Purpose: Neurotensin, a gut tridecapeptide, acts as a potent cellular mitogen for various colorectal and pancreatic cancers that possess high-affinity neurotensin receptors. Cytokine/ chemokine proteins are increasingly recognized as important local factors that play a role in the metastasis and invasion of multiple cancers. The purpose of this study was to (a) determine the effect of neurotensin on cytokine/chemokine gene expression and cell migration in human cancer cells and (b) assess the effect of curcumin, a natural dietary product, on neurotensinmediated processes. Experimental Design: The human colorectal cancer, HCT116, was treated with neurotensin, with or without curcumin, and interleukin (IL)-8 expression and protein secretion was measured. Signaling pathways, which contribute to the effects of neurotensin, were assessed. Finally, the effect of curcumin on neurotensin-mediated HCT116 cell migration was analyzed. Results: We show that neurotensin, acting through the native high-affinity neurotensin receptor, induced IL-8 expression in human colorectal cancer cells in a time-and dose-dependent fashion. This stimulation involves Ca 2+ -dependent protein kinase C, extracellular signal-regulated kinased ependent activator protein-1, and extracellular signal-regulated kinase^independent nuclear factor-nB pathways. Curcumin inhibited neurotensin-mediated activator protein-1 and nuclear factor-nB activation and Ca 2+ mobilization. Moreover, curcumin blocked neurotensin-stimulated IL-8 gene induction and protein secretion and, at a low concentration (i.e., 10 Amol/L), blocked neurotensin-stimulated colon cancer cell migration. Conclusions: Neurotensin-mediated induction of tumor cell IL-8 expression and secretion may contribute to the procarcinogenic effects of neurotensin on gastrointestinal cancers. Furthermore, a potential mechanism for the chemopreventive and chemotherapeutic effects of curcumin on colon cancers may be through the inhibition of gastrointestinal hormone (e.g., neurotensin)î nduced chemokine expression and cell migration.

Gastrin-releasing peptide (GRP) and its

Acute desensitization of many guanine nucleotide-binding protein-coupled receptors (GPCRs) requires receptor phosphorylation and subsequent binding of an arrestin. GPCRs are substrates for phosphorylation by several classes of kinases. Gastrin-releasing peptide receptor (GRPr) is phosphorylated by a kinase other than protein kinase C (PKC) after exposure to agonist and is also a substrate for PKC-dependent phosphorylation after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using GRPr mutants, we examined receptor domains required for agonist-and TPA-induced phosphorylation of GRPr and consequences of these phosphorylation events on GRPr signaling via G q . Agonist-and TPA-stimulated GRPr phosphorylation in cells require an intact carboxyl terminal domain (CTD). GRPr is phosphorylated in vitro by GPCR kinase 2 (GRK2) and multiple PKC isoforms. An intact DRY motif is required for agonist-stimulated phosphorylation in cells, and agonist-dependent GRK2 phosphorylation in vitro. Although GRPr CTD mutants do not show enhanced in vitro coupling to G q relative to intact GRPr, CTD mutants have more potent G q -dependent signaling in cells. Acute desensitization involves CTD-independent processes because desensitization can precede ligand binding in intact GRPr and CTD mutants. TPAmediated impairment of GRPr-G q signaling in cells also requires an intact CTD. Similar to GRK2 phosphorylation, PKC phosphorylation reduces GRPr-G q coupling by approximately 80% in vitro. Arrestin translocation to plasma membrane requires agonist, an intact DRY motif, and GRPr phosphorylation. Therefore, agonist-and PKC-induced GRPr phosphorylation sites are in nearby regions of the receptor, and phosphorylation at both sites has similar functional consequences for G q signaling.An activated GPCR catalyzes guanine nucleotide exchange on the ␣ subunit of a heterotrimeric G protein, leading to the formation of G␣-GTP. The subsequent dissociation of the heterotrimeric G protein leads to stimulation of signal transduction cascades mediated by the free G␣-GTP and G␤␥ subunits. Mechanisms have evolved that limit the amplitude and/or duration of signal transduction cascades and are collectively referred to as "desensitization". At the molecular level, desensitization may result from the degradation of ligand, changes in receptor availability or activity, or changes in the availability or activity of downstream effector molecules. The most extensively studied GPCRs have been rhodopsin, which signals through transducin (G t ), and the ␤ 2 -adrenergic receptor (␤ 2 AR), which signals through G s . In both cases, rapid agonist-induced receptor phosphorylation, along with subsequent binding of an arrestin to the receptor, play critical roles in receptor deactivation (Krupnick and Benovic, 1998).Arrestins directly interact with phosphorylated, agonistoccupied GPCRs. Visual arrestin uncouples rhodopsin from transducin and desensitizes rhodopsin signaling. The two mammalian nonvisual arrestins, arrestin2 (␤-arrestin1) and R.A.A. and K.L...

No abstract

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133− cells. We hypothesized that CD133+ cells, compared to CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma –associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and – cells using FACS. The CD133+ cells formed large tumors in NOD-SCID mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change, and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs. − cells. RT PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs − cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2 (FGFR2). The CAF highly express the respective ligands: SDF-1, vitronectin, and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in [Ca2+]I in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension, compared to only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew both more colonies compared to vehicle as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared to CD133−, cells is due to their increased ability to interact with their neighboring CAF.