ABSTRACT
In this study, neural stem cells (NSCs) were obtained from the hippocampus using the serum-free culturing. NSCs labeled with 5'-bromo-2'-deoxyuridine (BrdU) were transplanted into transected rat basal forebrain followed by the injection of brain-derived neurotrophic factor (BDNF) into the lateral ventricle. Nestin staining and double-labeling immunohistochemistry were used to detect cell survival and neuronal differentiation of the BrdU labeled cells in the basal forebrain and it was observed that labeled NSCs differentiated into neurons and astrocytes in the basal forebrain. Immunohistochemical detection of p75(NGFR) indicated that the number of cholinergic neurons of the combination groups treated by NSCs, BDNF, and NSCs groups had more significant improvement than that of the injured groups in medial septum (MS) and vertical diagonal branch (VDB). Learning and memory abilities were also measured by Y-maze test and the results support that BDNF can enhance the treatment effects of NSCs transplanted into brain lesion model.
Subject(s)
Alzheimer Disease/pathology , Alzheimer Disease/therapy , Brain-Derived Neurotrophic Factor/therapeutic use , Fornix, Brain/injuries , Neurons/drug effects , Stem Cells/drug effects , Alzheimer Disease/complications , Animals , Animals, Newborn , Behavior, Animal , Bromodeoxyuridine/metabolism , Cell Differentiation/drug effects , Disease Models, Animal , Fornix, Brain/drug effects , Functional Laterality , Hippocampus/cytology , Male , Maze Learning/drug effects , Maze Learning/physiology , Nerve Tissue Proteins/metabolism , Neurons/physiology , Rats , Rats, Sprague-Dawley , Receptor, Nerve Growth Factor/metabolism , Stem Cell Transplantation/methods , Stem Cells/physiologyABSTRACT
Butyrate has potent anti-tumorigenic effects on many colon cancer cell lines, including inhibition of growth and promotion of apoptosis in vitro. Nevertheless, despite the butyrate concentration in the colonic lumen being sufficient to result in the death of almost all cells in vitro, colon cancers still develop and grow in vivo, suggesting that cancer cells must develop mechanisms by which they escape the effects of butyrate observed in vitro. Insulin-like growth factor-II (IGF-II) is an autocrine growth factor in many colon cancer cells. The aim of this study was to determine whether IGF-II influences butyrate-mediated apoptosis in LIM 2405 human colon cancer cells. Butyrate and trichostatin A, both of which are histone deacetylase inhibitors although the latter is more specific, induced apoptosis as determined by floating cell counting, Hoechst 33258 staining, DNA laddering and a cell death detection ELISA. IGF-II inhibited the effects of both agents. Butyrate but not trichostatin A also induced LIM 2405 cell migration. In contrast to the above results, IGF-II enhanced butyrate-induced cell migration. Levels of IGF binding protein-3 (IGFBP-3), which may induce apoptosis by IGF-dependent or -independent mechanisms, were increased by butyrate and trichostatin A; IGF-II augmented this effect. It is therefore unlikely that IGFBP-3 mediates butyrate-induced apoptosis. We suggest that IGF-II inhibits the pro-apoptotic effect of butyrate downstream of histone deacetylase inhibition. In contrast, IGF-II promotes histone deacetylase-dependent IGFBP-3 expression and histone deacetylase-independent migration. IGF-II may promote tumour growth by mediating the development of resistance to the pro-apoptotic effects of butyrate.
Subject(s)
Apoptosis/drug effects , Butyrates/pharmacology , Colonic Neoplasms/pathology , Insulin-Like Growth Factor II/pharmacology , Bisbenzimidazole , Blotting, Western , Cell Movement/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation/drug effects , Histone Deacetylase Inhibitors , Humans , Hydroxamic Acids/pharmacology , Immunoblotting , Insulin-Like Growth Factor Binding Protein 3/metabolism , Insulin-Like Growth Factor II/metabolism , RNA, Messenger/analysis , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/pathologyABSTRACT
IGF-II is an autocrine growth factor for many colon cancer cells. This study aimed to determine the role of IGF-II in proliferation and adhesion of LIM 1215 colon cancer cells. RT-PCR demonstrated expression of IGF-I and IGF-II mRNA. Addition of IGF-I or -II increased monolayer proliferation in a dose-dependent manner. Although addition of IGFBP-6 had no effect on basal proliferation, coincubation of IGFBP-6 decreased IGF-II but not IGF-I-induced proliferation. Colony formation in agar was increased by IGF-II, an effect inhibited by coincubation with IGFBP-6. IGFBP-6 alone significantly decreased colony formation. Preincubation of cells with IGF-II increased adhesion to type IV collagen, fibronectin and laminin. IGFBP-6 had no effect on basal cell adhesion but completely inhibited the effects of IGF-II. LIM 1215 colon cancer cells are therefore IGF-responsive but IGF-II is not a major autocrine factor for these cells in monolayer, suggesting heterogeneity between colon carcinoma cell lines with respect to the role of the IGF system.
Subject(s)
Colonic Neoplasms/pathology , Insulin-Like Growth Factor Binding Protein 6/pharmacology , Insulin-Like Growth Factor II/antagonists & inhibitors , Autocrine Communication , Cell Adhesion/drug effects , Cell Division/drug effects , Extracellular Matrix/metabolism , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor II/genetics , RNA, Messenger/metabolism , Tumor Cells, CulturedABSTRACT
The predominant insulin-like growth factor binding protein (IGFBP) synthesized by PC12 rat phaeochromocytoma cells is IGFBP-6. Since cAMP agonists regulate IGFBP-6 in other cells, and they may increase neurite outgrowth and catecholaminergic enzyme expression in PC12 cells, we studied regulation of IGFBP-6 by these agents. After 72 h incubation, forskolin and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) both increased IGFBP-6 protein levels in conditioned media to maximum levels of 231 +/- 40 and 275 +/- 30%, respectively. Incubation with forskolin resulted in a small, transient rise in IGFBP-6 mRNA levels which was insufficient to account for the increased protein levels. The increased protein levels also could not be attributed to increased cell number, protection of IGFBP-6 from proteolysis or release of IGFBP-6 from a cell-associated reservoir. These findings suggest that increased protein levels may have been due to increased translation of mRNA. Co-incubation of forskolin with dexamethasone (which decreases IGFBP-6 protein and mRNA) demonstrated that the effects of the latter were dominant. The effects of cAMP agonists and IGF-II, which increases IGFBP-6 protein but not mRNA levels, were not inhibited by rapamycin, suggesting that p70 S6 kinase is not involved. The effects of cAMP agonists on IGFBP-6 levels were not directly correlated with neurite outgrowth. These findings extend our knowledge of the molecular basis of the regulation of IGFBP-6 by cAMP agonists, and indicate a novel action of these agents in PC12 cells.
Subject(s)
Cyclic AMP/analogs & derivatives , Cyclic AMP/agonists , Insulin-Like Growth Factor Binding Protein 6/drug effects , Insulin-Like Growth Factor Binding Protein 6/metabolism , Thionucleotides/pharmacology , Animals , Cell Division/drug effects , Colforsin/pharmacology , Cyclic AMP/pharmacology , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Insulin-Like Growth Factor Binding Protein 6/genetics , Insulin-Like Growth Factor II/pharmacology , Neurites/drug effects , PC12 Cells/drug effects , PC12 Cells/metabolism , Rats , Sirolimus/pharmacologyABSTRACT
PC12 rat phaeochromocytoma cells are widely used as a model of neuronal differentiation. They express IGF receptors and are responsive to IGFs. The main IGF-binding protein synthesized by these cells is IGFBP-6. Glucocorticoids induce differentiation of PC12 cells towards a chromaffin phenotype. The effect of dexamethasone on IGFBP-6 levels was therefore studied. Dexamethasone (500 nM) decreased IGFBP-6 protein in conditioned media and mRNA levels to 61 +/- 5% (P < 0.0001) and 34 +/- 14% (P = 0.03) of control levels respectively. Incubation of PC12 cells with IGF-II (100 ng/ml) for 72 h increased IGFBP-6 protein levels in media to 217 +/- 19% of control (P < 0.0001). IGFBP-6 mRNA levels, however, were unchanged. IGF-I had similar effects on IGFBP-6 protein and mRNA levels. IGFs increased cell number by 50-60%, but this was insufficient to explain the increases in protein levels. IGFBP-6 was not released from a cell-associated reservoir or protected from proteolysis by IGFs, excluding these post-translational mechanisms as explanations for the IGF effects on IGFBP-6 levels. The effects of IGF-II and dexamethasone on IGFBP-6 levels were independent. These results indicated that (1) dexamethasone decrease IGFBP-6 at the mRNA level, and (2) IGFs stimulate IGFBP-6 levels by a post-transcriptional mechanism.
