Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

BCL-2 is upregulated in human SH-SY5Y neuroblastoma cells differentiated by overexpression of fibroblast growth factor 1.

Fibroblast growth factor 1 (FGF1) is a multipotent factor in the development and differentiation of the central nervous system. Recent studies in PC12 cells attribute these effects to high endogenous FGF1 expression. To examine the differentiation mechanisms induced by FGF1, we performed studies in SH-SY5Y human neuroblastoma cells. We monitored the impact of FGF1 overexpression in SH-SY5Y either after addition of exogenous FGF1 and heparin or after stable transfection with the FGF1 eukaryotic expression vector. Under both conditions, the FGF1 endogenous rise caused SH-SY5Y cell differentiation with morphological changes (appearance of neuritic extensions), increased GAP-43 gene expression, decreased of N-myc gene expression, and prolonged long-term survival in serum-free media. These modifications were correlated with Bcl-2 upregulation. These results suggest that there is a link between the endogenous FGF1 signaling pathway and Bcl-2 in neuronal survival modulation.     

Production of IGF-II-related peptide by an anaplastic cells line (AT-3) established from the dunning prostatic carcinoma of rats

Summary AT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under serum-free conditions in a state of suspension detached from a substratum. Radioimmunoassays using monoclonal antibody against rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts extracsts of lyophilized serum-free media conditioned by AT-3 cell. The peptide contents in the culture media increased with increase in cell number; 71 ng at 3.0 × 10⁶ cells and 449 ng at 4.6 × 10⁷ cells. IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture. These results indicate that AT-3 cells produce IGF-II-related peptide ana may release it into the culture media. Editor's statement One or more members of the insulin-like growth factor family have been established previously as mitogen for isolated prostate cells. This report suggests that IGF-II member of the family may be involved in autocrine support of cells from highly malignant prostate tumors.     

Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma tumors

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and appears to arise from developing striated muscle-forming cells. Since insulin-like growth factor II (IGF-II) is involved in normal muscle growth and maturation and elevated IGF-II mRNA levels have previously been reported in rhabdomyosarcomas, we have been studying the possible role of IGF-II in the unregulated growth and invasive potential of these embryonal tumors. In this study, we demonstrate that 13 of 14 rhabdomyosarcoma tumors express high levels of IGF-II mRNA relative to normal adult muscle and also express mRNA for the type I IGF receptors on their cell surface, the receptor thought to mediate the effects of IGF-II on muscle cells. We have established several rhabdomyosarcoma cell lines in mitogen-free media and demonstrate that these cells express type I IGF receptors on their cell surface and secrete IGF-II into the media. Exogenous IGF-II is able to stimulate cellular motility in these cell lines as assayed in a modified Boyden chamber. Finally, alpha IR-3, a type I receptor antagonist, inhibits the growth of these cell lines in serum-free media but does not inhibit IGF-II-induced motility of these cells. These data suggest that endogenously produced IGF-II functions as an autocrine growth and motility factor in many rhabdomyosarcoma tumors. The mitogenic actions of IGF-II are mediated through a domain of the type I IGF receptor that is blocked by alpha IR-3. IGF-II-induced motility may be mediated through an alternative signaling pathway.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation

Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and beta-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13-acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to approximately 70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I.     

Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10⁶ cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10⁶ cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10⁶cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.     

Insulin-like growth factor I receptor involvement in proliferation of NOR-P1 cells in serum-free media

Insulin-like growth factor (IGF)-I is up-regulated in pancreatic cancer tissues. Pancreatic cancer cell lines were analyzed in serum-free media as a model of the fibrous tissues that these cells often invade. Pancreatic cancer surgical specimens were immunostained with anti-IGF-I receptor (IGF-IR)β antibody. The growth of pancreatic cancer cells in serum-free media was also analyzed. Cell lysates were analyzed for protein by western blot analysis. Cells cultured in the presence of picropodophyllin (PPP), LY294002, or PD98059, were subjected to cell proliferation and scratch assays. In addition, BrdU uptake and apoptosis were analyzed in these cells. IGF-IRβ was detected in pancreatic cancer cells invading fibrous tissues. NOR-P1 grew most rapidly in serum-free media. The concentrations of IGF-I and IGF-II in the media were higher in NOR-P1 than the other cell lines. Cell proliferation in NOR-P1 cells was enhanced by IGF-I or IGF-II treatment more than in MIA-Paca2 or PK-1 cells. PPP, LY294002, and PD98059 suppressed proliferation and motility of NOR-P1 cells and inhibited BrdU uptake, while PPP induced apoptosis. IGF-IRβ may be a potential therapeutic target to inhibit invasion of pancreatic cancer.     

Autocrine growth factors for human tumor clonogenic cells

A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.     

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When [125I]IGF-I or [125I]IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. The dissociation constants were determined to be 0.106 nM for IGF-I and 0.209 nM for IGF-II binding. Cross-linking of [125I]IGF-I or [125I]IGF-II to the CMs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range 24-32 kDa. The binding was competitively inhibited by addition of cold IGF-I and IGF-II but not by insulin. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

LncRNA MALAT1 targeting miR-124-3p regulates DAPK1 expression contributes to cell apoptosis in Parkinson's Disease

Death associated protein kinase 1 (DAPK1) was initially discovered in the progress of gamma-interferon induced programmed cell death, it is a key factor in the central nervous system, including Parkinson's disease (PD). However, the underlying mechanisms of DAPK1 in PD remain unclear and this research work aims to explore the potential mechanisms of DAPK1 in PD. In the study, we exposed SH-SY5Y cells to MPP⁺ and treated mice with MPTP to investigate the roles of DAPK1 in PD and the underlying mechanisms. The results indicated that the expression of DAPK1 is significantly upregulated and negatively correlated with miR-124-3p levels in SH-SY5Y cells treated by MPP⁺, and miR-124-3p mimics could effectively inhibit DAPK1 expressions and alleviate MPP⁺-induced cell apoptosis. In addition, knockdown MALAT1 reduces the levels of DAPK1 and the ratio of SH-SY5Y cell apoptosis, which is reversed via miR-124-3p inhibitor in vitro. Similarly, knockdown MALAT1 could improve behavioral changes and reduce apoptosis by miR-124-3p upregulation and DAPK1 downregulation in MPTP induced PD mice. Taken together, our data showed that lncRNA MALAT1 positively regulates DAPK1 expression by targeting miR-124-3p, and mediates cell apoptosis and motor disorders in PD. In summary, these results suggest that MALAT1/miR-124-3p /DAPK1 signaling cascade mediates cell apoptosis in vitro and in vivo, which may provide experimental evidence of developing potential therapeutic strategies for PD.     

Retinoic acid evoked-differentiation of neuroblastoma cells predominates over growth factor stimulation: an automated image capture and quantitation approach to neuritogenesis.

To facilitate the characterization of compounds that have positive growth factor mimetic effects on neuritogenesis, we have implemented a high-throughput functional assay which measures, in a multiparametric manner, the proliferation and differentiation characteristics of cells in a microtiter plate. Conditions were established using chronic incubation of SH-SY5Y human neuroblastoma cells with retinoic acid (RA) and/or nerve growth factor (NGF) in which discernible alterations in proliferation, growth, and differentiation of cells were induced. SH-SY5Y cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on Cellomics ArrayScanII was utilized to quantify the effects of these treatments on cell characteristics. NGF and retinoic acid were found to increase multiple parameters of SH-SY5Y differentiation, including an increased proportion of cells having neurites and increased extent of branching. However, marked differences in the effects of these compounds on SH-SY5Y growth and differentiation were also detected: whereas NGF increased cell number, RA treatment decreased cell number, and RA but not NGF caused significant elongation of neurites. This study quantifies and characterizes the effects of differentiating and proliferating agents on a human-derived neuroblastoma cell line. The high-content, rapid-throughput nature of this assay makes it ideal for functional identification and characterization of compounds regulating cell behavior.     

Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 +/- 3% reduction 96 h after adding 5 microM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (M(r)) IGF-II and higher M(r) forms of IGF-II. The levels of the higher M(r) and the mature form of IGF-II were decreased 50 +/- 3% and 22 +/- 2%, respectively, by 5 microM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.