o-(chloroacetylcarbamoyl)fumagillol and Glioblastoma

Tumor angiogenesis is a promising target for cancer treatment, because severing the supply of oxygen and nutrients to tumors halts tumor growth. Unfortunately, many anticancer drugs, including angiogenesis inhibitors, fail in clinical trials, despite showing high efficiency during in vitro and in vivo experiments. This inconsistency from in vitro and in vivo experiments to clinical trials represents a major obstacle in drug development and cancer treatment. Therefore, we set out to demonstrate how our rapid, stable, easy-to-use three-dimensional (3-D) in vitro angiogenesis model can be used to investigate tumor formation and implement drug screening. In this study, we utilized a 3-D in vitro angiogenesis model, based on gelatin methacrylate (GelMA) hydrogel microwells, to mimic the native microenvironment of tumor angiogenesis. Using this model, we were able to quantify the immigration of endothelial cells into a cancer spheroid during the angiogenic process. Next, we tested the anti-angiogenic effect of the angiogenesis inhibitor, TNP-470, on the cancer spheroids in the model. Based on our results, we believe that this novel in vitro system can be widely used for the high-throughput screening of other anti-angiogenic drugs, and could contribute to the development of personalized medicine in the future.

Topics: Angiogenesis Inhibitors; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Cyclohexanes; Drug Screening Assays, Antitumor; Gelatin; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; Methacrylates; Models, Biological; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Spheroids, Cellular; Tumor Cells, Cultured

This study investigated the effect of TNP-470 in combination with carmustine (BCNU) on the growth of subcutaneously implanted human glioblastoma xenografts in nude mice. Human glioblastoma U-251 cells (1×10(7)) were injected into 24 nude mice subcutaneously. The tumor-bearing mice were randomly divided into 4 groups on the seventh day following tumor implantation: TNP-470 group, in which TNP-470 was given 30 mg/kg subcutaneously every other day 7 times; BCNU group, in which 20 mg/kg BCNU were injected into peritoneal cavity per 4 days 3 times; TNP-470 plus BCNU group, in which TNP-470 and BCNU were coadministered in the same manner as in the TNP-470 group and the BCNU group; control group, in which the mice were given 0.2 mL of the mixture including 3% ethanol, 5% acacia and 0.9% saline subcutaneously every other day 7 times. The tumor size and weights were measured. The tumor microvessel density (MVD) was determined by immunostaining by using goat-anti-mouse polyclonal antibody CD105. The results showed that on the 21th day following treatment, the volume of xenografts in the TNP-470 plus BCNU group was (108.93±17.63)mm(3), markedly lower than that in the TNP-470 group [(576.10±114.29)mm(3)] and the BCNU group [(473.01±48.04)mm(3)] (both P<0.01). And the xenograft volume in these 3 treatment groups was even much lower than that in the control group [(1512.61±470.25) mm(3)] (all P<0.01). There was no significant difference in the volume of xenografts between the TNP-470 group and the BCNU group (P>0.05). The inhibition rate of the tumor growth in the TNP-470 plus BCNU group was (92.80±11.37)%, notably higher than that in the TNP-470 group [(61.91±6.29)%] and the BCNU group [(68.73±9.65)%] (both P<0.01) on the 21th day following treatment. There was no significant difference in the inhibition rate of tumor growth between the TNP-470 group and the BCNU group (P>0.05). The MVD of xenografts in the TNP-470 plus BCNU group was decreased significantly as compared with that in the TNP-470 group or the BCNU group (both P<0.05). The MVD of xenografts in the 3 treatment groups was markedly reduced as compared with that in the control group (all P<0.05). No significant changes in weights were observed before and after the treatment in each group (all P>0.05). It was concluded that the combination of TNP-470 and BCNU can significantly inhibit the growth of human glioblastoma xenografts in nude mice without evident side effects.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Carmustine; Cell Line, Tumor; Cyclohexanes; Female; Glioblastoma; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Xenograft Model Antitumor Assays

We examined the effect on tumor growth, vessel morphology, and expression of angiogenic factors of combining radiotherapy and antiangiogenesis in the human glioblastoma line U87 grown in the flank or intracranially in the nude mouse. The antiangiogenic agent TNP-470 was given 6.7 mg/kg s.c. daily on day 1-7 starting 1 week after transplantation. Irradiation (IR), 10 Gy x 1, was administered on day 7. A series of tumors were excised 8 and 48 h after the end of treatment. The vascular morphology was evaluated in CD31 immunostained cryosections and by electron microscopy, and the pattern of expression of angiogenic factors (mRNA and protein) was quantitatively analyzed by phosphorimaging of Northern blots and Western blots. Significant inhibition of s.c. flank tumor growth relative to untreated controls was achieved by monotherapy with both TNP-470 (P < 0.001) and IR (P < 0.001). A significant enhancement of this effect was obtained by combining TNP-470 and IR (P < 0.05). We saw no effect of TNP-470 either alone or in addition to the effect of IR on the survival of mice with intracranial tumors. CD31 immunostaining of s.c. tumors showed acute endothelial swelling and luminal protrusion in irradiated tumor vessels but never in tumors pretreated with TNP-470, and not in the untreated controls. The vessel density (Chalkley point counts) was unchanged by TNP-470 therapy. In the TNP-470-treated tumors, we observed a distinct broadening of the endothelial basement membrane by an approximately 400-700-nm-thick electron-dense yet uncharacterized fibrillar material. TNP-470 treated tumors +/- IR also had a significantly increased mRNA expression of angiopoietin-1, whereas angiopoietin-2, vascular endothelial growth factor and basic fibroblast growth factor mRNA were unchanged by the treatments. In conclusion, TNP-470 significantly enhanced the tumor effect of ionizing IR, and our findings strongly indicate that acute microvascular damage after IR is effectively prevented by concurrent TNP-470 treatment. A significant up-regulation of angiopoietin-1 seems to play a role in this protective mechanism, which as yet is not fully elucidated.

Topics: Angiogenesis Inhibitors; Angiopoietin-1; Angiopoietin-2; Animals; Antibiotics, Antineoplastic; Cell Division; Combined Modality Therapy; Cyclohexanes; Endothelial Growth Factors; Endothelium, Vascular; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Lymphokines; Male; Membrane Glycoproteins; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Proteins; RNA, Messenger; Sesquiterpenes; Survival Analysis; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Clinical Trials as Topic; Cyclohexanes; Female; Glioblastoma; Humans; Melanoma, Experimental; Mice; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Sesquiterpenes; Tumor Stem Cell Assay; Uterine Cervical Neoplasms

Glioblastoma is the most malignant primary brain tumor. Inhibition of angiogenesis is one potential strategy for treating this fatal hypervascular tumor. AGM-1470 (also called TNP-470), a novel, potent, fungus-derived inhibitor of angiogenesis, was tested on the growth of human glioblastoma cells in culture and on the growth of the tumor in nude mice. In nude mice with subrenally implanted U-87 MG glioblastomas, AGM-1470 significantly inhibited tumor growth (P < 0.01), and in nude mice with intracranial U-87 MG glioblastomas, AGM-1470 prolonged survival. In addition to its expected action as an angiogenesis inhibitor, AGM-1470 also directly inhibited U-87 MG cells in culture at concentrations similar to those that inhibited endothelial cells. The combined inhibition of glioblastoma cell mitosis and of glioblastoma-induced neovascularization suggests that AGM-1470 should be considered for further investigation in the treatment of this fatal tumor.

Topics: Animals; Biomarkers, Tumor; Brain Neoplasms; Cell Division; Cell Line; Cyclohexanes; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Glioblastoma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Subrenal Capsule Assay; Tumor Cells, Cultured; von Willebrand Factor