Rhabdomyosarcoma is the most common soft-tissue sarcoma of childhood. Rhabdomyosarcoma cell lines overexpress insulin-like growth factor-II (IGF-II), an autocrine growth factor that is inhibited by insulin-like growth factor binding protein-6 (IGFBP-6). IGFBP-6 is associated with myoblast quiescence, and expression in rhabdomyosarcoma cells is low. The effect of IGFBP-6 on 2 rhabdomyosarcoma cell lines, RD and Rh30, was studied. IGFBP-6 inhibited anchorage-dependent growth of RD and Rh30 cells in a dosedependent manner (p < 0.0001). IGFBP-6 also inhibited anchorage-independent growth of RD cells in soft agar in a dose-dependent manner (p < 0.01). Anchorage-independent growth of RD cells on polyhydroxyethylmethacrylate-coated plates was decreased to a minimum of 48% of control after treatment with IGFBP-6 (p < 0.001). In this system, IGFBP-6 increased apoptosis 4-fold (p < 0.001). IGF-II partially reversed the IGFBP-6-induced decrease in growth and increase in apoptosis. Rh30 cells were stably transfected with an IG-FBP-6 cDNA and subcutaneous xenografts established in BALB/c nude mice. After 18 days, sizes of 2 independent clones of IGFBP-6-overexpressing Rh30 cells were reduced to 12% and 26% of vector control-transfected tumors (p ‫؍‬ 0.0006 and 0.002, respectively). IGFBP-6 therefore inhibits proliferation and promotes apoptosis of rhabdomyosarcoma in vitro and dramatically inhibits xenograft growth in vivo, at least in part by inhibiting IGF-II. Low expression of IGFBP-6 may therefore contribute to rhabdomyosarcoma growth and metastasis.

The presence of advanced glycation end products (AGEs) formed because of hyperglycemia in diabetic patients has been strongly linked to the development of diabetic complications and disturbances in cellular function. In this report, we describe the isolation and identification of novel AGE-binding proteins from diabetic rat kidneys. The proteins were purified by cation exchange and AGE-modified bovine serum albumin (AGE-BSA) affinity chromatography.

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a HIF-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and VEGF, a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ∼40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.