Insulin-like growth factor binding protein-3 (IGFBP-3)is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of IGFBP-3 has been observed and nuclear binding partners for IGFBP-3 demonstrated. However, little is known about the mechanism of IGFBP-3 internalization. We hypothesized that IGFBP-3 is first secreted then taken up again into cells and that its internalization could occur via binding to transferrin or caveolin. Incubation of cells with an IGFBP-3-neutralizing antibody demonstrated that nuclear translocation of endogenous IGFBP-3 requires IGFBP-3 secretion and re-uptake. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3. Co-immunoprecipitation assays confirmed that IGFBP-3 binds transferrin but not directly to the transferrin receptor (TfR1); however, transferrin binds TfR1 and a ternary complex is formed. Specific binding to caveolin scaffolding docking sequence was confirmed utilizing radiolabeled IGFBP-3. Blocking TfR1-mediated endocytosis prevents both endogenous and exogenous IGFBP-3 re-uptake and inhibitors of caveolae formation also retard IGFBP-3 nuclear entry. Co-treatment with anti-transferrin receptor antibody and cholesterol depletion agents completely abolished endogenous and exogenous IGFBP-3 uptake. Suppression of IGFBP-3 internalization by TfR1 blockade inhibited IGFBP-3-induced apoptosis. Together, these data indicate that the actions of IGFBP-3 are mediated by internalization via distinct endocytic pathways.

We have previously identified the retinoid X receptor-a (RXRa) as an insulin-like growth factor binding protein-3 (IGFBP-3) nuclear binding partner, which is required for IGFBP-3-induced apoptosis. In the current study, we investigated the biological interactions of the RXR ligand, VTP194204 and rhIGFBP-3, in vitro and in vivo. In vitro, IGFBP-3 and VTP194204 individually induced apoptosis, and suppressed cell growth in prostate cancer cell lines in an additive manner. In vivo, LAPC-4 xenograft^bearing severe combined immunodeficiency mice treated daily with saline, IGFBP-3, and/or VTP194204 for 3 weeks showed no effect of individual treatments with IGFBP-3 or VTP194204 on tumor growth. However, the combination of IGFBP-3 and VTP194204 treatments inhibited tumor growth by 50% and induced a significant reduction in serum prostate-specific antigen levels. In terminal nucleotidyl transferase^mediated nick end labeling immunohistochemistry of LAPC-4 xenografts, there was modest induction of apoptosis with either IGFBP-3 or VTP194204 individual treatment, but combination therapy resulted in massive cell death, indicating that IGFBP-3 and VTP194204 have a synergistic effect in preventing tumor growth by apoptosis induction. In summary, this is an initial description of the successful therapeutic use of IGFBP-3 as a cancer therapy in vivo, and shows that combination treatment of IGFBP-3 and RXR ligand has a synergistic effect on apoptosis induction leading to substantial inhibition of prostate cancer xenograft growth. Taken together, these observations suggest that combination therapy with IGFBP-3 and RXR ligands may have therapeutic potential for prostate cancer treatment.The insulin-like growth factor (IGF) system is well recognized to play a critical role in cancer development. In particular, IGF binding proteins (IGFBP) have been proposed to act as growth inhibitory molecules through their IGF inhibitory effects. However, a direct IGF-independent role of IGFBPs, particularly IGFBP-3 in the regulation of apoptosis, has emerged over the last decade (1). A key intracellular role for IGFBP-3 was shown in 2000 with the discovery of the nuclear retinoid X receptor a (RXRa) as a binding partner for IGFBP-3. In that study, IGFBP-3 cotransfections potently and dose-dependently inhibited retinoic acid signaling via retinoic acid response element, but enhanced RXR-specific ligand signaling via the retinoid X response element, supporting a co-activator/repressor role for IGFBP-3 in transcription. In addition, IGFBP-3 had no discernible effects in an RXR knock-out line, indicating that RXR is required for IGFBP-3-induced apoptosis. In both androgen-dependent and -independent prostate cancer cell lines, additive effects of IGFBP-3 and RXR ligand on apoptosis were shown (2).Retinoids (including RXR ligands) are prime candidates for cancer chemoprevention because cancer is characterized by abnormal growth with lack of differentiation, which could be modified by retinoids. The nuclear retinoic acid receptors and RXRs media...