IGFBP-3 Protein Research Articles

Risk factors and molecular characterization of penile cancer: impact on prognosis and potential targets for systemic therapy.

To provide a comprehensive summary of risk factors, molecular machinery as well as potential therapeutic targets with a particular focus on literature published in the last 2 years on prognosis and treatment of penile cancer (PeCa). E2F, LAMC2, MAML2, ID1 and IGFBP2 proteins were demonstrated to play a critical role for aggressive tumor behavior and might predict poor survival in PeCa. PD-L1 axis was confirmed as a promising pathway to serve as a therapeutic target. A number of genetic alterations were illuminated. In clinical testing, pan-HER tyrosine kinase inhibitor dacomitinib provided promising results in chemo-naïve and EGFR monoantibody nimotuzumab in chemotherapy-failed PeCa patients. Knowledge of prognosis-relevant altered molecular pathways in PeCa is expanding paving the way for identification of potential therapeutic targets. Multicenter clinical trials in the setting of centralized PeCa care are warranted to foster effective marker-based individualized treatment strategies.

Abstract PD3-08: Effects of low dose tamoxifen on circulating risk biomarkers in a phase III trial in breast pre-invasive disease

Abstract Background: We recently showed that low dose (5mg/d) tamoxifen (babytam) for 3 years can halve the incidence of new breast neoplastic events in hormone sensitive or unknown breast pre-invasive neoplasia after surgery with limited toxicity (DeCensi et al JCO 2019; 37:1629-37). Here we report the results of circulating surrogate endpoint biomarkers of breast cancer risk with special attention to the risk of ovarian stimulation observed with the full dose in premenopausal women. Methods: Five hundred women 75 years old or younger were randomized to babytam or placebo (PLA). A subgroup of 406 women consented for collection of morning fasting serum at baseline (0), 1 (1Y) and 3 years (3Y) of treatment. There was a loss of about 25% of blood sampling at 3Y. Serum IGF-I, IGFBP-3, SHBG and C-reactive Protein (CRP) were performed on all available samples. Estradiol and testosterone were determined in a subsample (n=285) to determine the extent of ovarian stimulation by babytam in premenopausal women. We used Mann-Whitney test for univariate comparisons and linear regression modeling for multivariate analyses setting changes from baseline (1Y or 3Y minus baseline values) as dependent variable and treatment arm, age, BMI and baseline biomarkers values as explanatory factors. We tested treatment*menopausal status as interaction term. COX P-H model was used to calculate hazard ratio for CRP increase. Results: At baseline, all biomarkers were evenly balanced between arms (data not shown). IGF-I decreased significantly on babytam as compared to PLA. The difference of the changes between arms was -20 ng/mL after 1Y (p<0.001) and -23 after 3Y (p<0.001). A treatment by menopausal status interaction was observed after 1Y (p=0.017) and 3Y (p=0.058), with a steeper decrease in postmenopausal (-25, p<0.001 both after 1Y and 3Y) as compared to premenopausal women (-15, p=0.007 after 1Y and -17, p=0.009 after 3Y). IGFBP-3 increased significantly on babytam, without effect modification by menopausal status. The difference between arms was +0.26 ug/mL after 1Y (p=0.006) and +0.19 after 3Y (p=0.024). SHBG increased markedly on babytam, irrespective of menopausal status. The difference between arms from 0 to 1Y and 3Y was 24 nMol/L (p<0.001). CRP levels followed a trend towards a decrease with babytam (-0.172 mg/dL, p=0.057 after 1Y and -0.091, p=0.108 after 3Y), without effect modification by menopausal status. There was no significant increase in serum estradiol after 1Y with babytam overall (+12 pg/mL, p=0.106), but a trend to an interaction with menopausal status (p=0.079): +17 (p=0.361) in premenopausal women versus +3 (p=0.284) in postmenopausal women. Similarly, there was no overall effect of babytam on testosterone after 1Y (-0.008 ng/mL, p=0.518), but a significant effect modification by menopausal status (p=0.001), showing an increase of +0.056 (p=0.006) in premenopausal women, and a decrease of -0.045 (p=0.006) in postmenopausal women. Irrespective of treatment, the increase in CRP at 3Y was significantly higher in women who experienced recurrence compared to women who did not (Mann-Whitney p=0.009). An increase in CRP was associated with a HR of 2.9 (95% CI, 1.0-8.3, p=0.05) as compared to women with a decreased or stable CRP levels. Conclusions: Babytam for 3 years exhibits a favorable effect on sex hormones and IGFs with only a slight increase of estradiol in premenopausal women which is far below that observed with 20 mg and is well compensated by a significant increase in SHBG. These findings further support the use of babytam as an effective and safe treatment for high risk individuals. ClinicalTrials.gov Identifier: NCT01357772. Supported by the Italian Ministry of Health (RFPS-2006-339898), the Italian Association for Cancer Research (IG 2008 Grant No 5611) and the Italian League against Cancer (LILT 7-08). Citation Format: Harriet Johansson, Matteo Puntoni, Debora Macis, Valentina Aristarco, Aliana Guerrieri-Gonzaga, Davide Serrano, Silvia Caviglia, Laura Cortesi, Cristiana Taverniti, Antonio Ponti, Maria Grazia Pacquola, Marcella Gulisano, Fabio Falcini, Maria Digennaro, Anna Carriello, Katia Cagossi, Graziella Pinotti, Tania Buttiron Webber, Matteo Lazzeroni, Bernardo Bonanni, Luca Boni, Andrea DeCensi. Effects of low dose tamoxifen on circulating risk biomarkers in a phase III trial in breast pre-invasive disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD3-08.

Exploring the Mesenchymal Stem Cell Secretome for Corneal Endothelial Proliferation.

Ex vivo grown human corneal endothelial cells (HCEnC) are a new emerging treatment option to treat visually impaired patients aimed at alleviating the current global donor shortage. Expanding HCEnC is still challenging, and obtaining cells in sufficient quantities is a limiting factor. It is already known that conditioned medium obtained from bone marrow mesenchymal stem cells can stimulate the proliferation of endothelial cells. The aim of this study was to take this work a step further to identify some of the underlying factors responsible. We confirmed the stimulatory effect of the mesenchymal stem cell secretome seen previously and separated the exosomes from the soluble proteins using size exclusion chromatography. We demonstrated the presence of exosomes and soluble proteins in the early and late fractions, respectively, with transmission electron microscopy and protein assays. Proliferation studies demonstrated that growth stimulation could be reproduced with the later protein-rich fractions but not with the exosome-rich fraction. Antibody assays revealed the presence of the secreted proteins EGF, IGFBP2, and IGFBP6 in protein-high fractions, but the growth enhancement was not seen with purified protein formulations. In conclusion, we confirmed the stimulatory effect of stem cell-conditioned medium and have determined that the effect was attributable to the proteins rather than to the exosomes. We were not able to reproduce the growth stimulation, however, with the pure recombinant protein candidates tested. Specific identification of the underlying proteins using proteomics could render a bioactive protein that can be used for ex vivo expansion of cells or as an in vivo drug to treat early corneal endothelial damage.

Engineered scaffolds based on mesenchymal stem cells/preosteoclasts extracellular matrix promote bone regeneration

Recently, extracellular matrix-based tissue-engineered bone is a promising approach to repairing bone defects, and the seed cells are mostly mesenchymal stem cells. However, bone remodelling is a complex biological process, in which osteoclasts perform bone resorption and osteoblasts dominate bone formation. The interaction and coupling of these two kinds of cells is the key to bone repair. Therefore, the extracellular matrix secreted by the mesenchymal stem cells alone cannot mimic a complex bone regeneration microenvironment, and the addition of extracellular matrix by preosteoclasts may contribute as an effective strategy for bone regeneration. Here, we established the mesenchymal stem cell/preosteoclast extracellular matrix -based tissue-engineered bones and demonstrated that engineered-scaffolds based on mesenchymal stem cell/ preosteoclast extracellular matrix significantly enhanced osteogenesis in a 3 mm rat femur defect model compared with mesenchymal stem cell alone. The bioactive proteins released from the mesenchymal stem cell/ preosteoclast extracellular matrix based tissue-engineered bones also promoted the migration, adhesion, and osteogenic differentiation of mesenchymal stem cells in vitro. As for the mechanisms, the iTRAQ-labeled mass spectrometry was performed, and 608 differentially expressed proteins were found, including the IGFBP5 and CXCL12. Through in vitro studies, we proved that CXCL12 and IGFBP5 proteins, mainly released from the preosteoclasts, contributed to mesenchymal stem cells migration and osteogenic differentiation, respectively. Overall, our research, for the first time, introduce pre-osteoclast into the tissue engineering of bone and optimize the strategy of constructing extracellular matrix–based tissue-engineered bone using different cells to simulate the natural bone regeneration environment, which provides new sight for bone tissue engineering.

IGFBP-2 in cervical cancer development

ObjectiveIncreased expression of insulin-like growth factor binding protein 2, IGFBP-2, is associated with many cancers, though its role in cervical cancer is unclear. The aim of this study was to investigate the expression of IGFBP-2 protein and the transcriptomics profile of genes involved in the IGF signaling pathway during cervical cancer development. DesignImmunohistochemical expression of IGFBP-2 protein was semi-quantitatively assessed in tissue microarrays containing 9 normal cervix, 10 low grade cervical intraepithelial neoplasia (LGCIN), 10 high grade cervical intraepithelial neoplasia (HGCIN) and 42 squamous cell carcinoma (SCC) cases. The gene expression profiles of IGFBP-2, IGF-1, IGF-1R, PTEN, MDM2, AKT1 and TP53 were determined in three cervical tissue samples each from normal cervix, human papillomavirus (HPV)-infected LGCIN, HGCIN and SCC, using Human Transcriptome Array 2.0. ResultsIGFBP-2 protein was highly expressed in the cytoplasm of SCC cells compared to normal cervix (p = .013). The expression was not significantly associated with CIN grade or SCC stage. Transcriptomics profiling demonstrated upregulation of IGFBP-2 and TP53 in HGCIN and SCC compared to normal cervix. IGF-1, IGF-1R and PTEN genes were downregulated in all histological groups. IGF-1 gene was significantly downregulated in SCC (p = .031), while PTEN gene was significantly downregulated in HGCIN (p = .012), compared to normal cervix. MDM2 and AKT1 genes were downregulated in LGCIN and HGCIN, while upregulated in SCC. ConclusionIn cervical carcinogenesis, IGFBP-2 appears to play an oncogenic role, probably through an IGF-independent mechanism.

Elevated circulating HtrA4 in preeclampsia may alter endothelial expression of senescence genes

IntroductionPreeclampsia (PE) is a serious complication of human pregnancy. Women who have had PE, especially early-onset PE (EPE), have an increased risk of cardiovascular disease (CVD) later in life. However, how PE is linked to CVD is not well understood. We previously reported that HtrA4, a placenta-specific protease, is significantly elevated in EPE, and inhibits the proliferation of endothelial cells as well as endothelial progenitor cells (EPCs). This can potentially impair endothelial repair and regeneration, leading to endothelial aging, which is a major risk factor of CVD. In this study, we examined whether HtrA4 can alter endothelial expression of senescence genes. MethodsHuman umbilical vein endothelial cells (HUVECs) and primary EPCs isolated from cord blood of healthy pregnancies were used as in vitro models. Firstly, HUVECs were treated with HtrA4 at the highest levels detected in EPE for 48h and screened with a senescence PCR array. The results were then validated by RT-PCR and ELISA in HUVECs and EPCs treated with HtrA4 for 24 and 48h. ResultsWe observed that HtrA4 significantly up-regulated IGFBP3, SERPINE1 and SERPINB2, which all promote senescence. IGFBP-3 protein was also significantly elevated in the media of HtrA4-treated HUVECs. Conversely, a number of genes including CDKN2C, PCNA, CALR, CHEK2 and NOX4 were downregulated by HtrA4. Many of these genes also showed a similar trend of change in EPCs following HtrA4 treatment. DiscussionElevation of placenta-derived HtrA4 in PE alters the expression of endothelial genes to promote cellular senescence and may contribute to premature endothelial aging.

Carbon disulfide induced decidualization disorder in the mice uterus at the window of implantation.

Carbon disulfide (CS2) is regarded as a common occupational poison that is widely used in the textile industry in China. Our previous research suggests that CS2 can induce significant implantation disorders in pregnant mice; however, the specific mechanism remains unclear. Uterine conception in mice must undergo decidualization, which is the prerequisite for propitious blastocyst implantation into the endometrium. Therefore, in this study, we established models of pregnant mice to explore the toxic effects of CS2 on decidualization to elucidate the basic mechanism of implantation disorder after CS2 exposure. The uterine tissues were immediately collected according to the predetermined endpoints to measure the expression levels of IGFBP1 and PRL (markers of decidualization differentiation), IL-11 (representing the secretory function of decidual cells), AKT and pAKT by western blotting, RT-PCR, immunohistochemical staining, H&E staining and ELISA. N-carbamoyl glutamic acid (NCG) acted as an agonist of AKT to verify the upstream regulatory mechanism of decidualization disorder by CS2. The results showed that the normal reaction of decidual transformation was obviously disrupted by CS2 upon 3.5 dpc and 4.5 dpc exposure. The blastocyst did not adhere to the epithelium after 3.5 dpc-exposure and did not invade the endometrium after 4.5 dpc-exposure, resulting in its suspension in the uterine cavity, stagnation and eventual loss. The proteins expression levels were decreased by 95.2% for IGFBP1 and 76.2% for PRL at the 4.5 dpc endpoint after 3.5 dpc CS2 exposure compared with the control. Simultaneously, the mRNA and protein expression levels of IL-11 in uterine tissues were significantly reduced by CS2, and consistent decreasing trends over time were observed for IGFBP1 and PRL, compared with the control. Additionally, the ratio of pAKT/AKT protein expression was decreased by 72.2% and 94.8% at 12h and 18h after 3.5 dpc exposure and by 53.3% and 74.3% at 6h and 12h after 4.5 dpc exposure, respectively, compared with the corresponding controls. Furthermore, NCG could recover the IGFBP1 and PRL protein expression, which was increased by 27.5% and 52.3% at 4.5 dpc and 6.5 dpc, respectively, after 3.5 dpc exposure for IGFBP1 and by 30.3% at 6.5 dpc after 4.5 dpc exposure for PRL, compared with CS2 exposure alone. Collectively, this study suggested that the decidualization disorder caused by CS2 at the window of implantation in pregnant mice, which is triggered by pAKT, contributed to the implantation disorder and eventually led to embryo loss. It is worth noting that our study may provide a new perspective and reference for exploring the toxic mechanism of implantation disorder and even infertility in harmful circumstances.

Management of growth failure and growth hormone deficiency after pediatric allogeneic HSCT: Endocrinologists are of importance for further guidelines and studies

Growth failure (GF) is a frequent problem after pediatric allogeneic hematopoietic stem cell transplantation (HSCT). Growth hormone deficiency (GHD) occurs in 20 to 85%, but published data on the efficacy of growth hormone treatment (GHT) are conflicting. Currently, there are no recommendations on screening for and treatment of GHD after HSCT. We aimed to describe the management of endocrine follow-up (FU)and details of GHT within European Society for Blood and Marrow Transplantation (EBMT) centers.In a retrospective questionnaire study, all EBMT centers performing pediatric HSCT were invited. Results were evaluated in correlation with the structure of endocrine aftercare (HSCT-clinicians and endocrinologists).The majority of centers (80%) reported endocrine FU by an endocrinologist – either within the HSCT-center or in a separate endocrine clinic. Fifty-four percent reported FU outside of the HSCT-center. As diagnostic tests the insulin-like growth factor IGF-I and insulin-like growth factor binding protein IGFBP3, insulin tolerance test and arginine stimulation test were most frequently used. Sixty-four percent of centers performed GHT and endocrinologists were more likely to prescribe GH (74%) compared to HSCT-clinicians (33%). The most frequent indication for GHT was GHD in 60%, with a distinct different approach of endocrinologists in comparison with HSCT-clinicians.Our study reveals substantial variation in practice and emphasizes the need for endocrine aftercare performed by dedicated endocrinologists in close collaboration with the HSCT-center. Our results indicate that the management of GHT depends on the structure of endocrine aftercare, which is important for the future development and distribution of studies and guidelines.

Quantitative iTRAQ-based proteomic analysis of differentially expressed proteins in aging in human and monkey

BackgroundThe underlying physiological mechanisms associated with aging are still complex and unclear. As a very important tissue of human body, the circulatory system also plays a very important role in the process of aging. In this study, we use the isobaric tags for relative and absolute quantification (iTRAQ) method to identify differentially expressed proteins in plasma for humans and monkeys between young and aged. Western blotting and behavioral experiment in mice were performed to validate the expression of the candidate protein.ResultsBetween the young / the old humans and the young / the old monkeys 74 and 69 proteins were found to be differently expressed, respectively. For the human samples, these included 38 up-regulated proteins and 36 down-regulated proteins (a fold change ≥1.3 or ≤ 0.667, p value ≤0.05).For the monkey samples, 51 up-regulated proteins and 18 down-regulated proteins (a fold change ≥1.3 or ≤ 0.667, p value ≤0.05). KEGG pathway analysis revealed that phagosome, focal adhesion, ECM-receptor interaction and PI3K/AKT signaling pathway were the most common pathways involved in aging. We found only IGFBP4 protein that existed in up-regulated proteins in aged both for human and monkey. In addition, the differential expression of IGFBP4 was validated by western blot analysis and IGFBP4 treatment mimicked aging-related cognitive dysfunction in mice.ConclusionsThis first, the integrated proteomics for the plasma protein of human and monkey reveal one protein-IGFBP4, which was validated by western blotting and behavioral analysis can promote the process of aging. And, iTRAQ analysis showed that proteolytic systems, and inflammatory responses plays an important role in the process of aging. These findings provide a basis for better understanding of the underlying mechanisms involved in aging.

The Concept of Growth Hormone Deficiency Affecting Clinical Prognosis in IVF.

The current understanding of human growth hormone (hGH; here GH) action is that the molecule is a 191-amino acid, single-chain polypeptide that is synthesized, stored and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. It can be classified as a protein (comprising more than 50 amino acids) but true proteins have tertiary and quaternary chains creating a more complex structure, hence GH is usually classified as a polypeptide. GH is normally secreted at night during sleep and promotes skeletal, visceral and general body growth through the action of somatomedins or IGFs, notably IGF-1. In some tissues, GH action is directed via specific receptors GHRs; these are most abundant in liver, adipose and muscle tissues but have also been shown in granulosa cells, testicular tissues and on the oocyte, as well as in glandular cells of the luteal phase endometrium and decidua; such findings being recent and minimally researched to now. Following engagement with its receptor, the transduction process activates multiple signaling proteins. These all lead to extensive metabolic and mitogenic (growth promoting) responses. Clinically, GH is known to have an important role in pubertal development and is a key hormone for the vigor associated with adolescence and early adult life stages but has a faded presence and role for later adulthood, beyond age 30 years, and is minimally detected in advanced age, beyond 40 years. In association with the rapidly increasing trend for delaying reproduction beyond age 35 years, GH is being widely researched now as a potential adjuvant for infertility treatment in this group who, studies consistently show, have a poorer prognosis than younger females when relying on autologous oocytes. The idea that the age-related reduction in fertility prognosis is a feature of growth hormone deficiency is supported by our studies showing an elevated binding protein IGFBP-3/IGF-1 ratio and this can be reduced to a normal range (matching younger, good prognosis women) by the administration of GH as an adjuvant.

Immune and Inflammatory Proteins in Cord Blood as Predictive Biomarkers of Retinopathy of Prematurity in Preterm Infants

To determine whether elevated levels of immune/inflammatory proteins in cord blood, alone or in combination with conventional clinical parameters, can predict the occurrence and progression of retinopathy of prematurity (ROP) in preterm infants. This was a retrospective cohort study of 110 premature singleton infants who were born at ≤32.0 weeks. Cord plasma at birth was assayed for interleukin-6, C3a, C5a, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1, macrophage colony-stimulating factor, endostatin, a proliferation-inducing ligand, insulin-like growth factor-binding protein-1 (IGFBP-1), IGFBP-2, and calcium-binding protein A8/A9 complex levels. The primary outcome measures were the occurrence of any stage ROP, severe ROP (>stage 3), and vision-threatening type 1 ROP requiring laser treatment. ROP was diagnosed in 30 of 110 infants (27.3%), including 14 (12.7%) with severe ROP. Laser treatment was performed on 7 infants (6.4%). Multiple logistic regression analyses indicated that elevated levels of cord plasma IL-6 were significantly associated with severe ROP, whereas elevated levels of cord plasma C5a were significantly associated with ROP laser treatments. However, none of the proteins measured in the cord plasma were associated with ROP occurrence. Using a stepwise regression procedure, we developed a combined prediction model, which included high cord plasma IL-6 levels and low birth weight for severe ROP (area under the curve [AUC], 0.840), and high cord plasma C5a levels and low birth weight for laser treatment (AUC, 0.884). Elevated levels of cord plasma IL-6 and C5a could be used as independent markers to predict severe ROP and laser treatment, respectively, with combined models predicting ROP progression with good accuracy.

Abstract 1286: ONC201 inhibits RET and IGFBP2 signaling through ATF4 mediated- Integrated stress response in medullary thyroid cancer

Abstract Medullary thyroid cancer (MTC) is an aggressive tumor with frequent lymph node and distant metastasis at diagnosis. Activating mutations of the RET receptor tyrosine kinase occur frequently and prevent apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum stress. Prior studies have demonstrated shRNA depletion of ATF4 increases RET protein levels and cause resistance to tyrosine kinase inhibitor-mediated cell death. Conversely, forced expression of ATF4 in MTC cells promotes RET ubiquitination and degradation. Here, we report the anti-cancer efficacy of ONC201 in MTC models. ONC201 is a selective DRD2 antagonist that is in clinical trials for a range of advanced cancers. Downstream signaling studies have found that ONC201 induces ATF expression and activates the integrated stress response (ISR) in a variety of cancers. ONC201 decreased cell survival of MTC cell lines (TT and MZCRC1) harboring activating RET mutations and increased the percentage of sub-G1 DNA content. Moreover, ONC201 decreased, in a dose-dependent manner, the abundance of RET protein and induced the mRNA and protein levels of ATF4 and its target genes. In our attempt to uncover RET targets, we performed reverse phase protein array analysis using TT- shRNA-RET knockdown cells. Among the top 5 proteins regulated by RET in this screen, we found insulin-like growth factor-binding protein IGFBP2, to be downregulated in shRNA-RET cells. ELISA based cytokine array of MTC-conditioned media showed high levels of secreted IGFBP2. IGFBP2 have been reported to be an oncogene in many human cancers and we show that elevated IGFBP2 protein levels are associated with poor prognosis of primary MTC. The treatment of MTC cells with ONC201 or overexpression of ATF4 inhibited the secretion of IGFBP2 into the media and decreased IGFBP2 protein levels, suggesting that ONC201 targets IGF signaling through activation of ISR. To investigate the efficacy of ONC201 in vivo, established MTC xenografts (TT or MZCRC1) were treated with a weekly dose of 120 mg/kg orally for 8 weeks and tumor burden was assessed every week. We observed that ONC201 decreased tumor growth of TT and MZCRC1 xenografts by 80-90%. There was no evidence of toxicity, as indicated by weight loss in mice. Immunohistochemical and western blot analysis of tumors at the termination of the study demonstrated decreased IGFBP2 protein levels in mice treated with ONC201. These studies identify IGFBP2 as a novel target of the RET proto-oncogene and demonstrate that ONC201, through its effect on ATF4, reverses RET-mediated signaling. These results support the further investigation of ONC201 as a therapeutic agent for the treatment of MTC alone or in combination with tyrosine kinase inhibitors. Citation Format: Rozita Bagheri-Yarmand, Ling Li, Rohinton Tarapore, Joshua E. Allen, Steven I. Sherman, Robert F. Gagel. ONC201 inhibits RET and IGFBP2 signaling through ATF4 mediated- Integrated stress response in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1286.

Airway smooth muscle cells are insensitive to the anti-proliferative effects of corticosteroids: The novel role of insulin growth factor binding Protein-1 in asthma

Airway remodeling in asthma manifests, in part, as enhanced airway smooth muscle (ASM) mass, due to myocyte proliferation. While the anti-proliferative effects of glucocorticoid (GC) were investigated in normal ASM cells (NASMC), little is known about such effects in ASM cells derived from asthma subjects (AASMC). We posit that GC differentially modulates mitogen-induced proliferation of AASMC and NASMC. Cells were cultured, starved, then treated with Epidermal growth factor (EGF) (10 ng/ml) and Platelet-derived growth factor (PDGF) (10 ng/ml) for 24 h and/or fluticasone propionate (FP) (100 nM) added 2 h before. Cell counts and flow cytometry analyses showed that FP failed to decrease the cell number of and DNA synthesis in AASMC irrespective of mitogens used. We also examine the ability of Insulin Growth Factor Binding Protein-1 (IGFBP-1), a steroid-inducible gene that deters cell growth in other cell types, to inhibit proliferation of AASMC where FP failed. We found that FP increased IGFBP1 mRNA and protein levels. Interestingly, the addition of IGFBP1 (1 μg/ml) to FP completely inhibited the proliferation of AASMC irrespective to the mitogens used. Further investigation of different signaling molecules involved in ASM growth and GC receptor functions (Protein kinase B (PKB/AKT), Mitogen-activated protein kinases (MAPKs), Focal Adhesion Kinase (FAK)) showed that IGFBP-1 selectively decreased mitogen-induced p38 phosphorylation in AASMC. Collectively, our results show the insensitivity of AASMC to the anti-proliferative effects of GC, and demonstrate the ability of IGFBP1 to modulate AASMC growth representing, hence, a promising strategy to control ASM growth in subjects with GC insensitive asthma.

Prenatal vitamin D and cord blood insulin-like growth factors in Dhaka, Bangladesh.

Fetal growth restriction is linked to adverse health outcomes and is prevalent in low- and middle-income countries; however, determinants of fetal growth are still poorly understood. The objectives were to determine the effect of prenatal vitamin D supplementation on the insulin-like growth factor (IGF) axis at birth, to compare the concentrations of IGF-I in newborns in Bangladesh to a European reference population and to estimate the associations between IGF protein concentrations and birth size. In a randomized controlled trial in Dhaka, Bangladesh, pregnant women enrolled at 17–24 weeks of gestation were assigned to weekly oral vitamin D3 supplementation from enrolment to delivery at doses of 4200 IU/week, 16,800 IU/week, 28,000 IU/week or placebo. In this sub-study, 559 woman–infant pairs were included for analysis and cord blood IGF protein concentrations were quantified at birth. There were no significant effects of vitamin D supplementation on cord blood concentrations of IGF-I (P = 0.398), IGF-II (P = 0.525), binding proteins (BPs) IGFBP-1 (P = 0.170), IGFBP-3 (P = 0.203) or the molar ratio of IGF-I/IGFBP-3 (P = 0.941). In comparison to a European reference population, 6% of girls and 23% of boys had IGF-I concentrations below the 2.5th percentile of the reference population. IGF-I, IGF-II, IGFBP-3 and the IGF-I/IGFBP-3 ratio were positively associated with at least one anthropometric parameter, whereas IGFBP-1 was negatively associated with birth anthropometry. In conclusion, prenatal vitamin D supplementation does not alter or enhance fetal IGF pathways.

Preoperative plasma IGFBP2 is associated with nodal metastasis in patients with penile squamous cell carcinoma

PurposeThe nodal status is a strong predictor for clinical outcome in patients with penile cancer. We aimed to evaluate the association between preoperative plasma IGFBP2 levels and nodal status in patients with penile squamous cell carcinoma (PSCC). MethodsThis retrospective study enrolled 56 penile cancer patients who underwent penectomy between 2015 and 2017. Preoperative plasma IGFBP2 levels were detected by enzyme linked immunosorbent assay, which was analyzed in association with clinicopathological parameters (age, body mass index, pathological grade, phimosis, histological subtype, tumor stage, and nodal status). Univariable and Multivariable Cox regression analysis was conducted to identify the prognostic factors that influence disease free survival in PSCC. CCK8 assay and clonogenic assay were used to evaluate the cell viability and tumorigenic potential of PSCC cell line, respectively; wound healing assay, and transwell invasion assay were conducted to evaluate the effect of IGFBP2 depletion on cell migration and invasion in PSCC cells; IGFBP2 protein expression was analyzed by Western blotting. ResultsPlasma IGFBP2 levels were markedly higher in preoperative PSCC than those in healthy male subjects (P = 0.0007). Penectomy led to a significant reduction of plasma IGFBP2 levels in PSCC patients (P = 0.0098). Preoperative plasma IGFBP2 levels were significantly associated with nodal status of PSCC (P < 0.0001). At the cutoff value of 486.2 ng/ml, preoperative plasma IGFBP2 produced a sensitivity of 80.8% and a specificity of 86.7% to discriminate nodal metastasis. Preoperative plasma IGFBP2 levels could serve as independent prognostic factor for disease free survival in PSCC (P = 0.001). Further, knockdown of IGFBP2 expression suppressed cell growth, inhibited clonogenesis, and attenuated cell migration and invasion in Penl1 cells; depletion of IGFBP2 expression attenuated the levels of p-AKT and p-ERK1/2, while increased the expression of p16 and cleaved caspase-3 in Penl1 cells. Silencing IGFBP2 also led to a considerable decline of MMP2/9 levels in culture supernatant of Penl1 cells. ConclusionHigher preoperative plasma IGFBP2 was closely associated with nodal metastasis, which might serve as a useful diagnostic and prognostic biomarker for clinical management of PSCC. IGFBP2 might play an important role in the malignant progression of PSCC. Therapeutic strategies targeting IGFBP2-related signaling pathways may have a therapeutic benefit in PSCC patients.

Suppression of tumor growth via IGFBP3 depletion as a potential treatment in glioma.

Despite intensive medical treatment, patients with glioblastoma (grade IV glioma [GBM]) have a low 5-year survival rate of 5.5%. In this study, the authors tried to improve currently used therapies by identification of a therapeutic target, IGFBP3, for glioma treatment. IGFBP3 RNA expression in 135 patients newly diagnosed with glioma was correlated with clinicopathological factors. Immunohistochemical analysis was performed to determine IGFBP3 protein expression in glioma specimens. The effect of IGFBP3 depletion on cell proliferation was examined using IGFBP3 knockdown glioma cells. Intracranial infusion of IGFBP3 siRNAs was performed to evaluate the effect of IGFBP3 depletion in mouse intracranial xenograft models. We demonstrated higher IGFBP3 expression in GBM than in tumor margin and grade II glioma. IGFBP3 expression was not only positively correlated with tumor grades but also associated with tumor histology and IDH1/2 mutation status. Additionally, higher IGFBP3 expression predicted shorter overall survival in glioma and GBM proneural subgroup patients. In vitro cell culture studies suggested IGFBP3 knockdown suppressed cell proliferation and induced cell cycle G2/M arrest as well as apoptosis in glioma cells. Also, accumulation of DNA double-strand breaks and γH2AX was observed in IGFBP3 knockdown cells. IGFBP3 knockdown delayed in vivo tumor growth in mouse subcutaneous xenograft models. Furthermore, convection-enhanced delivery of IGFBP3 siRNA to mouse brain suppressed intracranial tumor growth and prolonged survival of tumor-bearing mice. Our findings suggest IGFBP3 predicts poor outcome of glioma patients and is a potential therapeutic target for which depletion of its expression suppresses tumor growth through inducing apoptosis and accumulation of DNA damage in glioma cells.

Changes in the Metastatic Properties of MDA-MB-231 Cells after IGFBP6 Gene Knockdown Is Associated with Increased Expression of miRNA Genes Controlling INSR, IGF1R, and CCND1 Genes.

Metastatic cascade is associated with the process of epithelial-mesenchymal transition accompanied by changes in cell proliferation, migration, adhesion, and invasiveness mediated by the insulin-like growth factor (IGF) signal pathway. IGFBP6 protein binds IGF and prevents its interaction with receptors. IGFBP6 gene knockdown through RNA-interference inhibits cell migration and increased the rate of proliferation of breast cancer MDA-MB-231 cells. IGFBP6 knockdown cells are characterized by increased expression of MIR100 and MIRLET7A2 genes encoding hsa-miR-100-3p, hsa-miR-100-5p, hsa-let-7a-5p, and hsa-let-7a-2-3p miRNA. The target genes of these microRNAs are IGF2, IGF1R, INSR, and CCND1 associated with IGF signaling pathway and proliferative and migratory activity during the metastatic cascade. A significant decrease in the expression of INSR and CCND1 genes was demonstrated by PCR and microarray analysis.

In vitro decidualisation of human endometrial stromal cells is enhanced by seminal fluid extracellular vesicles

ABSTRACTExtracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR−/−: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFβ: transforming growth factor β; uNK: uterine natural killer

An integrative systems approach identifies novel candidates in Marfan syndrome-related pathophysiology.

Marfan syndrome (MFS) is an autosomal dominant genetic disorder caused by mutations in the FBN1 gene. Although many peripheral tissues are affected, aortic complications, such as dilation, dissection and rupture, are the leading causes of MFS‐related mortality. Aberrant TGF‐beta signalling plays a major role in the pathophysiology of MFS. However, the contributing mechanisms are still poorly understood. Here, we aimed at identifying novel aorta‐specific pathways involved in the pathophysiology of MFS. For this purpose, we employed the Fbn1 under‐expressing mgR/mgR mouse model of MFS. We performed RNA‐sequencing of aortic tissues of 9‐week‐old mgR/mgR mice compared with wild‐type (WT) mice. With a false discovery rate <5%, our analysis revealed 248 genes to be differentially regulated including 20 genes previously unrelated with MFS‐related pathology. Among these, we identified Igfbp2, Ccl8, Spp1, Mylk2, Mfap4, Dsp and H19. We confirmed the expression of regulated genes by quantitative real‐time PCR. Pathway classification revealed transcript signatures involved in chemokine signalling, cardiac muscle contraction, dilated and hypertrophic cardiomyopathy. Furthermore, our immunoblot analysis of aortic tissues revealed altered regulation of pSmad2 signalling, Perk1/2, Igfbp2, Mfap4, Ccl8 and Mylk2 protein levels in mgR/mgR vs WT mice. Together, our integrative systems approach identified several novel factors associated with MFS‐aortic‐specific pathophysiology that might offer potential novel therapeutic targets for MFS.

Involvement and expression of growth hormone/insulin-like growth factor member mRNAs in the ovarian development of turbot (Scophthalmus maximus).

Accumulating evidence suggests that the growth hormone (GH)/insulin-like growth factor (IGF) system participates in fish reproduction. To understand the physiological functions of the GH/IGF system, the mRNA expression profiles of all known members of the GH/IGF system, including hepatic and ovarian gh, GH receptor (ghr), IGFs (igf-i, igf-ii), IGF-I receptor (igf-ir) and IGF binding protein (igfbp1, igfbp2), pituitary gh, and hepatic vitellogenin (vtg) were investigated during ovarian development in turbot Scophthalmus maximus. Results showed that ghr, igf-i, igf-ii, igf-ir, and igfbp2 were expressed in the liver and ovary, whereas igfbp1 and gh were undetected. The hepatosomatic index (HSI) and gonadosomatic index (GSI) gradually increased and peaked during the late vitellogenesis (Latvtg) and migratory nucleus (Mig-nucl) stages, respectively. The mRNA expression profiles of ovarian ghr, igf-ii, hepatic igf-ir, vtg, and pituitary gh were similar to the HSI; ovarian igf-i and igf-ir expression was close to the GSI. However, the hepatic mRNA levels of ghr, igf-i, and igf-ii peaked at the early vitellogenesis (Evtg) stage, and then drastically declined during ovarian development. The mRNA expression of hepatic igfbp2 decreased and reached the lowest at the atresia (Atre) stage, whereas that of ovarian igfbp2 increased and peaked at Latvtg stage. Furthermore, significant correlations between pituitary gh, ovarian ghr, igf-i, and igf-ii, and hepatic ghr, igf-i, igf-ir, and igf-ii were observed, respectively. These results suggest that GH/IGF members appear to play distinct roles in the regulation of ovarian development in turbot and will be valuable for fish reproduction and broodstock management of aqua-cultured fish species.