Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells.

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

Arachidonic Acid in Follicular Fluid of PCOS Induces Oxidative Stress in a Human Ovarian Granulosa Tumor Cell Line (KGN) and Upregulates GDF15 Expression as a Response.

Polycystic ovarian ovary syndrome (PCOS) is the main cause of ovulatory infertility and a common reproductive endocrine disease of women in reproductive age. In addition, nearly half of PCOS patients are associated with obesity, and their total free fatty acids tend to increase. Arachidonic acid (AA) is a polyunsaturated fatty acid. Oxidation products of AA reacting with various enzymes[cyclooxygenases (COX), lipoxygenases (LOX), cytochrome P450s (CYP)] can change cellular mitochondrial distribution and calcium ion concentration, and increase reactive oxygen species (ROS) production. In this study, we analyzed the follicular fluid fatty acids and found higher levels of C20:4n6 (AA) in PCOS patients than in normal control subjects. Also, to determine whether AA induces oxidative stress (OS) in the human ovarian granulosa tumor cell line (KGN) and affects its function, we treated KGN cells with or without reduced glutathione (GSH) and then stimulated them with AA. The results showed that AA significantly reduced the total antioxidant capacity (TAC) and activity of antioxidant enzymes and increased the malondialdehyde (MDA), ROS and superoxide anion(O2-)levels in KGN cells. In addition, AA was also found to impair the secretory and mitochondrial functions of KGN cells and induce their apoptosis. We further investigated the downstream genes affected by AA in KGN cells and its mechanism of action. We found that AA upregulated the expression of growth differentiation factor 15 (GDF15), which had a protective effect on inflammation and tissue damage. Therefore, we investigated whether AA-induced OS in KGN cells upregulates GDF15 expression as an OS response.Through silencing of GDF15 and supplementation with recombinant GDF15 (rGDF15), we found that GDF15, expressed as an OS response, protected KGN cells against AA-induced OS effects, such as impairment of secretory and mitochondrial functions and apoptosis. Therefore, this study suggested that AA might induce OS in KGN cells and upregulate the expression of GDF15 as a response to OS.

TGF-ß1, NAG-1, and antioxidant enzymes expression alterations in Cisplatin-induced nephrotoxicity in a rat model: Comparative modulating role of Melatonin, Vit. E and Ozone.

Cisplatin (CP) is an anticancer medication that is commonly used to treat solid tumors. Its use is, however, dose-restricted due to nephrotoxicity. We planned to compare the nephroprotective effects of three major compounds, including melatonin (MN), Ozone, or vitamin E, against the CP-induced renal damage in rats. CP was given once intraperitoneally (10 mg/kg,) eliciting acute kidney injury as assured by several adverse histological changes; glomerulopathy, tubulopathy, and vasculopathy, an inflammatory response including elevated TNF-α, IL-6, and IL-1ß. Furthermore, biochemical alterations including, elevated plasma levels of urea, uric acid, creatinine, phosphorous, decreased plasma calcium levels, and gene expression abnormalities; upregulation of N-acetyl-ß-d-glucosaminidase (NAG) and Transforming growth factor-ß1 (TGF-ß1), downregulation of CAT and SOD. Concurrent supplementation with either MN (10 mg/kg per os) or Ozone (1.1 mg/kg ip) and Vit E given by oral gavage (1 g/kg) for five consecutive days prior to CP injection and five days afterward displayed variable significant nephroprotective effects by mitigating the pro-inflammatory secretion, augmenting antioxidant competence, and modulating the gene expression in the renal tissue. The obtained biochemical, histological, and gene expression data suggested that MN had foremost rescue effects followed by Ozone then Vit E. MN's ameliorative effect was augmented in many indices including TNF-α, IL-6 , IL1-ß, uric acid, creatinine, sNGAL and GGT, more than observed in Ozone, and Vit E therapy. A combination of these medications is expected to be more useful in relieving the damaging renal effects of CP given to cancer patients, pending further toxicological and pharmacological research.

GDF15: a potential therapeutic target for type 1 diabetes.

INTRODUCTION: Current treatment for type 1 diabetes (T1D) is centered around insulin supplementation to manage the effects of pancreatic ß cell loss. GDF15 is a potential preventative therapy against T1D progression that could work to curb increasing disease incidence. AREAS COVERED: This paper discusses the known actions of GDF15, a pleiotropic protein with metabolic, feeding, and immunomodulatory effects, connecting them to highlight the open opportunities for future research. The role of GDF15 in the prevention of insulitis and protection of pancreatic ß cells against pro-inflammatory cytokine-mediated cellular stress are examined and the pharmacological promise of GDF15 and critical areas of future research are discussed. EXPERT OPINION: GDF15 shows promise as a potential intervention but requires further development. Preclinical studies have shown poor efficacy, but this result may be confounded by the measurement of gross GDF15 instead of the active form. Additionally, the effect of GDF15 in the induction of anorexia and nausea-like behavior and short-half-life present significant challenges to its deployment, but a systems pharmacology approach paired with chronotherapy may provide a possible solution to therapy for this currently unpreventable disease.

Qizhen capsule inhibits colorectal cancer by inducing NAG-1/GDF15 expression that mediated via MAPK/ERK activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Qizhen capsule (QZC) is a traditional Chinese medicine (TCM) preparation that has been widely used in clinical practice and exerts promising therapeutic effects against breast, lung, and gastric cancers. However, studies have not reported whether QZC inhibits colorectal cancer (CRC) development and progression. Meanwhile, the underlying molecular mechanisms of its anticancer activity have not been studied. AIM OF THE STUDY: To investigate the anticancer effects of QZC on CRC and the possible underlying molecular mechanisms of QZC in vitro and in vivo. MATERIALS AND METHODS: The MTT assay and flow cytometry were used to determine the viability and apoptosis of HCT116 and HT-29 cancer cells. A xenograft nude mouse model was used to study the antitumor effects of QZC in vivo. Western blotting was performed to determine the expression of key proteins responsible for the molecular mechanisms elicited by QZC. Immunofluorescence staining was performed to detect the expression of nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 or growth differentiation factor-15 (NAG-1/GDF15). Small interfering RNAs (siRNAs) were used to silence NAG-1/GDF15 in cells. RESULTS: In this study, QZC significantly reduced the viability of HCT116 and HT-29 cells and induced apoptosis in dose- and time-dependent manners, but displayed much less toxicity toward normal cells. QZC-induced apoptosis in HCT116 cells was accompanied by the deregulation of the expression of the Bcl-2, Bax, PARP, caspase-3, and caspase-9 proteins. Furthermore, QZC induced NAG-1/GDF15 expression in HCT116 cells, while silencing of NAG-1/GDF15 attenuated QZC-induced apoptosis and cell death. Next, QZC increased the phosphorylation of mTOR, AMPK, p38, and MAPK/ERK in HCT116 cells. We then demonstrated that QZC-induced apoptosis and NAG-1/GDF15 upregulation were mediated by MAPK/ERK activation. Moreover, QZC significantly inhibited HCT116 xenograft tumor growth in nude mice, which was accompanied by NAG/GDF15 upregulation and MAPK/ERK activation. QZC also prevented 5-FU-induced weight loss or cachexia in tumor-bearing mice. The expression of Ki67 and PCNA was suppressed, while cleaved caspase-3 level and TUNEL staining were increased in the tumor sections from QZC-treated mice compared to the control. CONCLUSION: QZC is a novel anticancer agent for CRC that targets NAG-1/GDF15 via the MAPK/ERK signaling pathway.

Growth differentiation factor 15 as a potential therapeutic for treating obesity.

BACKGROUND: Obesity is rapidly becoming one of the world's most critical health care concerns. Comorbidities accompanying excess weight include cardiovascular disease, diabetes, and certain cancers. These comorbidities result in greater hospitalization and other health care-related costs. Economic impacts are likely to be felt more acutely in developing countries, where obesity rates continue to rise and health care resources are already insufficient. Some of the more effective treatments are invasive and expensive surgeries, which some economies in the world cannot afford to offer to a broad population. Pharmacological therapies are needed to supplement treatment options for patients who cannot, or will not, undergo surgical treatment. However, the few drug therapies currently available have either limited efficacy or safety concerns. A possible exception has been glucagon-like peptide-1 analogs, although these have shown a number of adverse events. New drug therapies that are safe and produce robust weight loss are needed. SCOPE OF REVIEW: Herein, we review the role of growth differentiation factor 15 (GDF15) in feeding behavior and obesity, summarize some of the new and exciting biological discoveries around signaling pathways and tissue sites of action, and highlight initial efforts to develop GDF15-based therapies suitable for inducing weight loss in humans. MAJOR CONCLUSIONS: Within the last several years, great strides have been made in understanding the biology of GDF15. Recent developments include identification of an endogenous receptor, biological localization of the receptor system, impact on energy homeostasis, and identification of molecules suitable for administration to humans as anti-obesity treatments. New and exciting research on GDF15 suggests that it holds promise as a novel obesity treatment as new molecules progress toward clinical development.

Targeting MAPK (p38, ERK, JNK) and inflammatory CK (GDF-15, GM-CSF) in UVB-Activated Human Skin Cells with Vitis vinifera Seed Extract.

Ultraviolet B radiation (UVB) activates mitogen-activated protein kinases (MAPK): p38, extracellular signal regulated (ERK), and c-Jun N-terminal (JNK) kinases in human skin cells. Human keratinocytes (KC) exposed to UVB secrete several cytokines (CK), among which the growth differentiation factor-15 (GDF-15) is augmented in inflammatory and aging processes and the granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in cell proliferation, differentiation, and survival, and both CK have implications in skin carcinogenesis. We assessed p38, ERK, JNK, GDF-15, and GM-CSF in UVB-exposed skin cells and a red grape (Vitis vinifera) seed extract's (GSE) capacities to regulate these pathways in UVB-exposed KC. Two concentrations of the GSE extract were selected: GSE1 (37.5 µgEqGA/mL) and GSE2 (75 µgEqGA/mL) and a UVB dose (100 mJ/cm2) within the physiological range. Molecules were assessed with ELISA, semiquantitative results being confirmed by Western blot. UVB triggered the signaling molecules' phosphorylation and the concentrations of CK. All molecules but GM-CSF increased early, at 2 h, from UVB exposure while GM-CSF increased later (at 8 h). MAPK and GDF-15 were regulated by GSE1; GM-CSF, by the higher concentration, GSE2. The amplitude and kinetics of the responses were diverse according to time point, molecules, and the extract's concentration. GSE exerted beneficial effects on MAPK and CK triggered by UVB in human skin cells: reduction of phosphorylation of the assessed signaling molecules and anti-inflammatory effects. Targeting MAPK and specific inflammatory mediators such as GDF-15 and GM-CSF with GSE in UVB-induced skin cells represents a novel and a promising starting point for future photoprotection strategies.

Towards frailty biomarkers: Candidates from genes and pathways regulated in aging and age-related diseases.

OBJECTIVE: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel. METHODS: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers. RESULTS: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified. CONCLUSION: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFß (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Oral Treatment with the Ghrelin Receptor Agonist HM01 Attenuates Cachexia in Mice Bearing Colon-26 (C26) Tumors.

The gastrointestinal hormone ghrelin reduces energy expenditure and stimulates food intake. Ghrelin analogs are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to investigate whether oral treatment with the non-peptidergic ghrelin receptor agonist HM01 counteracts CACS in colon-26 (C26) tumor-bearing mice. The C26 tumor model is characterized by pronounced body weight (BW) loss and muscle wasting in the absence of severe anorexia. We analyzed the time course of BW loss, body composition, muscle mass, bone mineral density, and the cytokines interleukin-6 (IL-6) and macrophage-inhibitory cytokine-1 (MIC-1). Moreover, we measured the expression of the muscle degradation markers muscle RING-finger-protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx). After tumor inoculation, MIC-1 levels increased earlier than IL-6 and both cytokines were elevated before MuRF-1/MAFbx expression increased. Oral HM01 treatment increased BW, fat mass, and neuronal hypothalamic activity in healthy mice. In tumor-bearing mice, HM01 increased food intake, BW, fat mass, muscle mass, and bone mineral density while it decreased energy expenditure. These effects appeared to be independent of IL-6, MIC-1, MuRF-1 or MAFbx, which were not affected by HM01. Therefore, HM01 counteracts cachectic body weight loss under inflammatory conditions and is a promising compound for the treatment of cancer cachexia in the absence of severe anorexia.

Docosahexaenoic acid blocks progression of western diet-induced nonalcoholic steatohepatitis in obese Ldlr-/- mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a major public health concern in western societies. Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is characterized by hepatic steatosis, inflammation, oxidative stress and fibrosis. NASH is a risk factor for cirrhosis and hepatocellular carcinoma. NASH is predicted to be the leading cause of liver transplants by 2020. Despite this growing public health concern, there remain no Food and Drug Administration (FDA) approved NASH treatments. Using Ldlr -/- mice as a preclinical model of western diet (WD)-induced NASH, we previously established that dietary supplementation with docosahexaenoic acid (DHA, 22:6,ω3) attenuated WD-induced NASH in a prevention study. Herein, we evaluated the capacity of DHA supplementation of the WD and a low fat diet to fully reverse NASH in mice with pre-existing disease. METHODS: Ldlr -/- mice fed the WD for 22 wks developed metabolic syndrome (MetS) and a severe NASH phenotype, including obesity, dyslipidemia, hyperglycemia, hepatic steatosis, inflammation, fibrosis and low hepatic polyunsaturated fatty acid (PUFA) content. These mice were randomized to 5 groups: a baseline group (WDB, sacrificed at 22 wks) and 4 treatments: 1) WD + olive oil (WDO); 2) WD + DHA (WDD); 3) returned to chow + olive oil (WDChO); or 4) returned to chow + DHA (WDChD). The four treatment groups were maintained on their respective diets for 8 wks. An additional group was maintained on standard laboratory chow (Reference Diet, RD) for the 30-wk duration of the study. RESULTS: When compared to the WDB group, the WDO group displayed increased hepatic expression of genes linked to inflammation (Opn, Il1rn, Gdf15), hepatic fibrosis (collagen staining, Col1A1, Thbs2, Lox) reflecting disease progression. Mice in the WDD group, in contrast, had increased hepatic C20-22 ω3 PUFA and no evidence of NASH progression. MetS and NASH markers in the WDChO or WDChD groups were significantly attenuated and marginally different from the RD group, reflecting disease remission. CONCLUSION: While these studies establish that DHA supplementation of the WD blocks WD-induced NASH progression, DHA alone does not promote full remission of diet-induced MetS or NASH.

Potential of Horse Apple Isoflavones in Targeting Inflammation and Tau Protein Fibrillization.

In our ongoing search for anti-inflammatory and neuroprotective agents of natural origin, the total methanolic extract (MPE) of horse apple (Maclura pomifera) and its two major prenylated isoflavones, osajin (OSA) and pomiferin (POM), were evaluated in vitro for their ability to affect four mediators of inflammation and to inhibit tau protein fibrillization. The two isoflavones were effective in enhancing the activity of NSAID activated gene (NAG-1) at 2.5 pg/mL (1.5-1.8 fold increase) and inhibiting iNOS and NF-κB activity with IC50 values in the range of 6-13 µg/mL. Pomiferin also inhibited intracellular oxidative stress with IC50 of 3.3 µg/mL, while osajin did not show any effect. The extract activated NAG-1 and inhibited iNOS and oxidative stress without affecting NF-κB. As observed by transmission electron microscopy (TEM), MPE, OSA and POM also inhibited arachidonic acid-induced tau fibrillization in a concentration-dependent manner.

Taiwanin A targets non-steroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma.

Taiwanin A (α,ß-bis(piperonylidene)-γ-butyrolactone) is extracted from Taiwania cryptomerioides. Taiwanin A is extracted from tree bark and exhibits antitumor activity in breast, liver, and lung cancer cell lines. The objective of this study was to demonstrate the cytotoxicity of Taiwanin A against tumor cells by increasing the expression of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). NAG-1 has been reported to exhibit antitumor and proapoptotic activities, suggesting potential use in cancer therapy. Inhibiting NAG-1 mRNA expression in A549 reduced the cytotoxicity caused by Taiwanin A. Furthermore, the c-Jun-N-terminal kinase/Ste20-related protein proline/alanine-rich kinase (JNK/SPAK) pathway played a key role in the influence of NAG-1 on cell viability, whereas the addition of the JNK pathway inhibitor SP600125 resulted in an inhibitory effect on NAG-1 and recovery of Taiwanin-A-treated cells. A xenograft tumor model demonstrated that Taiwanin A dose-dependently significantly decreases tumor-mediated growth in nude mice by increasing the NAG-1 expression accompanying tumor apoptosis. These data supported the hypothesis that Taiwanin A inhibits lung carcinoma growth by increasing NAG-1 expression through the JNK pathway both in vivo and in vitro. This result can contribute to a compound design for increasing cytotoxicity activity in the future.

Nonsteroidal anti-inflammatory drug activated gene-1 (NAG-1) modulators from natural products as anti-cancer agents.

Natural products are rich sources of gene modulators that may be useful in prevention and treatment of cancer. Recently, nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) has been focused as a target of action against diverse cancers like colorectal, pancreatic, prostate, and breast. A variety of natural agents have been reported to play a pivotal role in regulation of NAG-1 through multiple transcriptional mechanisms. The aim of this paper is to review the NAG-1 modulators derived from natural products including plants, marine organisms, and microorganisms. Plant extracts belonging to the families of Fabaceae (Astragalus membranaceus), Ranunculaceae (Coptis chinensis), Menispermaceae (Coscinium fenestratum), Umbelliferae (Pleurospermum kamtschaticum), Lamiaceae (Marubium vulgare), and Rosaceae (Prunus serotina) increased the protein expression of NAG-1 in human colon cancer or hepatocarcinoma cells. Phytochemicals in the class of flavonoids (apigenin, quercetin, isoliquiritigenin, and 2'-hydroxyflavanone), isoflavonoids (formononetin and genistein), catechins (epigallocatechin gallate and epicatechin gallate), stilbenoids (resveratrol and pinosylvin), phenolics (6-gingerol), phloroglucinols (rottlerin and aspidin PB), terpenoids (18 α-glycyrrhetinic acid, platycodin D, pseudolaric acid B, and xanthorrhizol), alkaloids (berberine, capsaicin, and indole-3-carbinol), lignans (isochaihulactone), anthraquinones (damnacanthal), and allyl sulfides (diallyl disulfide) elicited NAG-1 overexpression in various cancer cells. Pectenotoxin-2 from marine organisms and prodigiosin and anisomycin from microorganisms were also reported as NAG-1 modulators. Several transcription factors including EGR-1, p53, ATF-3, Sp1 and PPARγ were involved in natural products-induced NAG-1 transcriptional signaling pathway.

Pro-osteogenic topographical cues promote early activation of osteoprogenitor differentiation via enhanced TGFß, Wnt, and Notch signaling.

OBJECTIVES: Titanium implant surfaces with modified topographies have improved osteogenic properties in vivo. However, the molecular mechanisms remain obscure. This study explored the signaling pathways responsible for the pro-osteogenic properties of micro-roughened (SLA) and chemically/nanostructurally (modSLA) modified titanium surfaces on human alveolar bone-derived osteoprogenitor cells (BCs) in vitro. MATERIALS AND METHODS: The activation of stem cell signaling pathways (TGFß/BMP, Wnt, FGF, Hedgehog, Notch) was investigated following early exposure (24 and 72 h) of BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture supplements. RESULTS: Key regulatory genes from the TGFß/BMP (TGFBR2, BMPR2, BMPR1B, ACVR1B, SMAD1, SMAD5), Wnt (Wnt/ß-catenin and Wnt/Ca(2+) ) (FZD1, FZD3, FZD5, LRP5, NFATC1, NFATC2, NFATC4, PYGO2, LEF1) and Notch (NOTCH1, NOTCH2, NOTCH4, PSEN1, PSEN2, PSENEN) pathways were upregulated on the modified surfaces. These findings correlated with a higher expression of osteogenic markers bone sialoprotein (IBSP) and osteocalcin (BGLAP), and bone differentiation factors BMP2, BMP6, and GDF15, as observed on the modified surfaces. CONCLUSIONS: These findings demonstrate that the activation of the pro-osteogenic cell signaling pathways by modSLA and SLA surfaces leads to enhanced osteogenic differentiation as evidenced after 7 and 14 days culture in osteogenic media and provides a mechanistic insight into the superior osseointegration on the modified surfaces observed in vivo.

Serum hepcidin levels and muscle iron proteins in humans injected with low- or high-dose erythropoietin.

Inhibition of hepcidin expression by erythropoietic signals is of great physiological importance; however, the inhibitory pathways remain poorly understood. To investigate (i) the direct effect of erythropoietin (Epo) and (ii) the contribution of putative mediators on hepcidin repression, healthy volunteers were injected with a single dose of Epo, either low (63 IU/kg, n = 8) or high (400 IU/kg, n = 6). Low-dose Epo provoked hepcidin down-modulation within 24 h; the effect was not immediate as hepcidin circadian variations were still present following injection. High-dose Epo induced no additional effect on the hepcidin response, that is hepcidin diurnal fluctuations were not abolished in spite of extremely high Epo levels. We did not find significant changes in putative mediators of hepcidin repression, such as transferrin saturation, soluble transferrin receptor, or growth differentiation factor 15. Furthermore, the potential hepcidin inhibitor, soluble hemojuvelin, was found unaltered by Epo stimulation. This finding was consistent with the absence of signs of iron deficiency observed at the level of skeletal muscle tissue. Our data suggest that hepcidin repression by erythropoietic signals in humans may not be controlled directly by Epo, but mediated by a still undefined factor.

2'-Hydroxyflavanone induces apoptosis through Egr-1 involving expression of Bax, p21, and NAG-1 in colon cancer cells.

SCOPE: Natural flavanones exhibit cancer preventive and/or therapeutic effects. The objective of this study was to investigate the molecular mechanism underlying the action of the antitumor activity of hydroxyflavanone using the HCT116 colon cancer cell line. METHODS AND RESULTS: We investigated the effect of hydroxyflavanones on antitumor activity. We found that 2'-hydroxyflavanone (2'-HF) potently inhibited the clonogenicity of HCT116 cells. 2'-HF triggered apoptosis in both wild-type and p53-null HCT116 cells, as revealed by DNA fragmentation and caspase activation. 2'-HF upregulated nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression through induction of Egr-1. Silencing of NAG-1 or Egr-1 using small interfering RNA (siRNA) could attenuate 2'-HF-induced apoptosis. Egr-1 also upregulated the proapoptotic gene Bax and the cell cycle inhibitor p21. CONCLUSION: Dietary 2'-HF may possess antitumor activity against human colon cancer.

Evaluation of the immunomodulatory properties in mice and in vitro anti-inflammatory activity of cycloartane type saponins from Astragalus species.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus roots are used to treat leukemia and for their wound healing properties in Southeast Anatolia-Turkey. MATERIALS AND METHODS: In vivo studies to investigate the effects of two Astragalus saponins were carried out on the immune response cytokines by using six to eight weeks old male Swiss albino mice. The production of IL-1ß, TGF-1ß, TNF-α, IL-2, IL-4 and IFN-γ cytokines was determined by ELISA. The spleen and lymph nodes, isolated from the mice subjects, were analyzed to realize induction of the surface antigen productions for IL-2Rα (CD25) and CD69. In addition, their effects on the targets of inflammation such as NF κB, iNOS and NAG-1 were investigated in cell-based assays. RESULTS: The results suggested that AST VII and Mac B had positive effect on Th1 cytokine release (IL-2 and IFN-γ), and suppression on Th2 cytokine production (IL-4). The immunohistochemical results exhibited induction of both IL-Rα (CD25) and CD69 surface receptors justifying the Th1 cytokine release. The compounds did not affect NF-κB or NAG-1 activity but iNOS activity was inhibited by Mac B with an IC(50) of 156 µg/ml. CONCLUSIONS: The results show that Ast VII and Mac B create powerful immunoregulatory effects without the stimulation of inflammatory cytokines in mice, and have no significant effect on the inflammatory cellular targets in vitro.

[Effect of GDF15 on iron overloading and erythropoiesis].

Ineffective erythropoiesis is recognized as the principal reason of non-transfusional iron overload. In the process of expanded erythropoiesis, the apoptosis of erythroblasts induces the up-regulation of GDF15. GDF15 suppresses hepcidin production by the hepatocytes. Subsequently, low hepcidin levels increase iron absorption from the intestine resulting in iron overload. Physiological dose of GDF15 can promote the growth and differentiation of erythroid progenitors, but the high dose of GDF15 can suppress the secretion of hepcidin. The regulation of GDF15 may also be related to iron levels, epigenetic regulation and hypoxia. In this article the GDF15 and its expression and distribution, roles of GDF15 in erythropoiesis and iron overload, as well as the regulation factors of GDF15 are reviewed.

Coptis chinensis inhibits hepatocellular carcinoma cell growth through nonsteroidal anti-inflammatory drug-activated gene activation.

Conventional chemotherapy of liver cancer fails to provide satisfactory remission and may cause serious side effects, thus it is crucial to derive alternative treatments that effectively inhibit cancer cell growth with known mechanisms of action. In the present study, we investigated the anti-carcinogenic effects of Coptis chinensis and its major constituent, berberine, in HepG2 hepatocellular carcinoma (HCC) cells and attempted to elucidate the underlying mechanism, including involvement of the nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1). Inhibition of cell proliferation, induction of apoptosis and cell cycle arrest at the G2/M phase were observed in HepG2 cells treated with Coptis chinensis or berberine. The pro-apoptotic effects were associated with corresponding down-regulation of Bcl-2, activation of procaspase-3 and -9 as well as cleavage of poly (ADP-ribose) polymerase. We further demonstrated the involvement of NAG-1 in the pro-apoptotic events following prior activation of its upstream transcriptional factor early growth response gene (Egr-1). This was confirmed by increased NAG-1 promoter activity preceded by the elevation of Egr-1/ DNA binding activity. Our results suggest that both Coptis chinensis and berberine are potential anti-carcinogenic agents in treating HCC by inducing cell cycle arrest and promotion of apoptosis, while NAG-1 is a molecular target during the drug-induced pro-apoptotic action in HepG2 cells.