Fumagillin suppresses angiogenesis in cancer models and clinical trials, but it is associated with neurotoxicity at systemic doses. In this study, alpha(nu)beta(3)-targeted fumagillin nanoparticles were used to suppress the neovasculature and inhibit Vx-2 adenocarcinoma development using minute drug doses. Tumor-bearing rabbits were treated on days 6, 9, and 12 postimplantation with alpha(nu)beta(3)-targeted fumagillin nanoparticles (30 microg/kg), alpha(nu)beta(3)-targeted nanoparticles without drug, nontargeted fumagillin nanoparticles (30 microg/kg) or saline. On day 16, MRI was performed with alpha(nu)beta(3)-targeted paramagnetic nanoparticles to quantify tumor size and assess neovascularity. Tumor volume was reduced among rabbits receiving alpha(nu)beta(3)-targeted fumagillin nanoparticles (470+/-120 mm(3)) compared with the three control groups: nontargeted fumagillin nanoparticles (1370+/-300 mm(3), P<0.05), alpha(nu)beta(3)-targeted nanoparticles without drug (1080+/-180 mm(3), P<0.05) and saline (980+/-80 mm(3), P<0.05). MR molecular imaging of control rabbits (no fumagillin) revealed a predominant peripheral distribution of neovascularity representing 7.2% of the tumor rim volume, which decreased to 2.8% (P<0.05) with alpha(nu)beta(3)-targeted fumagillin nanoparticle treatment. Microscopically, the tumor parenchyma tended to show T-cell infiltration after targeted fumagillin treatment, which was not appreciated in control animals. These results suggest that alpha(nu)beta(3)-targeted fumagillin nanoparticles could provide a safe and effective means to deliver MetAP2 inhibitors alone or in combination with cytotoxic or immunotherapy.

hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.

Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis. Integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. We describe the activities of two synthetic low-molecular-weight peptidomimetics of the ligand amino acid sequence arg-gly-asp (RGD) in integrin-based functional assays in vitro. We also evaluate efficacy and potential mechanisms of action in models of both spontaneous and experimental metastasis. Broad-spectrum potency against the family of alpha v subunit-containing integrins was observed, with significantly less potency against alpha5beta1 and alpha(IIb)beta3. Both endothelial and tumor cell migration mediated by alpha(v)beta3 was inhibited, whereas proliferation of endothelial cells but not tumor cells was diminished. Continuous infusion of compound by minipumps or oral administration twice daily significantly reduced metastatic tumor burden in the lungs of mice despite no reduction in growth of 435/HAL primary tumors, and only a slight reduction in tumor cells detected in circulating blood. Delaying treatment in this model until after extensive dissemination of tumor cells to the lungs had occurred, and after primary tumor resection, still produced significant efficacy. Conversely, administration of the agent for only the first 18 h after tumor-cell inoculation into the tail vein also resulted in decreased metastases observed after several weeks. These data suggest these compounds or their relatives have potential to interfere with both early and late steps of metastasis involving tumor and endothelial cell functions. Furthermore, the metastatic process can be effectively inhibited independently of primary tumor growth using integrin antagonists.

Purpose Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide, which may contribute to the aggressive and refractory nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide in the extracellular environment. The current work tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the redox balance in PDA. Experimental Design We evaluated the prognostic significance of EcSOD in a human tissue microarray of patients with PDA. EcSOD overexpression was performed in PDA cell lines and animal models of disease. The impact of EcSOD on PDA cell lines was evaluated with Matrigel invasion in combination with a superoxide-specific SOD mimic and a nitric oxide synthase inhibitor to determine the mechanism of action of EcSOD in PDA. Results Loss of EcSOD expression is a common event in PDA, which correlated with worse disease biology. Overexpression of EcSOD in PDA cell lines resulted in decreased invasiveness that appeared to be related to reactions of superoxide with nitric oxide. Pancreatic cancer xenografts overexpressing EcSOD also demonstrated slower growth and peritoneal metastasis. Over-expression of EcSOD or treatment with a superoxide-specific SOD mimic caused significant decreases in PDA cell invasive capacity. Conclusions These results support the hypothesis that loss of EcSOD leads to increased reactions of superoxide with nitric oxide which contributes to the invasive phenotype. These results allow for the speculation that superoxide dismutase mimetics might inhibit PDA progression in human clinical disease.

Recent studies have shown that Chinese hamster ovary (CHO) cells transfected with the FSH subunit genes secrete bioactive FSH. Here, we determined the in vitro and in vivo bioactivity of recombinant FSH produced by CHO mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase-I (NAGT-), resulting in glycoproteins with asparagine-linked (GlcNAc)2(Mannose)5 oligosaccharides, or mutant cells defective in sialic acid transport into the Golgi (ST-). In the latter, glycoproteins are secreted lacking terminal sialic acids. Determination of in vitro bioactivity, using the granulosa cell aromatase bioassay, indicated that both FSH variants are as active as FSH secreted by the wild type (WT) cells and purified pituitary FSH. Also, these normal and variant forms of FSH are equipotent in a radioligand receptor assay using rat testis membranes. However, the variant FSH molecules are more basic than the WT FSH as determined using a chromatofocusing column (pI: wild type 3.6-5.0, NAGT- greater than 7.0, ST- approximately 6.0 and greater than 7.0). Injection of immature estrogen-treated rats with WT FSH induced high aromatase activity in their granulosa cells whereas treatment with either one of the FSH variants was ineffective; the lack of in vivo activity of the FSH variants was correlated with rapid clearance of these molecules in serum. Thus, recombinant human FSH produced by cells deficient in NAGT-I or defective in sialic acid transport retains normal receptor binding and in vitro bioactivity, but exhibits minimal in vivo activity and a shortened half-life when compared to WT FSH, indicating the important role of terminal sugars for FSH action in vivo.