IGF-II Protein Levels Research Articles

High IGF1R protein expression correlates with disease-free survival of patients with stage III colon cancer

The aim of this study was to investigate the association between expression of insulin-like growth factor-1 receptor (IGF1R) and its ligand, IGF-II, and disease-free survival (DFS) in patients with stage III colon cancer (CC). In this retrospective study we included consecutive patients who underwent curative surgery for stage III CC. IGF1R and IGF-II/IGF2 status were evaluated in tumour samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Associations of markers with DFS were analysed using Cox proportional hazards models. Hundred and fifty-one CC patients were included (median age, 66.6years; female, 54.3%). Low levels of IGF1R and IGF-II protein expression were observed in 16.1% and 10.7% of the cases, respectively. No significant differences in clinicopathological characteristics between patients with tumours expressing low IGF1R or IGF-II protein levels and those with high levels were observed. A low IGF1R protein expression was found to be significantly associated with a shorter DFS (HR 3.32; 95% CI, 1.7-6.31; p= 0.0003), while no association was observed between IGF-II protein expression and DFS (HR 0.91; 95% CI, 0.28-2.96; p= 0.87). In a multivariate analysis, IGF1R protein status remained an independent prognostic factor for DFS (HR 2.73; 95% CI, 1.40-5.31; p= 0.003). Furthermore, we found that neither IGF1R nor IGF2 mRNA expression levels as measured by qRT-PCR correlated with the respective protein expression levels as assessed by immunohistochemistry. Neither of the mRNA expression levels was significantly associated with DFS. From our data we conclude that low IGF1R protein expression represents a poor prognostic biomarker in stage III colon cancer.

Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus.

Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short post-embryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes.

3-M syndrome: a growth disorder associated with IGF2 silencing.

3-M syndrome is an autosomal recessive disorder characterised by pre- and post-natal growth restriction, facial dysmorphism, normal intelligence and radiological features (slender long bones and tall vertebral bodies). It is known to be caused by mutations in the genes encoding cullin 7, obscurin-like 1 and coiled-coil domain containing 8. The mechanisms through which mutations in these genes impair growth are unclear. The aim of this study was to identify novel pathways involved in the growth impairment in 3-M syndrome. RNA was extracted from fibroblast cell lines derived from four 3-M syndrome patients and three control subjects, hybridised to Affymetrix HU 133 plus 2.0 arrays with quantitative real-time PCR used to confirm changes found on microarray. IGF-II protein levels in conditioned cell culture media were measured by ELISA. Of the top 10 downregulated probesets, three represented IGF2 while H19 was identified as the 23rd most upregulated probeset. QRT-PCR confirmed upregulation of H19 (P<0.001) and downregulation of IGF2 (P<0.001). Levels of IGF-II secreted into conditioned cell culture medium were higher for control fibroblasts than those for 3-M fibroblasts (10.2±2.9 vs 0.6±0.9 ng/ml, P<0.01). 3-M syndrome is associated with a gene expression profile of reduced IGF2 expression and increased H19 expression similar to that found in Silver–Russell syndrome. Loss of autocrine IGF-II in the growth plate may be associated with the short stature seen in children with 3-M syndrome.

The production and regulation of IGF and IGFBPs in human adipose tissue cultures

BackgroundAdipocytes express and secrete IGFs and IGFBPs; proteins with important effects on adipocyte homeostasis. However, the factors that control adipocyte generation of IGFs and IGFBPs are not clarified. AimTo identify regulators of the synthesis of IGFs and IGFBs in adipose tissue. MethodsSubcutaneous adipose tissue fragments (500mg) from 7 healthy lean women were incubated for 48h following addition of GH (50μg/l), dexamethasone (DXM, 20nM), insulin (100nM), interleukin (IL)-1β (50ng/l), IL-6 (50ng/l) and tumor-necrosis factor (TNF)-α (10ng/l). Outcome parameters included tissue mRNA and culture media IGF and IGFBP levels. ResultsAdipose tissue cultures secreted more IGF-II than IGF-I protein (1.14±0.41 vs. 0.26±0.09μg/l [mean±SEM]; P<0.02). IGF-I mRNA and protein levels were stimulated by GH (to 340% [153; 477] (median [interquartiles]) and 270±26%, respectively; P<0.003), and inhibited by IL-1β (to 28% [21; 77] and 68±11%, respectively; P<0.003). TNF-α reduced IGF-I and IGF-II protein levels to 51±8% and 69±8%, respectively (P≤0.002), without affecting mRNA levels. IGF protein levels were unaffected by DXM, insulin and IL-6. All IGFBPs IGFBP-1 were expressed. IGFBP-4 was by far the most predominant IGFBP by immunoassay and WLB revealed two bands at 28 and 24kDa, most likely representing glycosylated and non-glycosylated IGFBP-4. ConclusionAdipose tissue cultures secrete more IGF-II than IGF-I, and predominantly IGFBP-4. The secretion of IGF-I is affected by GH, IL-1β and TNF-α, whereas IGF-II is affected by TNF-α only. Hence, cytokines may control adipocyte homeostasis by affecting local IGF-generation.

Dose‐dependent growth inhibition in vivo of PC‐3 prostate cancer with a reduction in tumoral growth factors after therapy with GHRH antagonist MZ‐J‐7‐138

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers and affect tumoral growth factors. We investigated the effect of a new GHRH antagonist MZ-J-7-138 at doses of 1.25, 2.5, 5 and 10 microg/day s.c. on the growth of PC-3 human androgen independent prostate cancers xenografted s.c. into nude mice. Binding assays were used to investigate GHRH receptors. The levels of IGF-II and VEGF in tumors were measured by radioimmunoassays. Treatment with 2.5, 5, and 10 microg/day MZ-J-7-138 caused a significant dose-dependent growth reduction of PC-3 tumors. The greatest inhibition of 78% was obtained with 10 microg/day. The suppression of IGF-II protein levels in tumors was seen at all doses of MZ-J-7-138, but only 10 microg dose induced a significant inhibition. MZ-J-7-138 also reduced VEGF protein levels, the inhibition being significant at doses of 5 and 10 microg. Specific high affinity binding sites for GHRH were found on PC-3 tumors using (125)I-labeled GHRH antagonist JV-1-42. MZ-J-7-138 displaced radiolabeled JV-1-42 with an IC(50) of 0.32 nM indicating its high affinity to GHRH receptors. Real-time PCR analyses detected splice variant 1 (SV1) of GHRH receptor (GHRH-R) as well as pituitary type of GHRH-R and GHRH ligand. Our results demonstrate the efficacy of GHRH antagonist MZ-J-7-138 in suppressing growth of PC-3 prostate cancer at doses lower than previous antagonists. The reduction of levels of growth factors such as VEGF and IGF-II in tumors by GHRH antagonist was correlated with the suppression of tumor growth.

Dynamic regulation of estrogen receptor-α isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insulin-like growth factors

Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ERalpha isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ERalpha isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ERalpha was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ERalpha isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E(2) or PPT, an ERalpha agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E(2)- or PPT-induced downregulation of tubal ERalpha isoform expression in mice. However, alteration of ERalpha immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ERalpha isoform expression were found to be coupled to multiple E(2) effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E(2) exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E(2) of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ERalpha.

Involvement of insulin-like growth factors-I and -II and their receptors in medroxyprogesterone acetate-induced growth of mouse mammary adenocarcinomas

The role of the insulin-like growth factors (IGFs) system was investigated in hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. IGF-II protein and message showed a three- to four-fold increase in HD lines growing in MPA-treated mice, as compared with HD tumors growing in untreated mice. Progression to a hormone-independent phenotype in all these lines was accompanied by a high constitutive expression of IGF-II. Similar IGF-I mRNA levels were detected in HD and HI lines. Both IGF-I and -II messages arose from the malignant epithelial cells, as shown by in situ hybridization studies. A significant decrease in Man-6P/type II IGF-R content was detected in HD tumors growing in MPA-treated mice as compared with HD lines growing in untreated mice. On the other hand, in HI tumors, notwithstanding high IGF-II synthesis, the levels of Man-6P/type II IGF-R remain high. Competitive inhibition and affinity labeling studies showed an almost exclusive binding of IGF-II to Man-6P/type II IGF-R on tumor membranes. The involvement of IGFs in the growth of epithelial primary cultures of the C4-HD line was evaluated. Exogenous IGF-I potentiated MPA stimulatory effect at concentrations of 50–100 ng/ml. Treatment of C4-HD cells with antisense oligodeoxynucleotides (ASODNs) to type I IGF-R and to IGF-II RNA resulted in a dose-dependent inhibition of MPA-mediated cell proliferation. The inhibition caused by IGF-II ASODNs could not be overcome by the addition of IGF-II up to 150 ng/ml. ASODNs to type I IGF-R at 40 μg/ml reduced by 75% the number of type I IGF-R; ASODNs to IGF-II at 1 μM decreased by 83% the levels of IGF-II protein. Our results provide support for the involvement of IGF-I and -II in MPA-induced mammary tumor growth by autocrine pathways.

The effect on the insulin-like growth factor system in human prostate epithelial cells of immortalization and transformation by simian virus-40 T antigen.

The insulin-like growth factor (IGF) system has been demonstrated to be important for proliferation and differentiation in tissues. This system has also been demonstrated to be an important regulator of the growth of normal prostate epithelium and has been implicated in the process of transformation to human epithelial prostate cancer. This study examined the function of the various components of the IGF system in benign prostate epithelium (BPE), simian virus-40 (SV40)-T antigen-immortalized prostate epithelial cells, P69SV40-T (P69), and two sublines generated from the parental line by serial passage through athymic mice: one tumorigenic (M2182) and one metastatic (M12). IGF-II messenger ribonucleic acid (mRNA) and protein were detected in BPE cells, and each of the three P69 cell lines. IGF-II protein levels were significantly higher in medium collected from the P69, M2182, and M12 cells than in BPE. Proliferation in response to IGF was P69 > BPE > M2182 > M12. The proliferative responses in the four cell types were paralleled by an increase in c-jun. In addition, as the cells became progressively more tumorigenic, the basal level of c-jun mRNA increased. IGF-binding protein-2 (IGFBP-2), -3, -4, -5, and -6 could be detected in the primary epithelial cell medium; however, as the cells became progressively more tumorigenic, there was a decrease in IGFBP-2, -3, -5, and -6 in the medium. The type 1 IGF receptor (IGFr) also decreased as the cells became more tumorigenic. The M12 cells had 80% fewer receptors than the P69 cells and 70% fewer than M2182 cells. There was no change in the Kd for IGF between the cell lines. Based on these data it would appear that the difference in proliferation between the BPE cells and P69s may be due to an increased concentration of inhibitory IGFBPs in the P69 medium. The decrease in proliferation seen in response to IGF in M2182 and M12 cells compared to the P69s would appear at least in part to be due to a decreased IGFr number. IGFr mRNA is represented by 11.0- and 7.0-kilobase bands in the BPE and P69 cells, but only by an 11.0-kilobase band in M2182 and M12 cells. These data indicate that there are significant changes that occur in the IGF system during the process of malignant transformation of the prostate epithelium. The changes described in the P69 cell system are similar to those seen in vivo and suggest that an intact IGF system may be important in maintaining a differentiated epithelial cell.