correlate with their efficacy to suppress tumor angiogenesis and inflammation. The strongest antitumor effect is achieved by LCL-encapsulated BUP (LCL-BUP), due to the highest potency of BUP versus the other three GC types. The in vitro results presented herein suggest that LCL-BUP has strong cytotoxic effects on B16.F10 melanoma cells and the anti-proliferative effects of all LCL-GC towards angiogenic endothelial cells play a role in their antitumor activity.

Dopamine Increases the Efficacy of Anticancer Drugs in Breast and Colon Cancer Preclinical Models

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

A Role for PPARβ/δ in Tumor Stroma and Tumorigenesis

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is a transcription factor that is activated by endogenous fatty acid ligands and by synthetic agonists. Its role in the regulation of skeletal muscle fatty acid catabolism, glucose homeostasis, and cellular differentiation has been established in multiple studies. On the contrary, a role for PPARβ/δ in tumorigenesis is less clear because there are contradictory reports in the literature. However, the majority of these studies have not examined the role of PPARβ/δ in the tumor stroma. Recent evidence suggests that stromal PPARβ/δ regulates tumor endothelial cell proliferation and promotes differentiation leading to the properly orchestrated events required for tumor blood vessel formation. This review briefly summarizes the significance of these studies that may provide clues to help explain the reported discrepancies in the literature regarding the role of PPARβ/δ in tumorigenesis.

Nutrient Regulation of Tumor and Vascular Endothelial Cell Proliferation

Specific bioactive dietary components, such as the steroid receptor superfamily ligands vitamins A and D, have been studied extensively as potential cancer preventive and therapeutic agents due to their ability to regulate key processes in a variety of cell types which are dysregulated in neoplastic transformation namely, proliferation and differentiation. Alteration of one or more factors that regulate cell cycle control has been described as a predisposing event for early tumor development. In addition to tumor cell proliferation, the viability, growth and metastasis of solid tumors are also dependent on the vascularization of the tumor and establishment of blood flow. Both vitamins A and D exhibit anti-angiogenic properties which further strengthen their role as potential targets for the prevention and treatment of cancer. This review focuses on the role of vitamins A and D in preventing early tumor initiation and progression via control of the cell cycle in both tumor and vascular endothelial cells.