Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

Computational Investigation Identified Potential Chemical Scaffolds for Heparanase as Anticancer Therapeutics.

Heparanase (Hpse) is an endo-ß-D-glucuronidase capable of cleaving heparan sulfate side chains. Its upregulated expression is implicated in tumor growth, metastasis and angiogenesis, thus making it an attractive target in cancer therapeutics. Currently, a few small molecule inhibitors have been reported to inhibit Hpse, with promising oral administration and pharmacokinetic (PK) properties. In the present study, a ligand-based pharmacophore model was generated from a dataset of well-known active small molecule Hpse inhibitors which were observed to display favorable PK properties. The compounds from the InterBioScreen database of natural (69,034) and synthetic (195,469) molecules were first filtered for their drug-likeness and the pharmacophore model was used to screen the drug-like database. The compounds acquired from screening were subjected to molecular docking with Heparanase, where two molecules used in pharmacophore generation were used as reference. From the docking analysis, 33 compounds displayed higher docking scores than the reference and favorable interactions with the catalytic residues. Complex interactions were further evaluated by molecular dynamics simulations to assess their stability over a period of 50 ns. Furthermore, the binding free energies of the 33 compounds revealed 2 natural and 2 synthetic compounds, with better binding affinities than reference molecules, and were, therefore, deemed as hits. The hit compounds presented from this in silico investigation could act as potent Heparanase inhibitors and further serve as lead scaffolds to develop compounds targeting Heparanase upregulation in cancer.

Therapeutic potential of russelioside B as anti-arthritic agent in Freund's adjuvant-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma species are traditional edible herbs used in folkloric medicine as antidiabetic, antioxidant, antipyretic, antirheumatic, anti-inflammatory and anthelmintic agents. C. quadrangula was selected in this study to document the traditional use of the genus as anti-rheumatic treatment and the possible mechanisms of action. AIM OF THE STUDY: The higher mortality rates and shorter survival among the patients suffering from rheumatoid arthritis (RA) led to the increased interest on searching for new treatments for RA. Russelioside B (RB), a major pregnane glycoside found in C. quadrangula, was evaluated as a new anti-rheumatic agent. MATERIALS AND METHODS: The n-butanol fraction of C. quadrangula was chromatographed on a silica gel column to isolate RB. The adjuvant-induced arthritis (AIA) model was established in rats by intradermal injection of complete Freund's adjuvant (CFA) to evaluate its anti-arthritic effect. Ibuprofen was used as a reference drug. Forty rats were randomly divided into 5 groups (n = 8): normal (NOR); CFA model (CFA); ibuprofen, 5 mg/kg; RB, 25 mg/kg and RB, 50 mg/kg. The treatments were initiated from day 16 when AIA model was established and continued up to day 40. Serum diagnostic rheumatoid markers, inflammatory cytokines, oxidative stress biomarkers, cartilage and bone degeneration enzymes were assessed. RESULTS: RB at 50 mg/kg b. wt., showed significant decreases in the activities of hyaluronidase and ß-glucouronidase enzymes as well significant decreases in the levels of proinflammatory cytokines as nuclear factor-kappa-B (NF-κB), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) compared to the CFA group; 11.04 ± 0.61 pg/mg protein, 4.35 ± 0.25 pg/mg protein, 3.32 ± 0.13 pg/mg protein & 2.75 ± 0.14 pg/mg protein for RB, 50 mg/kg b. wt. group vs. 25.33 ± 2.13 pg/mg protein, 25.65 ± 2.1 pg/mg protein, 22.20 ± 1.34 pg/mg protein & 13.27 ± 1.40 pg/mg protein for the arthritic group, respectively. The total antioxidant capacity (TAC) was significantly restored to normal values in RB, 50 mg/kg treated rats (4.01 ± 0.09 nmol/mL vs. 3.71 ± 0.27 nmol/mL) and the levels of myeloperoxidase (MPO) reduced by 10-folds of the CFA arthritic group. Bone histomorphometry revealed that RB treatment significantly attenuated the CFA-induced bone loss in a dose-dependent manner. CONCLUSIONS: These findings suggested that the anti-arthritic effect of RB was mediated through the reduction of the rheumatoid markers, anti-inflammatory and antioxidant action, inhibition of cartilage and bone degenerative enzymes as well as attenuation of bone loss and osteoclastogenesis.

C-type natriuretic peptide stimulates osteoblastic proliferation and collagen-X expression but suppresses fibroblast growth factor-23 expression in vitro.

BACKGROUND: The effects of C-type natriuretic peptide (CNP) and fibroblast growth factor (FGF)-23 appear to oppose each other during the process of bone formation, whereas few studies exist on the interaction between CNP and FGF-23. The main objective of the present study is to probe whether CNP is directly responsible for the regulation of osteoblast or via antagonizing FGF-23. METHODS: Osteoblasts were cultured in the absence or presence of CNP (0, 10, and 100 pmol/L) for 24 h, 48 h and 72 h, respectively. RESULTS: The findings of the present study indicated that: (1) CNP significantly stimulated osteoblastic proliferation and collagen (Col)-X expression; (2) both osteoblastic (osteocalcin, procollagen type I carboxy-terminal propeptide, total alkaline phosphatase and bone-specific alkaline phosphatase) and osteolytic (tartrate-resistant acid phosphatase and cross-linked carboxyterminal telopeptide of type I collagen) bone turnover biomarkers were up-regulated by CNP in osteoblasts; (3) FGF-23 mRNA and protein were significantly down-regulated at 24 h by CNP in osteoblasts, but the expression of FGF receptor-1/Klotho had no significant change. CONCLUSIONS: CNP stimulates osteoblastic proliferation and Col-X expression via the down-regulation of FGF-23 possibly in vitro. However, the specific mechanisms of the interaction between CNP and FGF-23 in osteoblasts are still unclear according to our findings. A further study on osteoblasts cultured with CNP and FGF-23 inhibitor will be undertaken in our laboratory.

Targeted inhibition of gut bacterial ß-glucuronidase activity enhances anticancer drug efficacy.

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

[Potential application of fibroblast growth factor 23-klotho axis in chronic kidney disease]. / Potentielle application de l'axe fibroblast growth factor 23-Klotho dans la maladie rénale chronique.

Membrane Alpha Klotho (α-klotho) is expressed in the kidney and functions as a co-receptor of FGF receptors (FGFRs) to activate specific fibroblast growth factor 23 (FGF23) signal pathway. FGF23 is produced in bones and participates in mineral homeostasis. The extracellular domain of transmembrane αklotho can be cleaved by proteases and released into the circulation as soluble α-klotho. Klotho deficiency is a pathogenic factor for chronic kidney disease progression and cardiovascular diseases. The FGF23 excess may also contribute to cardiovascular diseases where its pathogenic effect acts via the FGFR4 and independently of α-klotho. The decline in serum α-klotho followed by a rise in serum FGF23 at an early stage of chronic kidney disease can serve as a robust predictor for risk of cardiovascular diseases and mortality in both CKD patients and the general population. The first randomized trials suggest the possibility to reduce FGF23 excess in chronic kidney disease by controlling the phosphate serum using phosphate binders and reducing PTH levels with calcimimetic drug. New strategies emerge, including the administration of α-klotho recombinant and the use of epidrugs in order to correct the klotho deficiency. The FGR4 inhibitors are promising to limit the development of left ventricular hypertrophy linked to FGF23 excess. Finally, a better understanding of the molecular mechanisms of FGF23/α-klotho axis will allow us to find new strategic approaches and improve the CKD patient's management and their outcomes.

FGF21 promotes functional recovery after hypoxic-ischemic brain injury in neonatal rats by activating the PI3K/Akt signaling pathway via FGFR1/ß-klotho.

Perinatal asphyxia often results in neonatal cerebral hypoxia-ischemia (HI), which is associated with high mortality and severe long-term neurological deficits in newborns. Currently, there are no effective drugs to mitigate the functional impairments post-HI. Previous studies have shown that fibroblast growth factor 21 (FGF21) has a potential neuroprotective effect against brain injury. However, the effect of FGF21 on neonatal HI brain injury is unclear. In the present study, both in vivo and in vitro models were used to assess whether recombinant human FGF21 (rhFGF21) could exert a neuroprotective effect after HI and explore the associated mechanism. The results showed that the rhFGF21 treatment remarkably reduced the infarct volume, ameliorated the body weight and improved the tissue structure after HI in neonatal rats. In addition, the rhFGF21 treatment lengthened the running endurance times in the rotarod test and decreased the mean escape latencies and increased the number of platform crossings in the Morris water maze test at 21 d post-HI insult. In contrast, the FGFR1 inhibitor PD173074 and PI3K inhibitor LY294002 partially reversed these therapeutic effects. In isolated primary cortical neurons, the rhFGF21 treatment protected primary neurons from oxygen-glucose deprivation (OGD) insult by inhibiting neuronal apoptosis and promoting neuronal survival. Both our in vivo and in vitro results reveal that rhFGF21 could inhibit neuronal apoptosis by activating the PI3K/Akt signaling pathway via FGF21/FGFR1/ß-klotho complex formation. Therefore, rhFGF21 may be a promising therapeutic agent for promoting functional recovery after HI-induced neonatal brain injury.

Daidzein upregulates anti-aging protein Klotho and NaPi 2a cotransporter in a rat model of the andropause.

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.

Bicarbonate Supplement Restores Urinary Klotho Excretion in Chronic Kidney Disease: A Pilot Study.

OBJECTIVE: We tested the hypothesis that correcting acidosis may improve urinary Klotho excretion and serum α-Klotho. DESIGN: This is a prospective, interventional, nonrandomized, open-label trial study. In this study setting, metabolic acidosis is commonly observed during chronic kidney disease (CKD). We reported a positive relationship between serum bicarbonate (Sbicar) and serum α-Klotho in these patients. SUBJECTS: The study involved 20 patients with a known kidney disease referred for renal checkup. Inclusion criteria were age ≥ 18 years, CKD stage 3-5 non dialysis, Sbicar < 22 mmol/L, and not receiving bicarbonate supplementation. INTERVENTION: Patients were then prescribed 1 g of oral sodium bicarbonate 3 times per day for 4 weeks. MAIN OUTCOME MEASURE: Patients were evaluated at two and 4 weeks by blood and urine measurements. RESULTS: Mean serum Klotho was 615 ± 287 pg/mL, and mean serum Sbicar was 19.3 ± 1.7 mmol/L at baseline. Sbicar increased from baseline at two (23.9 ± 2.9 mmol/L, P < .001) and 4 weeks (23.4 ± 1.9 mmol/L, P < .001). There was no change in serum Klotho at two (630 ± 333 mmol/L) and 4 weeks (632 ± 285 mmol/L). By contrast, urine Klotho/creatinine ratio, which was very low at baseline (34.6 ± 31.6 pg/mmoL), increased by 320% at two weeks (P < .005) and by 280% at 4 weeks (P < .01). CONCLUSIONS: Correcting acidosis by oral administration of sodium bicarbonate rapidly increases the urine excretion of soluble α-Klotho in CKD patients. However, a 4-week bicarbonate treatment was not able to increase serum α-Klotho. A longer study may confirm this pilot observation and increase serum Klotho, which has been shown to exert a protective cardiovascular effect during CKD.

Vitamin D Attenuates Endothelial Dysfunction in Uremic Rats and Maintains Human Endothelial Stability.

Background Dysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease ( CKD ). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD , not only by direct effects on the endothelium but also by an increment of α-Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia-induced endothelial damage and the extent to which this was dependent on increased α-Klotho concentrations. Methods and Results In a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial-gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α-Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real-time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro-permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial-cadherin-based cell-cell junctions and diminished F-actin stress fiber organization, preventing the formation of endothelial intracellular gaps. Conclusions Our results demonstrate that paricalcitol attenuates the CKD -induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell-cell interactions.

Physicochemical and nutraceutical properties of moringa (Moringa oleifera) leaves and their effects in an in vivo AOM/DSS-induced colorectal carcinogenesis model.

Moringa (Moringa oleifera) is a plant that has generated great interest in recent years because of its attributed medicinal properties. The aim of this study was to characterize the bioactive compounds of moringa leaves (MO) and evaluate their effect on a colorectal carcinogenesis model. Twenty-four male CD-1 mice were divided into 4 groups: Group 1 fed with basal diet (negative control/NC); Group 2 received AOM/DSS (positive control); Groups 3 and 4 were fed with basal diet supplemented with moringa leaves (2.5% w/w and 5% w/w, respectively) for 12weeks. Moringa leaves exhibited a high content of dietary fiber (~18.75%) and insoluble dietary fiber (2.29%). There were identified 9 phenolic compounds whereas the chlorogenic and ρ-coumaric acid showed the higher contents (44.23-63.34µg/g and 180.45-707.42µg/g, respectively). Moringa leaves decreased the activity of harmful fecal enzymes (ß-glucosidase, ß-glucuronidase, tryptophanase and urease up to 40%, 43%, 103% and 266%, respectively) as well tumors incidence in male CD1-mice (~50% with 5% w/v of moringa dose). These findings suggest that the bioactive compounds of moringa such as total dietary fiber and phenolic compounds may have chemopreventive capacity. This is the first study of the suppressive effect of moringa leaves in an in vivo model of AOM/DSS-induced colorectal carcinogenesis.

Thyroid Hormone-Induced Expression of the Hepatic Scaffold Proteins Sestrin2, ß-Klotho, and FRS2α in Relation to FGF21-AMPK Signaling.

Thyroid hormone (3,3',5-triiodothyronine, T3) accelerates energy metabolism in the liver through mechanisms involving upregulation of AMP-activated protein kinase (AMPK). This study aims to assess the influence of T3 on the expression of the scaffold proteins ß-Klotho, fibroblast growth factor receptor substrate 2α (FRS2α), and Sestrin2 in relation to FGF21-AMPK signaling. Male Sprague-Dawley rats were given 0.1 mg T3/kg or hormone vehicle (controls) and studies were done 24 h after treatment. These include measurements of the mRNA expression (qPCR) of hepatic ß-Klotho, FGF21, FGF21 receptor-1 (FGFR1), extracellular-signal-regulated kinase 1/2 (ERK1/2), FRS2α, ribosomal S6 kinase-1 (RSK1), liver kinase B1 (LKB1), AMPK, and Sestrin2. Also, protein levels of FGF21, FGFR1 (ELISA), and ERK1/2 (Western blot) were measured. T3 elicited a calorigenic response with higher hepatic mRNA expression of ß-Klotho, FRS2α, and FGF21, increased serum FGF21, without changes in liver FGFR1 mRNA and its plasma levels. In addition, T3 enhanced ERK1/2 phosphorylation and the mRNA expression of ERK1/2, RSK1, LKB1, AMPK, and Sestrin2. T3 administration enhances liver FGF21-AMPK signaling involving upregulation of the scaffold proteins ß-Klotho, FRS2α, and Sestrin2. ß-Klotho and FRS2 induction favours the operation of the FGF21-FGFR1-ß-Klotho complex as evidenced by the enhancement in ERK1/2 phosphorylation, whereas that of Sestrin2 recruits LKB1 to achieved AMPK activation, thus supporting a higher energy expenditure condition that may be desirable in some metabolic disorders.

Effects of Angiotensin II Receptor Blockade on Soluble Klotho and Oxidative Stress in Calcineurin Inhibitor Nephrotoxicity in Rats.

INTRODUCTION: Calcineurin inhibitor nephrotoxicity is major problem after organ transplantation. It is multifactorial, but oxidative stress may have an important role in this process. It has been shown that angiotensin II receptor blockers have renoprotective effects but their molecular mechanism is largely unknown. Antioxidative effect is an important role of the recently known anti-aging protein, klotho. This study aimed to evaluate effect of valsartan in alleviation of cyclosporine A nephrotoxicity via a probable increase in serum klotho levels or decreasing oxidative stress. MATERIALS AND METHODS: Thirty-two Sprague-Dawley rats were divided into 4 groups to receive 1 mL/kg/d of olive oil as control; 30 mg/kg/d of cyclosporine; 30 mg/kg/d of cyclosporine and 50 mg/kg/d of valsartan; and 50 mg/kg/d of valsartan. After the 6 weeks of administration period, serum levels of klotho and 8-hydroxydeoxyguanosine were measured using an enzyme-linked immunosorbent assay. Serum malondialdehyde level was measured spectrophotometrically. RESULTS: The mean serum level of klotho was significantly lower in the cyclosporine group compared with control and valsartan groups. Klotho level in the valsartan group was significantly higher than those in the other groups. The cyclosporine group was detected to have significantly higher serum 8-hydroxydeoxyguanosine and malondialdehyde levels compared with the other study groups. The levels of klotho were negatively correlated with 8-hydroxydeoxyguanosine and malondialdehyde levels. CONCLUSIONS: Administration of valsartan may lead to attenuation of the nephrotoxic side effect of cyclosporine via enhancing klotho and decreasing oxidative stress levels.

Klotho sensitive regulation of dendritic cell functions by vitamin E.

BACKGROUND: Dendritic cells (DCs) are the most potent professional antigen-presenting cells for naive T cells to link innate and acquired immunity. Klotho, an anti-aging protein, participates in the regulation of Ca2+ dependent migration in DCs. Vitamin E (VitE) is an essential antioxidant to protect cells from damage and elicits its inhibitory effects on NF-κB-mediated inflammatory response. However, the roles of VitE on mouse DC functions and the contribution of klotho to those effects both are unknown. The present study explored the effects of VitE on klotho expression, maturation, ROS production and migration in DCs. METHODS: The mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were stimulated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM). RT-PCR and immunoprecipitation methods were employed to determine klotho expression, ELISA to determine cytokine release, flow cytometry to analyze number of CD86+CD11c+ cells, the intracellular expression of cytokines and reactive oxygen species (ROS) production and a transwell migration assay to trace migration. RESULTS: Klotho transcript level and this hormone secretion in DC supernatant were enhanced by VitE treatment and further increased in the presence of NF-κB inhibitor Bay 11-7082 (10 µM). Moreover, VitE treatment inhibited IL-12p70 protein expression of, ROS accumulation in and CCL21-dependent migration of LPS-triggered mature DCs, these effects were reversed following klotho silencing. CONCLUSION: The up-regulation of klotho by VitE could contribute to the inhibitory effects of VitE on NF-κB-mediated DC functional maturation. The events might contribute to immunotherapeutic effect of VitE on the pathophysiology of klotho-related disease.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

Long-term sevelamer treatment lowers serum fibroblast growth factor 23 accompanied with increasing serum Klotho levels in chronic haemodialysis patients.

AIMS: Fibroblast growth factor 23 (FGF23) and Klotho are associated with vascular calcification and cardiovascular disease in dialysis patients. Sevelamer has been shown to reduce progression of vascular calcification. This study aimed to determine the long-term effect of sevelamer treatment on serum FGF23 and Klotho levels in chronic haemodialysis (HD) patients. METHODS: In the post-hoc analysis, we measured serum FGF23, Klotho and other biochemical factors (Ca, P, i-PTH, hsCRP, LDL-C) in 50 haemodialysis patients, who completed a 48-week, open-Label, controlled randomized parallel-group study. Twenty-three patients received sevelamer and 27 patients received calcium carbonate. RESULTS: After 48-week sevelamer treatment, there were significant changes with lower LDL-C (from 2.82 ± 0.78 to 1.65 ± 0.53 mmol/L, P = 0.000), lower FGF23 (from 2465.97 (2568.88) to 795.61 (1098.39), P = 0.000) and higher s-Klotho levels (from 189.35 (161.88) to 252.94 (517.80) pg/mL, P = 0.000). In calcium carbonate group, there were no significant changes of LDL-C and FGF23, but with a borderline significant increase of s-Klotho level (from 142.34 (265.24) to 188.57 (252.38) pg/mL, P = 0.054). Multivariate analysis showed that FGF23 decrement was associated with sevelamer treatment (ß = -0.277, P = 0.005), change of serum phosphate (ß = 0.609, P = 0.000) and calcium levels (ß = 0.635, P = 0.000). The increase of serum Klotho was associated with the decrease of serum phosphate (ß = 0.490, P = 0.019). CONCLUSION: Maintenance HD patients had lower serum FGF23 levels, accompanied with significantly increased serum Klotho levels, after 48-week sevelamer treatment. The FGF23 decrement was associated with sevelamer use, the change of serum phosphate and calcium levels. The serum Klotho increment was proportional to the phosphate-lowering power of the binders.

Changes chemopreventive markers in colorectal cancer development after inulin supplementation.

BACKGROUND: Natural dietary compounds such as prebiotics modulate microbial composition and could prevent the colon cancer development as potential chemopreventive agent. OBJECTIVES: Effect of prebiotic-inulin on biochemical, microbial and chemopreventive markers were examined in Sprague-Dawley rats during experimental chemically dimethylhydrazine induced colon cancer development. METHODS: Rats were divided to 3 groups: control group (CG), group with dimethylhydrazine (DMH) and group with DMH and prebiotic (DMH+PRE). The efficacy of the prebiotic inulin (PRE) on the activities of ß-glucuronidase, short chain fatty acids (SCFAs), counts of coliforms and lactobacilli, immunoreactivity of cyclooxygenase-2 (COX-2), transcription nuclear factor kappa beta (NFκB) and inducible nitric oxide synthase (iNOS) in colon tissue were examined. RESULTS: Inulin significantly decreased coliforms counts (p<0.01), increased lactobacilli counts (p<0.001), and decreased activity of ß-glucuronidase (p<0.01) in fresh caecal digesta. Butyric and propionic acids concentrations were increased after inulin supplementation in comparison to DMH group. Application of inulin decreased immunoreactivity and numbers of COX-2, NFκB and iNOS positive cells in colon tissue in comparison to DMH group. CONCLUSION: Inulin suppressed expression observed markers, which play an important role in carcinogenesis and in the inflammatory process, which predisposes to the use of inulin in the prevention or treatment of inflammatory bowel disease (Tab. 1, Fig. 2, Ref. 17).

The potential of heparanase as a therapeutic target in cancer.

Heparanase has generated substantial interest as therapeutic target for antitumor therapy, because its activity is implicated in malignant behavior of cancer cells and in tumor progression. Increased heparanase expression was found in numerous tumor types and correlates with poor prognosis. Heparanase, an endoglucuronidase responsible for heparan sulfate cleavage, regulates the structure and function of heparan sulfate proteoglycans, leading to disassembly of the extracellular matrix. The action of heparanase is involved in multiple regulatory events related, among other effects, to augmented bioavailability of growth factors and cytokines. Inhibitors of heparanase suppress tumor growth, angiogenesis and metastasis by modulating growth factor-mediated signaling, ECM barrier function and cell interactions in the tumor microenvironment. Therefore, targeting heparanase has potential implications for anti-tumor, anti-angiogenic and anti-inflammatory therapies. Current approaches for heparanase inhibition include development of chemically modified heparins, small molecule inhibitors and neutralizing antibodies. The available evidence supports the emerging utility of heparanase inhibition as a promising antitumor strategy, specifically in rational combination with other agents. The recent studies with compounds designed to block heparanase (e.g., modified heparins) provide a rational basis for their therapeutic application and optimization.

ß-Glucuronidase-responsive prodrugs for selective cancer chemotherapy: an update.

The design of novel antitumor agents allowing the destruction of malignant cells while sparing healthy tissues is one of the major challenges in medicinal chemistry. In this context, the use of non-toxic prodrugs programmed to be selectively activated by beta-glucuronidase present at high concentration in the microenvironment of most solid tumors has attracted considerable attention. This review summarizes the major progresses that have been realized in this field over the past ten years. This includes the new prodrugs that have been designed to target a wide variety of anticancer drugs, the prodrugs employed in the course of a combined therapy, the dendritic glucuronide prodrugs and the concept of ß-glucuronidase-responsive albumin binding prodrugs.

[Assessment of in vitro impact of low molecular weight heparin on expression of heparanase and heparin binding growth factors in the endometrium of women with impaired reproduction]. / Ocena wplywu in vitro niskoczasteczkówej heparyny na ekspresje heparanazy i czynników wzrostu wiazacych heparyne w endometriumn kobiet z zaburzonym rozrodem.

UNLABELLED: Heparin has a beneficial effect in the treatment of recurrent miscarriages and positively affects implantation rates in the IVF procedure in women with reproductive disorders not associated with thrombophilia. Several studies have indicated that heparin, by blocking the enzymatic activity of heparanase, may affect the structure and function of the extracellular matrix (ECM) and related growth factors. Disturbances in the remodeling (ECM) are believed to be the potential cause of implantation disorders and recurrent miscarriages. OBJECTIVES: The aim of the study was the evaluation, on an in vitro model, of the effect of low molecular weight heparin (LMWH) on the expression of heparanase (HPSE) and, important for successful implantation and invasion of trophoblast, heparan sulfate (HS)--binding growth factors, i.e., heparin-binding epidermal growth factor-like (HB-EGF), vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF2) in the endometrium, during the implantation window in women with recurrent miscarriage. METHOD: Biopsy samples, obtained from 10 patients with two or more unexplained miscarriages, were used to construct a co-culture of glandular epithelial cells and stroma. Endometrium in vitro model was supplemented with steroid hormones and enoxaparin 5 ug/ml. Using the qPCR, we assessed, relative levels of the HPSE, HB-EGF, VEGF-A and FGF2 transcripts in glandular epithelium and stroma in cell culture. Using ELISA, we measured con- centrations of the mentioned above factors in culture medium. RESULTS: A statistically significant increase in the relative level of HPSE, HB-EGF VEGF-A, FGF2 transcripts in the cells of the glandular epithelium and stroma (p<0.001), as well as their increased concentration in the medium of cultures treated with steroid hormones (p<0.001) were observed. However we found no effect of LMWH supple- mentation on the level of the investigated factors. CONCLUSIONS: Our results show that the importance of the HPSE hydrolytic activity in the endometrium, during the implantation window, may have a secondary function, and/or that beneficial effects of LMWH in women with impaired reproduction have no significant, direct connection with the, catalyzed by HPSE, reconstruction of the ECM and with release of heparin-binding growth factors.