Anticancer properties of dried-pericarp water extracts of Camellia japonica L. fermented with Aspergillus oryzae through regulation of IGFBP-2/mTOR pathway.

This study aimed to investigate the anticancer activity of dried-pericarp water extract of fermented C. japonicus (CJ). The dried-pericarp water extracts of CJ were fermented using Aspergillus oryzae and Saccharomyces cerevisiae at 30 °C and 35 °C. The anticancer activities of both water extracts fermented at 30 °C and 35 °C using A. oryzae against FaDu cells were remarkably changed compared with unfermented dried-pericarp water extract of CJ, which has no anticancer activity. Cleaved-PARP, caspase 3, and apoptotic cells stained with annexin V/PI were significantly increased by treatment with A. oryzae extracts fermented at 30 °C. The insulin-like growth factor-binding protein 2 (IGFBP-2) protein level and mTOR phosphorylation by A. oryzae fermented extracts (AOFE) were dramatically reduced, and the expression levels of IGFBP-2 and phosphorylated mTOR were significantly increased depending on the glucose concentrations in FaDu cells. These results suggested that the cell viabilities in AOFE were restored as the glucose concentrations increased. Furthermore, it was confirmed LC/MS/MS that the content of gallic acid was increased by fermentation of Aspergillus oryzae (5.596 ± 0.1746 µg/mg) compared to the unfermented extract (1.620 ± 0.0432 µg/mg). Based on these results, the anticancer effect of AOFE was achieved through inhibition of the IGFBP-2/mTOR signaling pathway. These results suggest that AOFE may be a potential treatment for head and neck cancer.

Prominence of IL6, IGF, TLR, and Bioenergetics Pathway Perturbation in Lung Tissues of Scleroderma Patients With Pulmonary Fibrosis.

Scleroderma-associated pulmonary fibrosis (SSc-PF) and idiopathic pulmonary fibrosis (IPF) are two of many chronic fibroproliferative diseases that are responsible for nearly 45% of all deaths in developed countries. While sharing several pathobiological characteristics, they also have very distinct features. Currently no effective anti-fibrotic treatments exist that can halt the progression of PF or reverse it. Our goal is to uncover potential gene targets for the development of anti-fibrotic therapies efficacious in both diseases, and those specific to SSc-PF, by identifying universal pathways and molecules driving fibrosis in SSc-PF and IPF tissues as well as those unique to SSc-PF. Using DNA microarray data, a meta-analysis of the differentially expressed (DE) genes in SSc-PF and IPF lung tissues (diseased vs. normal) was performed followed by a full systems level analysis of the common and unique transcriptomic signatures obtained. Protein-protein interaction networks were generated to identify hub proteins and explore the data using the centrality principle. Our results suggest that therapeutic strategies targeting IL6 trans-signaling, IGFBP2, IGFL2, and the coagulation cascade may be efficacious in both SSc-PF and IPF. Further, our data suggest that the expression of matrikine-producing collagens is also perturbed in PF. Lastly, an overall perturbation of bioenergetics, specifically between glycolysis and fatty acid metabolism, was uncovered in SSc-PF. Our findings provide insights into potential targets for the development of anti-fibrotic therapies that could be effective in both IPF and SSc-PF.

Cloning, molecular characterization and expression analysis of insulin-like growth factor binding protein-2 (IGFBP-2) cDNA in goldfish, Carassius auratus.

In the present study, a full-length cDNA encoding the insulin-like growth factor binding protein-2 (IGFBP-2) was cloned from the liver of goldfish (Carassius auratus) by rapid amplification of cDNA ends technique. The goldfish IGFBP-2 cDNA sequence was 1,513 bp long and had an open reading frame of 825 bp encoding a predicted polypeptide of 274 amino acid residues. Semi-quantitative RT-PCR results revealed that goldfish IGFBP-2 mRNA was expressed in all detected tissues. In liver, central nervous system and pituitary gland, goldfish IGFBP-2 expressed at high levels, followed by anterior intestine, middle intestine and kidney. In posterior intestine, ovary, skin, fat, spleen, muscle and gill, the goldfish IGFBP-2 expression levels were very low. Fasting and refeeding experiment showed that the mRNA expression of goldfish IGFBP-2 was up-regulated significantly in liver compared to the fed group and restored rapidly to normal level after refed. However, the mRNA expressions of IGFBP-2 in hypothalamus and pituitary of goldfish were insensitive to fasting. Furthermore, the mRNA expressions of IGFBP-2 in hypothalamus, pituitary and liver were varied in periprandial changes and significantly down-regulated at 2 and 4 h after meal. These results imply that the IGFBP-2 mRNA expression may be associated with anabolic and catabolic metabolism and regulated by metabolic factors in goldfish.

Hepatic molecular responses to Bifidobacterium pseudocatenulatum CECT 7765 in a mouse model of diet-induced obesity.

BACKGROUND AND AIMS: Bifidobacterium pseudocatenulatum CECT 7765 moderates body weight gain and metabolic parameters in high-fat diet-(HFD)-fed mice but, the mechanisms of action are not yet understood. To further understand the effects of this bacterial strain, we have investigated the molecular changes in the liver of mice fed a HFD and supplemented with the bacteria. METHODS AND RESULTS: Gene expression and protein levels were measured in the liver of C57BL/6 male mice following sub-chronic consumption of a HFD and B. pseudocatenulatum CECT 7765. Our results show that the consumption of this bacterial strain modulated the expression of key genes involved in the regulation of energy metabolism and transport of lipids that were affected by the HFD.B. pseudocatenulatum CECT 7765 significantly counteracted the effects caused by the HFD on the fatty acid transporter CD36, the transcription regulator of lipid biosynthesis EGR1 and the regulators of glucose metabolism, IGFBP2 and PPP1R3B, both at the mRNA and protein levels. The bacterial strain slightly induced the transcript levels of PNPLA2, a lipase that hydrolyses triglycerides in lipid droplets. In the standard diet (SD)-fed mice, the administration of B. pseudocatenulatum CECT 7765 donwregulated the expression of INSIG1 and HMGCR critically involved in the regulation of cholesterol levels. CONCLUSION: B. pseudocatenulatum CECT 7765 modified the expression of key regulators of fatty acid and cholesterol metabolism and transport, lipid levels and glucose levels in the liver which supports the beneficial metabolic effects of this bacterial strain.

Insulin-like growth factor binding protein 2 (IGFBP-2) promotes growth and survival of breast epithelial cells: novel regulation of the estrogen receptor.

In breast tumors IGF binding protein-2 (IGFBP-2) is elevated, and the presence of IGFBP-2 has been shown to correlate with malignancy. However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity. Exogenous IGFBP-2 acting via integrin receptors to reduce phosphatase and tensin homolog deleted from chromosome 10 (PTEN) levels protected these cells against death induced by various chemotherapeutic agents. This was dependent on a functional estrogen receptor (ER)-α because silencing ER-α blocked the ability of IGFBP-2 to confer cell survival. Loss of IGFBP-2 increased levels of PTEN and improved chemosensitivity of the cells, confirming its role as a survival factor. Silencing IGFBP-2 had no effect on the response to IGF-II, but responses to estrogen and tamoxifen were no longer observed due to loss of ER-α, which could be prevented by the inhibition of PTEN. Conversely, exogenous IGFBP-2 increased ER-α mRNA and protein in both normal and cancer cells via its interaction with integrin receptors. These actions of IGFBP-2 on ER-α involved the IGF-I receptor and activation of phosphatidylinositol 3-kinase in the cancer cells but were independent of this in normal breast cells. The production of IGFBP-2 by breast cancer cells enhances their proliferative potential, increases their survival, and protects them against chemotherapy-induced death. IGFBP-2 not only modulates IGFs and directly regulates PTEN but also has a role in maintaining ER-α expression.

Isolated isoflavones do not affect the circulating insulin-like growth factor system in men at increased colorectal cancer risk.

Epidemiological studies show that increased insulin-like growth factor (IGF)-I concentrations are related to increased colorectal cancer risk. A reduced colorectal cancer risk has been associated with isoflavones, which might affect the IGF-system because of their weak estrogenic activity. We conducted a randomized, placebo-controlled, double-blind crossover study to investigate the effect of an 8-wk isolated isoflavone supplementation (84 mg/d) on serum concentrations of total IGF-I, free IGF-I, total IGF-II, IGF binding protein (BP)-1, IGFBP-2, and IGFBP-3. Additionally, we investigated whether IGF-system component differences were related to concentrations of the more potent estrogenic isoflavone metabolite, equol. Our study population consisted of 37 men with a family history of colorectal cancer or a personal history of colorectal adenomas. Isoflavone supplementation did not significantly affect serum total IGF-I concentrations (relative difference between serum total IGF-I concentrations after isoflavone supplementation and after placebo: -1.3%, 95% CI -8.6 to 6.0%). Neither free IGF-I, nor total IGF-II, IGFBP-1, IGFBP-2, or IGFBP-3 concentrations were significantly altered. Interestingly, the change in serum IGF-I concentrations after isoflavone supplementation was negatively associated with serum equol concentrations (r=-0.49, P=0.002). In conclusion, isolated isoflavones did not affect the circulating IGF-system in a male high-risk population for colorectal cancer. However, to our knowledge, this is the first study that suggests isoflavones might have an IGF-I lowering effect in equol producers only. This underlines the importance of taking into account equol status in future isoflavone intervention studies.

Structural analysis of human growth hormone with respect to the dominant expression of growth hormone (GH) mutations in isolated GH deficiency type II.

Human GH protein consists of four alpha-helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32-71, which comprise the complete loop between alpha-helices 1 and 2. The mutant GH protein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure-function relationship of GH mutants concerning the dominant negative effect, expression vectors harboring mutated GH cDNAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding beta-galactosidase, luciferase, or IGF-binding protein-2 were cotransfected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32-46 or 32-52. Deletion of more aa (32-53, 32-63, 32-69, 32-71) ascendingly decreased GH secretion and content in parallel with the increasing length of the deleted stretch. An inhibitory dose-dependent effect of del32-69GH and del32-71GH on the activity/amount of coexpressed beta-galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant negative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels.

Expression of insulin-like growth factors (IGF)-1 and -2, IGF-binding proteins-2 and -3, and receptors for growth hormone, IGF type-1 and -2 and insulin in the gastrointestinal tract of neonatal calves.

Growth hormone (GH), insulin-like growth factors (IGFs) and insulin influence post-natal gastrointestinal development and function. We have measured by real-time PCR the mRNA levels of IGF-1 and -2, IGF-binding proteins (IGFBPs)-2 and -3, and receptors for GH, IGF type-1 and -2, and insulin in esophagus, rumen, fundus, pylorus, duodenum, jejunum, ileum and colon of calves on days 1 and 5 of life. Levels of mRNA of measured traits were different (P < 0.05) at different gastrointestinal sites. Furthermore, mRNA levels of IGFs, IGFBPs and of receptors for GH and IGF type-1 and -2 and insulin differed (P < 0.05) on days 1 and 5. Differences in mRNA abundance of IGFs, IGFBPs and of receptors for GH, IGFs, and insulin among gastrointestinal sites on days 1 and 5 of life suggest site-specific functional importance and demonstrate that changes are the consequence of ontogenetic development and/or due to feeding.

Effects of colostrum feeding and dexamethasone treatment on mRNA levels of insulin-like growth factors (IGF)-I and -II, IGF binding proteins-2 and -3, and on receptors for growth hormone, IGF-I, IGF-II, and insulin in the gastrointestinal tract of neonatal calves.

The somatotropic axis regulates growth of the gastrointestinal tract (GIT). In addition, colostrum feeding and glucocorticoids affect maturation of the GIT around birth in mammals. We have measured mRNA levels of members of the somatotropic axis to test the hypothesis that colostrum intake and dexamethasone treatment affect respective gene expression in the GIT. Calves were fed either colostrum or an isoenergetic milk-based formula, and in each feeding group, half of the calves were treated with dexamethasone (DEXA; 30 microg/kg body weight per day). Individual parameters of the somatotropic axis differed (P < 0.05) among different GIT sections and formula feeding increased (P < 0.05) mRNA levels of individual parameters at various sites of the GIT. Effects of DEXA on the somatotropic axis in the GIT partly depended on different feeding. In colostrum-fed calves, DEXA decreased (P < 0.05) mRNA levels of IGF-I (esophagus, fundus, duodenum, and ileum), IGF-II (fundus), IGFBP-2 (fundus), IGFBP-3 (fundus), IGF1R (esophagus, ileum, and colon), IGF2R (fundus), GHR (fundus), and InsR (esophagus, fundus), but in formula-fed calves DEXA increased mRNA levels of IGF-I (esophagus, rumen, jejunum, and colon). Furthermore, DEXA increased (P < 0.05) mRNA levels of IGF-II (pylorus), IGFBP-3 (duodenum), IGF2R (pylorus), and GHR (ileum), but decreased mRNA levels of IGFBP-2 (ileum), and IGF1R (fundus). Whereas formula feeding had stimulating effects, effects of DEXA treatment on the gene expression of parameters of the somatotropic axis varied among GIT sites and partly depended on feeding.

Prenatal exposure of rats to Ginkgo biloba extract (EGb 761) increases neuronal survival/growth and alters gene expression in the developing fetal hippocampus.

Hippocampal neuron survival/growth and gene expression have been examined after prenatal (in utero) exposure of rats to EGb 761, a leaf extract of Ginkgo biloba. Oral administration of EGb 761 (100 or 300 mg/kg/day) to pregnant dams for 5 days increased the number of hippocampal neurons (maintained in culture) of their fetuses, indicating a neurotrophic effect of the extract. Using large-scale oligonucleotide microarrays containing over 8000 combined rat genes and expressed sequence tag clusters, it was shown that treatment of pregnant dams with EGb 761 (25, 50 or 100 mg/kg/day for 5 days) altered the expression of 187 genes in the hippocampi of male fetuses and 160 genes in those of female fetuses. Using gene-cluster analysis, these genes were grouped into 18 distinct clusters for males and 17 distinct clusters for females. Among these clusters, 35 genes shared a common expression pattern in male and female hippocampal development. Of these genes, the changes observed in insulin growth factor II, insulin growth factor binding protein 2, testosterone repressed prostate message-2, glutathione-dependent dehydroascorbate reductase, lipoprotein lipase, guanylate cyclase and DNA binding protein Brn-2 were confirmed by real-time quantitative polymerase chain reaction. These findings, which have provided the first genetic profile of the effects of EGb 761 on the developing rat hippocampus, increase our understanding of the molecular and genetic programs that are activated by the extract. These effects of EGb 761 may underlie its neuroprotective properties.

Dehydroepiandrosterone regulates insulin-like growth factor-1 system in adult rat hypothalamus.

Dehydroepiandrosterone (DHEA) and its sulfate ester (DHEA-S) exert multiple effects in rodent and human brain. Several findings suggest that insulin-like growth factor-1 (IGF-1) is involved in the actions of DHEA. In this study, we assessed whether systemic administration of DHEA regulates the IGF-1 system in the hypothalamus, hippocampus, cerebral cortex, and cerebellum of adult rats. DHEA resulted in a significant reduction in IGF-1 receptor protein levels. This effect was dose dependent and restricted to the hypothalamus. In contrast to IGF-1 receptor, IGF-1-binding protein 2 levels were unaffected by DHEA treatment. IGF-1 levels were significantly increased in the hypothalamus of the rats treated with DHEA, whereas IGF-1 serum levels were not affected by DHEA. The effects of DHEA on the hypothalamic IGF-1 system may be highly relevant to the control and maintenance of hypothalamic neuroendocrine function.

Insulin-like growth factor-I, insulin-like growth factor-binding proteins, and gonadotropins in the hypothalamic-pituitary axis and serum of nutrient-restricted ewes.

Body condition scores (BCS) of ovariectomized estradiol-treated ewes were controlled to examine effects of suboptimum BCS on insulin-like growth factor (IGF)-I, IGF-binding proteins (IGFBPs), and LH in the anterior pituitary gland, hypophyseal stalk-median eminence (SME), and circulation. Serum LH increased in ewes with BCS (1 = emaciated, 9 = obese) > 3 (HIGH-BCS), but not in ewes with BCS