The angiogenesis inhibitor AGM-1470 selectively increases E-selectin

The levels of E-selectin mRNA and protein were analyzed in bovine capillary cells treated with or without the angiogenesis inhibitor AGM-1470 (also known as TNP-470). Cells treated with AGM-1470 had a two- to sevenfold (median fivefold) increase in E-selectin mRNA compared with little or no increase in P-selectin, PECAM-1 and VCAM-1 mRNA. E-selectin protein was also significantly increased after exposure to AGM-1470. In contrast, there was no detectable effect on PECAM-1 protein. The increase in E-selectin mRNA and protein was always greater with subconfluent growing cells than with confluent cells. This apparent resistance of confluent endothelial cells to AGM-1470 may be relevant to its specificity in vivo. The fact that the effect of AGM-1470 on E-selectin is relatively selective for subconfluent growing cells may provide a clue as to how AGM-1470 is able to both reversibly inhibit endothelial cell proliferation in vitro and inhibit tumor growth in vivo without apparent effects to quiescent endothelium.     

Telomerase activity correlates with growth of transplantable osteosarcomas in rats treated with cis-diammine dichloroplatinum or the angiogenesis inhibitor AGM-1470

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase-positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis-diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillol (AGM-1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM-1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non-treated (T/C) tumor size ratio (r = 0.96, P < 0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM-1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM-1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re-growth (r = 0.73, P < 0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.     

Suppression of collagen-induced arthritis using an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer, taxol

Collagen-induced arthritis (CIA) is a T-cell-dependent rat model of rheumatoid arthritis (RA) that is induced by injection of collagen type II in incomplete Freund's adjuvant. Neovascularization within the synovium is a prominent feature of CIA and RA. The novel angiogenesis inhibitor AGM-1470 and the microtubule-stabilizing agent Taxol represent two new classes of agents with specific mechanisms of action. AGM-1470 inhibits fibroblast growth factor-induced stimulation of endothelial cell migration, endothelial cell proliferation, and capillary tube formation, resulting in effective suppression of new blood vessel formation. By enhancing microtubule polymerization, Taxol interferes with normal microtubule function in cell mitosis, migration, chemotaxis, and intracellular transport. Using a suppression protocol in established CIA, the effects of AGM-1470 and Taxol as single agents and in combination were evaluated. Combination therapy significantly reduced clinical arthritis compared to control rats (P < 0.00001). The combination therapy group also experienced earlier and significantly greater reduction of clinical arthritis compared to either single agent-treated groups (P < 0.05). Blinded radiographic scores at the end of the study demonstrated less soft tissue swelling and joint destruction using combination therapy than either single agent. This is the first use of AGM-1470 and Taxol in combination therapy. Further study of agents with distinct mechanisms of action may lead to more effective treatment options in chronic inflammatory arthritis and to a better understanding of the pathophysiologic processes of pannus formation.     

Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats

OBJECTIVE: To analyse the longitudinal relationships between body mass index (BMI)/sum of skinfolds (SSF) and biological and lifestyle risk factors for coronary heart disease (CHD). DESIGN: An observational longitudinal study; that is, the Amsterdam Growth and Health Study. SUBJECTS: 181 males and females, initially aged 13 y. Over a period of 15 y, six repeated measurements were carried out. MEASUREMENTS: BMI and SSF, biological CHD risk factors; that is, total cholesterol (TC), high density lipoprotein (HDL), TC:HDL ratio, systolic/diastolic blood pressure (SBP/DBP) and cardiopulmonary fitness (VO2-max) and lifestyle CHD risk factors (that is, daily physical activity, dietary parameters, smoking, and alcohol consumption). The longitudinal relationships were analysed by an autoregressive model, in which the value of the outcome variable at time-point t is not only related to the value of the predictor variable at t, but also to the value of the outcome variable at t-1. RESULTS: Both BMI and SSF were positively related to TC and the TC:HDL ratio. Only BMI was positively related to SBP and only SSF was negatively related to VO2-max. Physical activity was negatively related to SSF. None of the other lifestyle parameters were related to SSF and/or BMI. CONCLUSIONS: Both BMI and SSF were related to a high risk profile regarding CHD. Different relationships for SSF and BMI are found, because BMI not only reflects body fatness, but also lean body mass. Analyses with BMI as an indicator for body fatness should therefore be interpreted cautiously.     

Inhibition of tumor growth and angiogenesis by vitamin D3 agents in murine renal cell carcinoma

PURPOSE: To investigate the effect of active vitamin D3(VD) agents on tumor growth and metastasis. MATERIALS AND METHODS: BALB/c mice were inoculated with murine renal cancer Renca and graded doses of 1,25-dehydrovitamin D3 or 1- hydrovitamin D3 were given intraperitoneally every other day beginning on day 1, 3, or 7 and ending on day 9, 11, or 15. Direct cytocidal activity and angiogenic activity were evaluated by 48-hour MTT assay and by the colorimetric method, respectively. RESULTS: Both VD agents inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner and both suppressed tumor growth in athymic mice and euthymic mice with eliminated NK activity. Marginal body-weight loss without appreciable hypercalcemia was observed in mice given VD agents. When treatment was delayed on day 7, the VD agents failed to inhibit tumor growth. The MTT assay showed no direct cytotoxicity of VD agents on Renca. Tumor angiogenesis was inhibited to 46 to 30% of the control level by VD agents. Furthermore, VD agents reduced pulmonary and hepatic foci in the metastatic models. CONCLUSIONS: These results suggest that VD agents may be effective as a treatment for renal cell carcinoma, especially when micrometastases are involved.     

Antitumor activity of a medium-chain triglyceride solution of the angiogenesis inhibitor TNP-470 (AGM-1470) when administered via the hepatic artery to rats bearing Walker 256 carcinosarcoma in the liver

The antitumor effect of an angiogenesis inhibitor, TNP-470 (AGM-1470, 6-0-(N-chloroacetylcarbamoyl)-fumagillol), administered via the hepatic artery in a medium-chain triglyceride (MCT) solution, in which TNP-470 is very stable, was examined in rats bearing Walker 256 carcinosarcoma in the liver. The MCT solution containing 0.1 mg of TNP-470 completely suppressed tumor growth after a single arterial injection, and the solutions containing 0.5 approximately 5 mg of TNP-470 caused tumor regression function. These antitumor effects lasted for at least 2 weeks. Moreover, the administration of the MCT solution containing 5 mg of TNP-470 also caused remarkable regression of well-developed enlarged tumors 2 weeks after inoculation, indicating potential in the treatment of unresectable hepatic cancer. When the MCT solution containing radiolabeled TNP-470 was injected via the hepatic artery, the initial radioactivity in the tumor was 22 times that in the normal part of the liver and 5.7 times that in the tumor when an aqueous solution of radiolabeled TNP-470 was injected. Also, in the case of the MCT solution, the radioactivity in the tumor was maintained at a relatively high level for over 2 weeks after injection. These results indicate that the remarkable antitumor effect resulted from the selective delivery and prolonged retention of TNP-470 at the tumor site.     

Inhibition of liver metastasis of human pancreatic carcinoma by angiogenesis inhibitor TNP-470 in combination with cisplatin

The anti-tumor and anti-metastatic effects of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), an angiogenesis inhibitor, and cisplatin (CDDP), an anti-neoplastic agent, were investigated using our established liver-metastasizing pancreatic carcinoma line, HPC-3H4. HPC-3H4 was injected into the spleens of nude mice. Mice were randomly divided into 5 groups; a control group given saline solution, a group receiving 45 mg/kg TNP-470, a group receiving 90 mg/kg TNP-470, a group receiving 90 mg/kg TNP-470 in combination with 0.25 mg/kg CDDP, and a group receiving 0.25 mg/kg CDDP. In the control group, liver metastasis developed in 14 of 15 mice (93.3%). Liver metastasis developed in 9 of 11 mice (81.8%) receiving 0.25 mg/kg CDDP. It developed in 11 of 15 mice (73.3%) receiving 45 mg/kg TNP-470, in 17 of 18 mice (94.4%) receiving 90 mg/kg TNP-470, and in 4 of 10 mice (40%) receiving 90 mg/kg TNP-470 in combination with 0.25 mg/kg CDDP. TNP-470 in combination with CDDP displayed a significant inhibitory effect on liver metastasis compared to the control. Although TNP-470 alone and CDDP alone had no effect on the tumor growth in vivo, 90 mg/kg TNP-470 in combination with 0.25 mg/kg CDDP had a significant effect. In vitro examinations demonstrated that the growth of HPC-3H4 cells was only mildly inhibited by TNP-470, but the production of vascular endothelial growth factor (VEGF) by HPC-3H4 was clearly inhibited by TNP-470. The inhibitory effect on the production of VEGF was not strong with CDDP treatment. These results indicate that the angiogenesis inhibitor TNP-470 in combination with low-dose CDDP has inhibitory activity against liver metastasis of human pancreatic carcinoma.     

Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2

A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.     

Inhibition of platelet-derived growth factor autocrine growth stimulation by a monoclonal antibody to the human alpha platelet-derived growth factor receptor

A potent neutralizing monoclonal antibody to the human alpha platelet-derived growth factor (PDGF) receptor (alpha PDGFR) was raised by immunizing BALB/c mice with 32D cells expressing the human alpha PDGFR. This monoclonal antibody, designated alpha R1, immunoprecipitated human, monkey, rabbit, pig, dog, and cat, but not hamster, rat, or mouse alpha PDGFRs. Comparison with PR292, a monoclonal antibody previously generated against the alpha PDGFR, showed that both recognized alpha PDGFR extracellular domains, but neither demonstrated reactivity against the beta PDGFR. In vitro binding studies revealed that alpha R1, but not PR292, detection of the alpha PDGFR was blocked by either PDGF AA or PDGF BB. These results strongly suggest that the receptor ligand-binding domain spatially overlapped with the alpha R1 epitope. Monoclonal antibody alpha R1 also inhibited PDGF stimulation of [3H]thymidine uptake by 32D cells expressing the alpha PDGFR (32D alpha R) as well as autocrine growth stimulation of 32D alpha R cells transfected with and expressing PDGF AA or PDGF BB. Therefore, monoclonal antibody alpha R1 may be useful in the detection and growth inhibition of malignancies in which PDGF autocrine stimulation and/or alpha PDGFR overexpression plays an important role(s).     

Inhibition of angiogenesis by thalidomide requires metabolic activation, which is species-dependent

Thalidomide has been shown to be an inhibitor of angiogenesis in a rabbit cornea micropocket model; however, it has failed to demonstrate this activity in other models. These results suggest that the anti-angiogenic effects of thalidomide may only be observed following metabolic activation of the compound. This activation process may be species specific, similar to the teratogenic properties associated with thalidomide. Using a rat aorta model and human aortic endothelial cells, we co-incubated thalidomide in the presence of either human, rabbit, or rat liver microsomes. These experiments demonstrated that thalidomide inhibited microvessel formation from rat aortas and slowed human aortic endothelial cell proliferation in the presence of human or rabbit microsomes, but not in the presence of rat microsomes. In the absence of microsomes, thalidomide had no effect on either microvessel formation or cell proliferation, thus demonstrating that a metabolite of thalidomide is responsible for its anti-angiogenic effects and that this metabolite can be formed in both humans and rabbits, but not in rodents.     

Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth

Previously, we found a positive correlation between the expression of platelet-type 12-lipoxygenase (12-LOX) and the progression of human prostate adenocarcinoma (PCa; Gao et al., Urology, 46: 227-237, 1995). To determine the role of 12-LOX in PCa progression, we generated stable 12-LOX-transfected PC3 cells, which synthesize high levels of 12-LOX protein and 12(S)-hydroxyeicosatetraenoic acid metabolite. In vitro, 12-LOX-transfected PC3 cells demonstrated a proliferation rate similar to neo controls. However, following s.c. injection into athymic nude mice, 12-LOX-transfected PC3 cells formed larger tumors than did the controls. Decreased necrosis and increased vascularization were observed in the tumors from 12-LOX-transfected PC3 cells. Both endothelial cell migration and Matrigel implantation assays indicate that 12-LOX-transfected PC3 cells were more angiogenic than their neo controls. These data indicate that 12-LOX stimulates human PCa tumor growth by a novel angiogenic mechanism.     

Inhibition of angiogenesis by antitumor antibiotic C1027 and its effect on tumor metastasis

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na+/H+ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes.     

Inhibition of human brain tumor cell growth by a receptor-operated Ca2+ channel blocker

SK&F 96365, a reported receptor-operated Ca2+ channel blocker, inhibited the growth of U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines in a dose-dependent manner. Carbachol and serum which act as growth factors for these cells induced a rapid, transient increase of intracellular Ca2+ concentration without a sustained increase. SK&F 96365 also exerted a significant inhibition of carbachol or serum-induced intracellular Ca2+ mobilization. These results suggest that SK&F 96365 is a potent inhibitor of brain tumor cell growth and that its effect may be mediated by the inhibition of agonist-induced intracellular Ca2+ mobilization.     

A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1

Concomitant tumor resistance refers to the ability of some large primary tumors to hold smaller tumors in check, preventing their progressive growth. Here, we demonstrate this phenomenon with a human tumor growing in a nude mouse and show that it is caused by secretion by the tumor of the inhibitor of angiogenesis, thrombospondin-1. When growing subcutaneously, the human fibrosarcoma line HT1080 induced concomitant tumor resistance, preventing the growth of experimental B16/F10 melanoma metastases in the lung. Resistance was due to the production by the tumor cells themselves of high levels of thrombospondin-1, which was present at inhibitory levels in the plasma of tumor-bearing animals who become unable to mount an angiogenic response in their corneas. Animals carrying tumors formed by antisense-derived subclones of HT1080 that secreted low or no thrombospondin had weak or no ability to control the growth of lung metastases. Although purified human platelet thrombospondin-1 had no effect on the growth of melanoma cells in vitro, when injected into mice it was able to halt the growth of their experimental metastases, providing clear evidence of the efficacy of thrombospondin-1 as an anti-tumor agent.     

Inhibition of motor axon growth by T-cadherin substrata

As spinal motor neurons project to their hindlimb targets, their growth cones avoid particular regions along their pathway. T-cadherin is discretely distributed in the avoided caudal sclerotome and on extrasynaptic muscle surfaces (B. J. Fredette and B. Ranscht (1994) J. Neurosci. 14, 7331-7346), and therefore, the ability of T-cadherin to inhibit neurite growth was tested in vitro. T-cadherin inhibited neurite extension from select neuron populations both as a substratum, and as a soluble recombinant protein. Anti-T-cadherin antibodies neutralized the inhibition. Spinal motor neurons were inhibited only during the stages of axon growth across the sclerotome and muscle innervation. Inhibitory responses corresponded to neuronal T-cadherin expression, suggesting a homophilic binding mechanism. These results suggest that T-cadherin is a negative guidance cue for motor axon projections.     

Thalidomide inhibits corneal angiogenesis induced by vascular endothelial growth factor

BACKGROUND: Ocular diseases caused by neovascularization are among the leading causes of blindness. No specific pharmacological treatment is available. Among potential drugs, thalidomide deserves special interest since a wide body of clinical experience exists. However, its antiangiogenic effect is controversial. We therefore investigated the effect of thalidomide on corneal angiogenesis induced by vascular endothelial growth factor (VEGF), which has a special role among angiogenic growth factors. METHODS: Corneal neovascularization was induced in NZW rabbits by an intrastromal pellet loaded with 500 or 750 ng VEGF. Animals received two daily feedings of 200 mg/kg thalidomide. RESULTS: Significant inhibition of corneal angiogenesis (P < 0.0001) was caused by the teratogenic dose of thalidomide after the 5th day of treatment and persisted for more than 16 days. No obvious side effects were recorded. CONCLUSIONS: Thalidomide has a significant antiangiogenic effect against VEGF-induced neovasclar growth. Together with earlier findings this observation indicates that the drug inhibits two angiogenic pathways which are mediated through integrin adhesion molecules.     

Inhibition of human caspases by peptide-based and macromolecular inhibitors

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.     

Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells

Inadequate presentation of tumor antigens by host professional antigen-presenting cells (APCs), including dendritic cells (DCs), is one potential mechanism for the escape of tumors from the host immune system. Here, we show that human cancer cell lines release a soluble factor or factors that dramatically affect DC maturation from precursors without affecting the function of relatively mature DCs. One factor responsible for these effects was identified as vascular endothelial growth factor (VEGF). Thus, VEGF may play a broader role in the pathogenesis of cancer than was previously thought, and therapeutic blockade of VEGF action may improve prospects for immunotherapy as well as inhibit tumor neovasculature.