A Chimeric Signal Peptide-Galectin-3 Conjugate Induces Glycosylation-Dependent Cancer Cell-Specific Apoptosis.

PURPOSE: Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide-Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. EXPERIMENTAL DESIGN: sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. RESULTS: We found sGal-3 preferentially binds to ß1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-ß1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. CONCLUSIONS: We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin ß1-dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Poor immune responses of newborn rhesus macaques to measles virus DNA vaccines expressing the hemagglutinin and fusion glycoproteins.

A vaccine that would protect young infants against measles could facilitate elimination efforts and decrease morbidity and mortality in developing countries. However, immaturity of the immune system is an important obstacle to the development of such a vaccine. In this study, DNA vaccines expressing the measles virus (MeV) hemagglutinin (H) protein or H and fusion (F) proteins, previously shown to protect juvenile macaques, were used to immunize groups of 4 newborn rhesus macaques. Monkeys were inoculated intradermally with 200 µg of each DNA at birth and at 10 months of age. As controls, 2 newborn macaques were similarly vaccinated with DNA encoding the influenza virus H5, and 4 received one dose of the current live attenuated MeV vaccine (LAV) intramuscularly. All monkeys were monitored for development of MeV-specific neutralizing and binding IgG antibody and cytotoxic T lymphocyte (CTL) responses. These responses were poor compared to the responses induced by LAV. At 18 months of age, all monkeys were challenged intratracheally with a wild-type strain of MeV. Monkeys that received the DNA vaccine encoding H and F, but not H alone, were primed for an MeV-specific CD8(+) CTL response but not for production of antibody. LAV-vaccinated monkeys were protected from rash and viremia, while DNA-vaccinated monkeys developed rashes, similar to control monkeys, but had 10-fold lower levels of viremia. We conclude that vaccination of infant macaques with DNA encoding MeV H and F provided only partial protection from MeV infection.

16-kDa prolactin inhibits endothelial cell migration by down-regulating the Ras-Tiam1-Rac1-Pak1 signaling pathway.

Angiogenesis plays a key role in promoting tumorigenesis and metastasis. The 16-kDa fragment of prolactin (16k PRL) is an NH(2)-terminal natural breakdown fragment of the intact 23-kDa prolactin and has been shown to have potent antiangiogenic and antitumor activities. The mechanism(s) involved in the action of 16k PRL in endothelial cells remains unclear. In this study, we showed that 16k PRL reduced rat aortic endothelial cell (RAEC) migration in a wound-healing assay and in a Matrigel tube formation assay, suggesting that 16k PRL inhibits endothelial cell migration, an important activity involved in angiogenesis and tumorigenesis. We further investigated how 16k PRL attenuates endothelial cell migration. We first showed that RAEC migration is mediated through the Rho GTPase Rac1, as Rac1 inhibition by the Rac1-specific inhibitor NSC27366 or Rac1 knockdown by small interfering RNA both blocked RAEC migration. We next showed that 16k PRL reduced the activation of Rac1 in a concentration-dependent manner. Furthermore, 16k PRL inhibition of Rac1 is mediated through the suppression of T lymphoma invasion and metastasis 1 (Tiam1) and its upstream activator Ras in a phosphoinositide-3-kinase-independent manner. 16k PRL also down-regulated the phosphorylation of the downstream effector of Rac1, p21-activating kinase 1 (Pak1), and inhibited its translocation to the leading edge of migrating cells. Thus, 16k PRL inhibits cell migration by blocking the Ras-Tiam1-Rac1-Pak1 signaling pathway in endothelial cells.

16-kDa prolactin down-regulates inducible nitric oxide synthase expression through inhibition of the signal transducer and activator of transcription 1/IFN regulatory factor-1 pathway.

Angiogenesis plays a key role in promoting tumorigenesis and metastasis. Several antiangiogenic factors have been shown to inhibit tumor growth in animal models. Understanding their mechanism of action would allow for better therapeutic application. 16-kDa prolactin (PRL), a NH2-terminal natural breakdown fragment of the intact 23-kDa PRL, exerts potent antiangiogenic and antitumor activities. The signaling mechanism involved in 16-kDa PRL action in endothelial cells remains unclear. One of the actions of 16-kDa PRL is to attenuate the production of nitric oxide (NO) through the inhibition of inducible NO synthase (iNOS) expression in endothelial cells. To delineate the signaling mechanism from 16-kDa PRL, we examined the effect of 16-kDa PRL on interleukin IL-1beta-inducible iNOS expression, which is regulated by two parallel pathways, one involving IFN regulatory factor 1 (IRF-1) and the other nuclear factor-kappaB (NF-kappaB). Our studies showed that 16-kDa PRL specifically blocked IRF-1 but not NF-kappaB signaling to the iNOS promoter. We found that IL-1beta regulated IRF-1 gene expression through stimulation of p38 mitogen-activated protein kinase (MAPK), which mediated signal transducer and activator of transcription 1 (Stat1) serine phosphorylation and Stat1 nuclear translocation to activate the IRF-1 promoter. 16-kDa PRL effectively inhibited IL-1beta-inducible p38 MAPK phosphorylation, resulting in blocking Stat1 serine phosphorylation, its subsequent nuclear translocation and activation of the Stat1 target gene IRF-1. Thus, 16-kDa PRL inhibits the p38 MAPK/Stat1/IRF-1 pathway to attenuate iNOS/NO production in endothelial cells.