IGFBP-3 Expression Levels Research Articles

Insulin-like growth factor binding protein-3 (igfbp-3) and igfbp-5 in yellowtail kingfish (Seriola lalandi): molecular characterization and expression levels under different nutritional status and stocking density.

Insulin-like growth factor-binding proteins (IGFBPs) play important roles in regulating growth and development by binding to IGF, where IGFBP-3 and IGFBP-5 are the main binding carriers of IGF in the circulation system. In the present study, the gene sequences of igfbp-3, igfbp-5a, and igfbp-5b were cloned from the liver of yellowtail kingfish (Seriola lalandi). The ORF sequences of igfbp-3, igfbp-5a, and igfbp-5b were 888, 801, and 804bp in length, which encoded 295, 266, and 267 amino acids, respectively. The above three genes were widely expressed in yellowtail kingfish tissues, with igfbp-3 being the most highly expressed in the heart, brain, and gonads, while igfbp-5a and igfbp-5b were both most highly expressed in the liver and kidney. The expression levels of igfbp-3, igfbp-5a, and igfbp-5b were detected throughout the embryonic and larval stages, suggesting their roles in early development and growth regulation of yellowtail kingfish. Besides, igfbp-3 and igfbp-5a were significantly up-regulated in the liver under food deprivation and high-density rearing conditions, which was exactly opposite to the growth performance of yellowtail kingfish, implying that they may serve as biomarkers of adverse culture conditions. Overall, the above results initially identified the molecular characteristics of igfbp-3/-5a/-5b in yellowtail kingfish and implied that they might play important roles in the growth and development, providing a basis for further research on underlying regulatory mechanisms.

Molecular evidence of IGFBP-3 dependent and independent VD3 action and its nonlinear response on IGFBP-3 induction in prostate cancer cells

BackgroundClinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells.MethodsThe molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment.ResultsChanges in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner.ConclusionThese findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material.

BackgroundA previously reported expression signature of three genes (IGFBP3, F3 and VGLL3) was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA) samples.MethodsWe carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004–2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE) prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3) were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared.ResultsWhen combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone.ConclusionThe assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.

A129: Analysis of Igf axis in development of HCC

IGF signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, apoptosis and survival. Deregulation of this pathway has been frequently identified in the development of hepatocellular carcinoma (HCC). IGF signaling pathway consists of IGF ligands (IGF-I and IGF-II), IGF binding proteins (IGFBP 1-7), and membrane-bound IGF receptors (IGF-1R, IGF-II/M6PR, and IGF-2R). The insulin-like growth factor binding proteins (IGFBPs) have several functions, such as IGF transporting, accumulation of IGF at the specific cell pools, inhibition and activation of ligand–receptor interaction. Furthermore, the Igfbps may act independently of IGF-pathway.Diethylnitrosamine (DEN) was used for induction of hepatic tumorigenesis. The male mice, 2weeks old, were injectedintraperitoneally with 20mg/kg body weight of DEN. The liver tissue samples were collected 4, 6, 8, 12months after injection. We performed a comprehensive histological analysis of all kinds of liver samples: tumors, tumor surrounding tissue and normal tissue. Dysplasia was observed 4months after injection, at hepatocellular adenomas were identified 6–8months after injection. In the experimental group, 100% of mice had multifocal HCC 12months after injection. Evaluation of samples from the control group showed normal liver architecture and histology.Western blot analysis showed the presence the HCC tumor marker alfa-fetoprotein only in tumor samples. High levels of Igf-1R and FoxO3 proteins were observed in tumor tissue in contrast to the surrounding and normal tissues 12months after injection. The expression profiles of Igfbp-1,-2,-3,-4,-5,-6,-7, Igf-1R, FoxO3 were analyzed by real time PCR. The most significant results were observed 8 and 12months after DEN injection. RT-PCR showed the dynamic increase in Igf-1R expression in tumor. Expression pattern of Igfbps has shown a high level of Igf-1,-2,-5,-6,-7 mRNA in surrounding tissue and Igfbp-1,-3,-5 in tumor compared to control. The expression of Igfbp-2,-4,-5,-6,-7 was up-regulated, whereas expression of Igfbp-1,-3 was down-regulated in surrounding tissue. The expression of Igfbp-4 was decreased in tumor samples. The expression levels of Igfbp-3,-4 in surrounding tissue and Igfbp-7 in tumor were the same as in the control. We detected high level of mRNA of FoxO3 in tumor.In conclusion, these data confirm previously existing assumption about paracrine effect of Igfbps on tumor growth and progression. Phosphorylation of FoxO3 protein may play an important role in survival of cancer cells. Our results showed up-regulation of Igfbp-2 and Igfbp-5 in tumor and tumor surrounding tissue. Igfbp-2 and Igfbp-5, well known activators of Igfs, can be pro-tumorigenic factors, which are important for hepatocarcinogenesis. Hence, therapeutic targeting of these proteins may offer options for intervention in human HCC.Project supported by Russian Foundation for Basic Research (Russia) No. (113-04-01459).

Operator dependent choice of prostate cancer biopsy has limited impact on a gene signature analysis for the highly expressed genes IGFBP3 and F3 in prostate cancer epithelial cells.

BackgroundPredicting the prognosis of prostate cancer disease through gene expression analysis is receiving increasing interest. In many cases, such analyses are based on formalin-fixed, paraffin embedded (FFPE) core needle biopsy material on which Gleason grading for diagnosis has been conducted. Since each patient typically has multiple biopsy samples, and since Gleason grading is an operator dependent procedure known to be difficult, the impact of the operator's choice of biopsy was evaluated.MethodsMultiple biopsy samples from 43 patients were evaluated using a previously reported gene signature of IGFBP3, F3 and VGLL3 with potential prognostic value in estimating overall survival at diagnosis of prostate cancer. A four multiplex one-step qRT-PCR test kit, designed and optimized for measuring the signature in FFPE core needle biopsy samples was used. Concordance of gene expression levels between primary and secondary Gleason tumor patterns, as well as benign tissue specimens, was analyzed.ResultsThe gene expression levels of IGFBP3 and F3 in prostate cancer epithelial cell-containing tissue representing the primary and secondary Gleason patterns were high and consistent, while the low expressed VGLL3 showed more variation in its expression levels.ConclusionThe assessment of IGFBP3 and F3 gene expression levels in prostate cancer tissue is independent of Gleason patterns, meaning that the impact of operator's choice of biopsy is low.

Linking Pre-Diabetes with Benign Prostate Hyperplasia. IGFBP-3: A Conductor of Benign Prostate Hyperplasia Development Orchestra?

Benign prostatic hyperplasia (BPH) represents a pattern of non-malignant growth of prostatic fibromuscular stroma. Metabolic disturbances such us pre-diabetes and metabolic syndrome may have a role in BPH pathophysiology. A potential explanation for the above relationship involves the insulin-like growth factor (IGF) axis as well as IGF binding proteins, (IGFBPs) of which the most abundant form is IGFBP-3. Therefore, the aim of the present study was to investigate the association between intra-prostatic levels of IGF-1, IGF-2 as well as to evaluate the role of locally expressed IGFBP-3 in BPH development in pre-diabetes. A total of 49 patients admitted to the Urology department of a tertiary urban Greek hospital, for transurethral prostate resection, or prostatectomy and with pre-diabetes [impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) or both] were finally included. The majority of the sample consisted of subjects with IGT (51.0%), followed by IFG and IGT (32.7%) and isolated IFG (16.3%). For all participants a clinical examination was performed and blood samples were collected. In addition, total prostate (TP) volume or transitional zone (TZ) volume were estimated by transrectal ultrasonography. The results of the multivariate analysis regarding TP volume showed that higher PSA (p<0.001), larger waist circumference (p=0.007) and higher IGFBP-3 expression levels (p<0.001) independently predicted higher TP volume. The results regarding the volume of the TZ showed that higher PSA (p<0.001), larger waist circumference (p<0.001) and higher IGFBP-3 expression levels (p=0.024) were independently associated with higher TZ volume. Our findings show that intra-prostatic levels of IGFBP-3, PSA and waist circumference, but not overall obesity, are positively associated with prostate volume. IGFBP-3 seems to be a multifunctional protein, which can potentiate or inhibit IGF activity.

Human Parathyroid Hormone Increases the mRNA Expression of the IGF System and Hematopoietic Growth Factors in Osteoblasts, but Does Not Influence Expression in Mesenchymal Stem Cells

Osteoblasts, which are derived from pluripotent mesenchymal stem cells (MSCs), play an important role in hematopoiesis. Human parathyroid hormone (hPTH) induces osteoblasts to produce many factors that are essential to hematopoietic stem cells. However, little is known about the impact of hPTH on MSCs to enhance hematopoiesis. We determined the optimal dose of hPTH that was necessary in vitro for increased osteoblast function. In addition, we compared MSC and osteoblast function to explore the role of hPTH in hematopoiesis. The mRNA expression levels of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 6, stromal cell-derived factor 1, insulin-like growth factor 1 (IGF-1), IGF-2, insulin-like growth factor-binding protein 1 (IGFBP-1), IGFBP-2, and IGFBP-3 were comparable in osteoblasts and human cord blood-derived MSCs. However, G-CSF, GM-CSF, IGF-2, IGFBP-1, IGFBP-2, and IGFBP-3 expression levels in osteoblasts were markedly increased after treatment with 50 or 100 nM of hPTH. In conclusion, hPTH does not affect the ability of MSCs to differentiate into osteoblasts. In addition, hPTH may enhance hematopoiesis by activating the IGF system (IGF-2, IGFBP-1, IGFBP-2, and IGFBP-3) and hematopoietic growth factors (G-CSF and GM-CSF) in osteoblasts, but not in MSCs.

Higher Expression of mRNA and Protein of Insulin‐Like Growth Factor Binding Protein‐3 in Old Rat Penile Tissues: Implications for Erectile Dysfunction

Previous studies have confirmed the gene transfer of insulin-like growth factor-1 (IGF-1) and the IGF-1 protein can improve the erectile function in aging rats. IGF binding protein (BP)-3 can regulates the availability of IGF-I. The higher expression of IGFBP-3 may play an important role in erectile dysfunction (ED). The study aimed to investigate the mRNA and protein expression of IGFBP-3 in young and old rat penile tissues and assess the alteration of the penile structure and the NO-guanosine 3',5'-cyclic-monophosphate (cGMP) signaling pathways-related marker in ED associated with aging. The main outcome measures for this study were the expression of IGFBP-3, morphological changes, NO-cGMP signaling pathways-related marker, erectile responses were determined. Traditional reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR were performed to examine the mRNA expression of the IGFBP-3. The Western blot was used to confirm the protein expression. Immunohistochemistry was also performed to identify the cellular localization of the encoded protein. The percentage of smooth muscle in corpus cavernosum tissue, the activity of nitric oxide synthase (NOS), and concentration of cGMP in penile tissue were also analyzed. The expression levels of IGFBP-3 of mRNA and protein were greatly increased in aging rats compared with young control rats, which is confirmed by traditional RT-PCR, real-time PCR, and Western blot (P < 0.01, respectively). Increased IGFBP-3 protein was localized to the epithelium of the urethra, penile endothelium, and smooth muscle in the corpus cavernosum. Significant depletion of the smooth muscle density relative to the connective tissue was also observed in the penis of the aged rats, and the lower activity of NOS and lower concentration of cGMP was also demonstrated accompanied with a significant reduction in the intracavernous pressure. Our data suggest that the increased mRNA and protein expression of IGFBP-3 in old rats may play a role in ED.

Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Gene Expression In Myeloma Cell Lines and Patients

AbstractAbstract 2489 Introduction:Epigenetic aberrations play an important role in the development and progression of Multiple Myeloma (MM). 5-aza-2-deoxycytidine (5Aza-dC) and Trichostatin A (TSA) have been studied in reactivating the expression of epigenetically-silenced genes. IGFBP3 gene is a member of the insulin-like growth factor binding protein (IGFBP) family and can regulate cell growth and death by the ability to bind insulin-like growth factors (IGFs) as well as its IGF-independent effects involving binding to other molecules. Several in vitro, in vivo studies as well as clinical evidence point to IGFBP-3 as an anti-cancer molecule. In our study we analyzed global changes in gene expression profiles of MM cell lines, responding to 5Aza-dC and TSA and we evaluated the IGFBP3 expression in three myeloma cell lines and in samples from myeloma patients. Methods:Human MM cell lines U266 and H929 were treated either with 0.5 micromol/L 5Aza-dC for 7 days or with 100 ng/mL TSA for 24 h or with the combination of 0.5 micromol/L 5Aza-dC for 7 days and 100 ng/mL TSA for additional 24 h. Control cells received no drug treatment. Applied Biosystems microarray platform ABI 1300 was used for carrying out microarray profiling and analysis. To classify up-regulated genes into functional categories the PANTHER Classification System was used (http://www.pantherdb.org). Stained or unstained bone marrow slides were collected from archival samples of 179 myeloma patients. For validation of microarray results, real-time reverse transcription-PCR (RT-PCR) was performed using Taqman Gene Expression Assays (Applied Biosystems). Results:After treatment with 5Aza-dC there was up regulation of gene expression in 698 genes in H929 cell line and 258 genes in U266 cell line. After treatment with TSA 719 genes were up regulated in H929 cell line and 742 genes in U266 cell line. The exposure to the combination of 5Aza-dC/TSA resulted in up-regulation of 921 genes in H929 cell line and 615 genes in U266 cell line. By using Panther classification system we classified up-regulated genes into functional categories and we identified several 5Aza-dC or TSA up-regulated genes that are involved in important cancer-related pathways including cell cycle, apoptosis, cell adhesion, oncogenesis and cell metabolism, including DNA repair and nucleosome assembly. Between these genes we particularly found interesting the expression changes of IGFBP-3 which was up regulated after treatment with 5-azacitidine in U266 cell line (level of induced expression was 5,16). The microarray data were validated in 3 MM cell lines and 179 patients samples by analysis of relative changes in IGFBP3 expression through ΔΔCt method. We found that the expression levels of IGFBP3 were significantly lower in U266 and RPMI myeloma cell lines, but not in H929 cell line and interestingly levels were lower in 54 percent of patients (Fig 1). It has been demonstrated decreased IGFBP-3 expression is associated with cancer progression and in our study we have shown that the down-regulation of this gene may be involved also in myeloma pathogenesis and mediate progression events, hence levels of IGFBP3 may be considered as a biomarker for disease staging. From a therapeutic point of view, up-regulation of IGFBP3 might be considered as target therapeutic strategy for monoclonal gammopathies/ smouldering Myelomas at high risk of progression to active myeloma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increased insulin‐like growth factor binding protein‐3 expression level in psoriatic tissue before and after systemic treatment with cyclosporine A and methotrexate

Insulin-like growth factor (IGF) and its binding proteins (BPs) are candidates to play a role in the pathogenesis of psoriasis. IGF-I and -II and their binding proteins (IGFBPs) have both growth inhibitory and mitogenic effects on a multitude of cell types. Current studies have explored the potential role of IGFBP-3 in keratinocyte growth and differentiation. To gain more insight into the role of IGFBP-3 in the pathogenesis of psoriasis, we investigated the changes in IGFBP-3 expression levels in psoriatic plaque and compared these expressions with levels in other inflammatory skin diseases. In particular, we investigated whether or not the changes in IGFBP-3 were affected by systemic treatment with cyclosporine A (CsA) or methotrexate (Mtx). We found significantly increased IGFBP-3 expression levels in the psoriatic group compared with levels in patients with other, nonproliferative inflammatory skin diseases, and we demonstrated differences in distribution pattern before and after systemic treatment with Mtx or CsA. Mtx and CsA had no effect on tissue IGFBP-3 expression levels. Before treatment with Mtx or CsA, IGFBP-3 expression was limited to the basal layer and suprapapillary region. Unlike with Mtx, CsA significantly changed the IGFBP-3 distribution pattern.

Relation between Insulin-like growth factor-1 receptor (IGF-1R) expression and the efficacy of trastuzumab (T) monotherapy for hormone-resistant HER2-postitive metastatic breast cancer (MBC)

9578 Background: T is an active drug for HER2-positive MBC with excellent toxicity profile, but its anti-tumor effect as monotherapy is limited and the mechanism of T resistance is not elucidated. Lu Y et al. recently demonstrated in human breast cancer cell-lines that increased level of IGF-1R signaling adversely interferes with T's action on cell growth (JNCI, 2001). We assessed the predictive value of IGF-1R protein expression for T therapy in tumor samples using immunohistochemistry(IHC). Methods: Between June 2001 and May 2003, 39 patients without visceral crisis were treated with T monotherapy as the first line therapy for hormone-resistant MBC. All tumors were HER2 positive either by Herceptest(3+) or FISH. Among them, 26 formalin-fixed paraffin embedded tissue of primary or metastatic lesions were available. Mouse monoclonal antibody (clone 24–31) specific for alpha subunit was used for IGF-1R IHC. ER, PgR, IGF-BP3 was also evaluated by IHC. IGF-1R and IGFBP3 expression levels were measured from its staining on the membrane and cytoplasm, respectively, as mode-intensity (MI) by automated imaging analyzer(Ariol SL-50). T monotherapy was continued until clinical progression. Results: Median duration of T monotherapy was 78 days (range 20–375). The range of MI for IGF-1R and IGF-BP3 was 0–166.7 (mean 86.0, SD 40.8) and 0–86.4 (mean 57.3, SD 28.3), respectively. Neither IGF-1R MI nor IGF-BP3 MI correlated with duration of T monotherapy (p=.692 and .791, respectively). High IGF-1R MI (>50.0) was observed in 5 out of 6 patients who received T monotherapy more than 12 weeks without progression (1 CR, 5 MR), and 4 of the 5 patients showed high IGFBP3 MI(>50.0). Conclusions: In contrast to previously reported in vitro experiment, IGF-1R protein expression did not correlate with resistance to T monotherapy. Further studies are necessary to clarify the role of IGF-1R axis in mechanism of action of T. No significant financial relationships to disclose.

Ionic Products of Bioactive Glass Dissolution Increase Proliferation of Human Osteoblasts and Induce Insulin-like Growth Factor II mRNA Expression and Protein Synthesis

Bioglass 45S5 is an osteoproductive material, which resorbs by releasing its constitutive ions into solution. Treatment with the ionic products of Bioglass 45S5 dissolution in DMEM for 4 days increased human osteoblast proliferation to 155% of control. Two days after treatment, differential gene expression was analyzed by cDNA microarrays. Expression of a potent osteoblast mitogenic growth factor, insulin-like growth factor II (IGF-II), was increased to 290%. Additionally, there was a 168% increase in the concentration of unbound IGF-II protein in the conditioned media of treated osteoblasts. Expression levels of IGFBP-3, an IGF-II carrier protein, metalloproteinase-2 and cathepsin-D were also increased to 200, 340, and 310% of control levels, respectively. Metalloproteinase-2 and cathepsin-D are proteases that cleave IGF-II from its carrier proteins, resulting in the release of the unbound biologically active IGF-II. We suggest that the stimulatory effect of the ionic products of Bioglass 45S5 dissolution on osteoblast proliferation may be mediated by IGF-II.

Immunolocalization and expression of insulin-like growth factor I (IGF-I) in the mammary gland during rat gestation and lactation

We have studied the physiology and tissue expression of IGF-I and IGF-BP3 in pregnant and lactating rats. Specific assays (radioimmunoassays and a binding protein assay) were used to measure serum IGF-I, IGF-II, and IGF-BP levels. IGF-I and IGF-BP3 expression levels were determined in mammary gland and liver by slot-blotting. A sensitive and IGF-I-specific ribonuclease (RNAse) protection assay was further used to detect RNAs transcribed from the IGF-I gene. In the first half of pregnancy, the maternal serum IGF-I concentration rises while the IGF-BP level decreases. This may modify IGF-I availability, thus promoting rapid tissue growth and differentiation. In the second half of pregnancy, the mean serum IGF-I concentration falls sharply from 1140 ± 150 ng/ml at seven days of pregnancy to 470 ± 85 ng/ml at 20 days. Post-partum, serum IGF-I increases back to the level obtained in non-pregnant controls within 5 days. Serum levels of IGF-BP, during the same two periods, follow a similar pattern, decrease during pregnancy and increase after parturition. No IGF-II was detected at any time. From the onset of pregnancy to term, IGF-I gene expression in the mammary gland diminishes. In the liver, on the other hand, expression increases during very early pregnancy and diminishes thereafter, remaining below the level measured in non-pregnant animals from mid-pregnancy to term. The pattern of IGF-BP3 expression followed was similar in both organs, with a decrease during gestation. In lactating animals, expression of both the IGF-I and IGF-BP3 genes remains stable in the liver, at the level reached in late pregnancy. Not so in the mammary gland, where expression of both genes drops sharply. IGF-I was immunolocalized in the mammary gland to myoepithelial cells in non-pregnant, pregnant, and lactating animals. Our data suggest that IGF-I, both produced within the mammary gland and reaching it via the bloodstream, has a role in this organ's early development at the onset of pregnancy. IGF-I found in milk, on the other hand, is probably produced in the maternal liver rather than in the mammary gland.