ObjectiveTo evaluate whether gastrin-releasing peptide (GRP) and GRP receptor (GRP-R) expression correlate with tumor behavior and to examine the mitogenic actions of GRP on neuroblastomas. Summary Background DataNeuroblastoma is the most common solid tumor of infants and children. Despite recent advances in multimodality treatment regimens, the survival for advanced-stage tumors remains dismal. Neuroblastomas are known to produce GRP; however, the proliferative effects of GRP on neuroblastomas have not been elucidated. MethodsSections of paraffin-embedded neuroblastomas from 33 patients were analyzed for GRP and GRP-R protein expression by immunohistochemistry. Functional binding of GRP-R to the Ca 2ϩ signaling pathway was examined. In addition, the proliferative effect of GRP on neuroblastoma cells (SK-N-SH, IMR-32, SH-SY5Y, LAN-1) was determined. ResultsImmunohistochemical analysis showed GRP and GRP-R protein expression in neuroblastomas; an increased expression of GRP-R was noted in a higher percentage of undifferentiated tumors compared with tumors that were benign. GRP-R mRNA was confirmed in neuroblastoma cell lines. GRP treatment resulted in intracellular calcium [Ca 2ϩ ] i mobilization in two cell lines (SK-N-SH, LAN-1). GRP treatment stimulated growth of all four neuroblastoma cell lines; this effect was inhibited in SK-N-SH cells by pretreatment with GRP antibody. ConclusionsThese findings show increased GRP-R expression in the more aggressive and undifferentiated neuroblastomas. The synchronous expression of GRP and its receptor, GRP-R, suggests a role for these proteins in tumor growth. Moreover, these findings show enhanced proliferation of neuroblastoma cells in vitro after GRP treatment, suggesting that GRP may act as an autocrine and/or paracrine growth factor for neuroblastomas. Treatment with specific GRP-R antagonists may provide novel adjuvant therapy for neuroblastomas in children.
Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are cytoplasmic adapter proteins that link a wide variety of cell surface receptors to the apoptotic signaling cascade. The purpose of this study was to delineate the signaling pathways and TRAF1 promoter elements responsible for phorbol ester-mediated TRAF1 induction in human colon cancers. Here, we found that the PKC activators, phorbol 12-myristate 13-acetate (PMA) and bryostatin I, induced TRAF1 mRNA expression; pretreatment with actinomycin D blocked PMA-mediated TRAF1 expression suggesting induction at the transcriptional level. In contrast, expression of other TRAFs (TRAF2, 3 and 4) was minimally altered by PMA. Various PKC isoform-selective inhibitors blocked PMAmediated TRAF1 mRNA and promoter stimulation; rottlerin, a selective PKCd inhibitor, had no effect suggesting that Ca 2 þ -dependent PKC isoforms (e.g., PKCa and bI) play a role in TRAF1 regulation. In addition, the MEK/ERK inhibitors, PD98059 and UO126, suppressed PMA-stimulated TRAF1 promoter activity indicating a role for ERK in TRAF1 induction. Moreover, cotransfection of a dominant-negative Raf-1 (Raf-C4) significantly reduced PMA-stimulated TRAF1 promoter activity whereas transfection of dominantnegative Ras or treatment with Ras inhibitors had minimal to no effect on TRAF1 induction suggesting dependence on Raf, but not Ras, activation. Finally, site-specific mutagenesis of functional NF-jB sites (particularly the most proximal site) in the TRAF1 promoter significantly decreased PMA-mediated promoter activity. In conclusion, our results demonstrate selective induction of TRAF1 in human colon cancer cells through a Ca 2 þ -dependent PKC/Raf-1/ERK/NF-jB-dependent pathway.
Neuroblastomas are the most common extracranial solid tumors of childhood. These tumors are associated with an overall poor prognosis, particularly for advanced stage disease. The benzoquinone ansamycin antibiotic, geldanamycin (GA), exhibits potent antitumor activity in certain cancer cell lines by destabilizing important signal transduction proteins (e.g., Raf-1 and Akt). The purpose of our study was to determine whether GA can alter the expression of Raf-1 and Akt, which have been shown to be critical for neuronal cell survival, and induce apoptosis of neuroblastoma cells. Human neuroblastoma cells (SH-SY5Y, SK-N-SH and LAN-1) were treated with GA for a variable period of time. Cell viability was assessed with MTT assays. Apoptosis was assessed with DNA fragmentation ELISA, TUNEL-flow cytometric assay, Western blot and caspase activities. We found that GA decreases cell viability and induces apoptosis in the SH-SY5Y human neuroblastoma cell line. These effects were mediated through activation of caspase-9 and -3, mitochondrial release of cytochrome c and subsequent PARP cleavage. GA-induced apoptosis was associated with a reduction in the level and activity of Raf-1 and Akt. The importance of these proteins was further demonstrated by induction of apoptosis in SH- SY5Y cells by a combination of U0126 (MEK1/2 inhibitor) and LY294002 (an inhibitor of PI3K). Similar to SH-SY5Y cells, other human neuroblastoma cells (SK-N-SH and LAN-1)were sensitive to the effects of GA-induced apoptosis. Taken together, our findings suggest that GA may be a novel therapeutic agent, which may be effective in the treatment of neuroblastomas.
The embryo is a natural allograft and is the only exception to immune rejection, which reflects maternal immune tolerance towards the embryo. However, pregnancy loss is primarily caused by maternal immune rejection of the embryo. The aim of the present study was to explore the effects of combined treatment of programmed death-ligand 1 (Pd-l1) immunoglobulin (ig) and cd40-ligand (cd40l) monoclonal antibody (mab) on immune tolerance in an abortion-prone mating model. Mice were divided into the normal, spontaneous abortion, Pd-l1 ig, cd40l mab and the Pd-l1 ig + cd40l mab groups. on day 14 of gestation, the embryo resorption abortion rates of all the groups was observed. The maternal hypo-responsiveness to paternal antigens was determined using a mixed lymphocyte response and the splenic cd4 + cd25 + T-cell population, major histocompatibility complex (MHc)-ii + , cd80 + and cd86 + cell populations in pregnant female cBa/J mice were analyzed using flow cytometry. The expression levels of intracellular cytokines in the splenic tissues of pregnant cBa/J female mice were analyzed using western blotting. The Pd-l1 ig + cd40l group displayed the lowest resorption rate compared with the other groups. A significant decrease in the proliferative response of maternal splenic immunocompetent cells against paternal antigens, and a significant increase in the proliferative response of maternal splenic cd4 + cd25 + T cells was observed in the Pd-l1 ig + cd40l group compared with the spontaneous abortion group. The number of MHc-ii + , cd80 + and cd86 + bone marrow-derived dendritic cells (dcs) generated by female mice, and the levels of tumor necrosis factor-α and interferon-γ in the spleens of female mice were significantly decreased in the PD-L1 Ig + CD40L mAb group compared with the spontaneous abortion group. By contrast, interleukin-4 levels were significantly increased in the PD-L1 ig + cd40l mab group compared with the spontaneous abortion group. The results suggested that the administration of Pd-l1 ig + cd40l mab on day 4 of gestation, the period of peri-implantation, may induce paternal antigen-specific immunotolerance, leading to the embryo resorption rate of the abortion-prone model being similar to that of the normal pregnancy model. The results indicate that the combined treatment of Pd-l1 ig and anti-cd40l mabs may serve as a potential therapeutic for pregnancy loss.
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