semaxinib and Glioblastoma

Antiangiogenic drugs encompass many of the different cancer drugs currently under clinical investigation. One of the drawbacks of antiangiogenic therapy, though, is that upon cessation of drug treatment tumors can recur with an accelerated growth rate. In this study we investigate the capacity of using affinity interactions between a polymer made from cyclodextrin and four antiangiogenic drugs, tranilast, SU5416, 2-methoxyestradiol, and silibinin, with the ultimate goal of creating delivery profiles on the order of antiangiogenic processes (needing weeks, rather than hours of delivery). In these systems, release rate is dependent on affinity, so using in silico molecular docking studies followed by surface plasmon resonance we determined that silibinin possesses the highest affinity among the drugs screened. Silibinin also showed a differential binding affinity among various cyclodextrins tested, with a greater affinity toward the larger molecular pocket of γ-cyclodextrin than for β-cyclodextrin. Release studies confirmed this affinity to translate into a slower, more sustained release of silibinin. Similarly we found this trend in the release of tranilast. Then using U87 human glioblastoma cells in a mouse xenograft model, we showed that affinity-based cyclodextrin polymers loaded with silibinin showed substantially longer release rates than nonaffinity control polymers; however, both were capable of inhibiting tumor growth in the time frame studied. From this work we showed three different, but chemically similar, polymers, each with a different release rate. Future work is on evaluating longer term tumor models where this longer release rate from affinity delivery systems might have additional advantages over polymers dependent only on diffusion.

Topics: 2-Methoxyestradiol; Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cellulose; Cyclodextrins; Drug Delivery Systems; Estradiol; Female; Glioblastoma; Humans; Indoles; Mice; Mice, Nude; ortho-Aminobenzoates; Pyrroles; Silybin; Silymarin

The combination of irradiation and the antiangiogenic compound SU5416 was tested and compared with irradiation alone in a human glioblastoma tumor line xenografted in nude mice. The aim of this study was to monitor microenvironmental changes and growth delay.. A human glioblastoma xenograft tumor line was implanted in nude mice. Irradiations consisted of 10 Gy or 20 Gy with and without SU5416. Several microenvironmental parameters (tumor cell hypoxia, tumor blood perfusion, vascular volume, and microvascular density) were analyzed after imunohistochemical staining. Tumor growth delay was monitored for up to 200 days after treatment.. SU5416, when combined with irradiation, has an additive effect over treatment with irradiation alone. Analysis of the tumor microenvironment showed a decreased vascular density during treatment with SU5416. In tumors regrowing after reaching only a partial remission, vascular characteristics normalized shortly after cessation of SU5416. However, in tumors regrowing after reaching a complete remission, permanent microenvironmental changes and an increase of tumor necrosis with a subsequent slower tumor regrowth was found.. Permanent vascular changes were seen after combined treatment resulting in complete remission. Antiangiogenic treatment with SU5416 when combined with irradiation has an additive effect over treatment with irradiation or antiangiogenic treatment alone.

Topics: Angiogenesis Inhibitors; Animals; Cell Hypoxia; Cell Line, Tumor; Combined Modality Therapy; Dose-Response Relationship, Radiation; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Indoles; Mice; Mice, Inbred BALB C; Mice, Nude; Microcirculation; Necrosis; Pyrroles; Recurrence; Remission Induction; Transplantation, Heterologous

Neovascularization and invasion are key features of malignant gliomas. Matrix metalloproteinases (MMPs) are supposed to play a major role mediating these processes. To analyze the expression patterns of MMPs in microvascular human cerebral endothelial cells (HCEC), we isolated endothelial cells from normal human brain microvessels. Characterization of cellular origin was performed by immunostaining, using the endothelial cell markers Ulex europaeus Agglutinin-1, von-Willebrand-Factor and Glucose-transporter-1. Contamination by other cell types was tracked by immunohistochemistry for GFAP (astrocytes), ASM (pericytes) and CD68 (macrophages). Secretion of MMPs was evaluated by ELISA and zymography. To determine whether HCEC show any difference in MMP expression compared to endothelial cells of other origin we analyzed human umbilical vein endothelial cells (HUVEC). HCEC show a decrease of MMP-3 and MMP-2 protein when treated with SU5416, a VEGF-R2 (KDR/flk-1) inhibitor, whereas MMP expression remained unchanged in HUVEC. To determine whether these findings show any effect in the motility of these cells we used a three-dimensional co-culture assay of avascular glioblastoma spheroids with primary HCEC spheroids. Untreated controls showed invasion of both cell populations into each other whereas treatment of the co-cultures with SU5416 resulted in complete inhibition of endothelial cell invasion hence indicating that flk-1 related motility of endothelial cells is critically involved in this process and can be studied with this assay. The results of different effects of anti-angiogenic treatment on proteolytic properties of two endothelial cell populations suggest that neovascularization of human brain tumors in vitro is dependent on the surrounding endothelial cell type and should therefore be studied with organ-specific human microvascular cerebral endothelial cells.

Topics: Angiogenesis Inhibitors; Brain Neoplasms; Cell Movement; Cerebral Cortex; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glioblastoma; Humans; Immunoenzyme Techniques; In Vitro Techniques; Indoles; Interleukin-10; Matrix Metalloproteinases; Microcirculation; Neovascularization, Pathologic; Protein-Tyrosine Kinases; Pyrroles; Spheroids, Cellular; Tumor Cells, Cultured; Umbilical Veins; Vascular Endothelial Growth Factor Receptor-2

The multifaceted nature of the angiogenic process in malignant neoplasms suggests that protocols that combine antiangiogenic agents may be more effective than single-agent therapies. However it is unclear which combination of agents would be most efficacious and will have the highest degree of synergistic activity while maintaining low overall toxicity. Here we investigate the concept of combining a "direct" angiogenesis inhibitor (endostatin) with an "indirect" antiangiogenic compound [SU5416, a vascular endothelial growth factor receptor 2 (VEGFR2) receptor tyrosine kinase (RTK) inhibitor]. These angiogenic agents were more effective in combination than when used alone in vitro (endothelial cell proliferation, survival, migration/invasion, and tube formation tests) and in vivo. The combination of SU5416 and low-dose endostatin further reduced tumor growth versus monotherapy in human prostate (PC3), lung (A459), and glioma (U87) xenograft models, and reduced functional microvessel density, tumor microcirculation, and blood perfusion as detected by intravital microscopy and contrast-enhanced Doppler ultrasound. One plausible explanation for the efficacious combination could be that, whereas SU5416 specifically inhibits vascular endothelial growth factor signaling, low-dose endostatin is able to inhibit a broader spectrum of diverse angiogenic pathways directly in the endothelium. The direct antiangiogenic agent might be able to suppress alternative angiogenic pathways up-regulated by the tumor in response to the indirect, specific pathway inhibition. For future clinical evaluation of the concept, a variety of agents with similar mechanistic properties could be tested.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Movement; Cell Survival; Drug Synergism; Endostatins; Endothelium, Vascular; Female; Glioblastoma; Humans; Indoles; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasms; Neovascularization, Pathologic; Prostatic Neoplasms; Pyrroles; Ultrasonography; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Certain refractory neoplasms, such as glioblastoma multiforme (GBM) and melanoma, demonstrate a resistant tumor phenotype in vivo. We observed that these refractory tumor models (GBM and melanoma) contain blood vessels that are relatively resistant to radiotherapy. To determine whether the vascular endothelial growth factor receptor-2 (Flk-1/KDR) may be a therapeutic target to improve the effects of radiotherapy, we used the soluble extracellular component of Flk-1 (ExFlk), which blocks vascular endothelial growth factor binding to Flk-1 receptor expressed on the tumor endothelium. Both sFlk-1 and the Flk-1-specifc inhibitor SU5416 eliminated the resistance phenotype in GBM and melanoma microvasculature as determined by both the vascular window and Doppler blood flow methods. Human microendothelial cells and human umbilical vein endothelial cells showed minimal radiation-induced apoptosis. The Flk-1 antagonists sFlk-1 and SU5416 reverted these cell models to apoptosis-prone phenotype. Flk-1 antagonists also reverted GBM and melanoma tumor models to radiation-sensitive phenotype after treatment with 3 Gy. These findings demonstrate that the tumor microenvironment including the survival of tumor-associated endothelial cells contributes to tumor blood vessel resistance to therapy.

Topics: Angiogenesis Inhibitors; Animals; Cell Survival; Dose-Response Relationship, Radiation; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Inhibitors; Glioblastoma; Indoles; Lymphokines; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Pyrroles; Radiation Tolerance; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Vascular endothelial growth factor (VEGF)-Flk-1/KDR tyrosine kinase signaling pathway plays a pivotal role in tumor angiogenesis. Targeting this angiogenic signaling pathway presents a promising alternative for the treatment of neoplasms. However, recent experimental and clinical studies have suggested that VEGF-Flk-1/KDR activity is unevenly distributed throughout the tumor microvasculature. To further evaluate this phenomenon, the regional differences in VEGF-Flk-1/KDR signaling activities in vivo were studied using intravital fluorescence videomicroscopy in an experimental murine brain tumor model. Regional VEGF-Flk-1/KDR was assessed using the small molecule inhibitor SU5416, which selectively inhibits the tyrosine kinase receptor Flk-1. C(6) glioblastoma cells were implanted into the dorsal skinfold chamber preparation of nude mice. The process of tumor vascularization was repeatedly assessed over 22 days. SU5416 treatment resulted in a significant reduction in tumor vascular density (p<0.05). Regional microvascular evaluation indicated that the magnitude of this antiangiogenic effect was pronounced in the more angiogenic and better vascularized peritumoral areas than in the intratumoral areas of the tumor microvasculature. These results demonstrate regional differences in Flk-1 activity in vivo that may have significant impact on the susceptibility of tumors to compounds that target VEGF-Flk-1/KDR. This finding should be considered in upcoming clinical trials targeting individual signal transduction systems in cancer patients.

Topics: Analysis of Variance; Angiogenesis Inhibitors; Animals; Disease Models, Animal; Endothelial Growth Factors; Enzyme Inhibitors; Glioblastoma; Indoles; Lymphokines; Male; Mice; Mice, Nude; Microcirculation; Microscopy, Fluorescence; Microscopy, Video; Neoplasm Transplantation; Neovascularization, Pathologic; Protein Isoforms; Protein-Tyrosine Kinases; Pyrroles; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Mitogen; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Skin Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Vascular endothelial growth factor (VEGF) plays a fundamental role in mediating tumor angiogenesis and tumor growth. Here we investigate the direct effect of a novel small molecule inhibitor of the Flk-1-mediated signal transduction pathway of VEGF, SU5416, on tumor angiogenesis and microhemodynamics of an experimental glioblastoma by using intravital multifluorescence videomicroscopy. SU5416 treatment significantly suppressed tumor growth. In parallel, SU5416 demonstrated a potent antiangiogenic activity, resulting in a significant reduction of both the total and functional vascular density of the tumor microvasculature, which indicates an impaired vascularization as well as significant perfusion failure in treated tumors. This malperfusion was not compensated for by changes in vessel diameter or recruitment of nonperfused vessels. Analyses of the tumor microcirculation revealed significant microhemodynamic changes after angiogenesis blockage such as a higher red blood cell velocity and blood flow in remnant tumor vessels when compared with controls. Our results demonstrate that the novel antiangiogenic concept of targeting the tyrosine kinase of Flk-1/KDR by means of a small molecule inhibitor represents an efficient strategy to control growth and progression of angiogenesis-dependent tumors. This study provides insight into microvascular consequences of Flk-1/KDR targeting in vivo and may have important implications for the future treatment of angiogenesis-dependent neoplasms.

Topics: Animals; Antineoplastic Agents; Enzyme Inhibitors; Glioblastoma; Indoles; Male; Mice; Mice, Nude; Microcirculation; Neovascularization, Pathologic; Pyrroles; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor