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Chronic kidney disease (CKD) is associated with elevated plasma fibrinogen concentration. However, the underlying molecular mechanism for elevated plasma fibrinogen concentration in CKD patients has not yet been clarified. We recently found that HNF1α was significantly upregulated in the liver of chronic renal failure (CRF) rats, an experimental model of CKD in patients. Given that the promoter region of the fibrinogen gene possesses potential binding sites for HNF1α, we hypothesized that the upregulation of HNF1α can increase fibrinogen gene expression and consequently plasma fibrinogen concentration in the experimental model of CKD. Here, we found the coordinated upregulation of Aα-chain fibrinogen and Hnfα gene expression in the liver and elevated plasma fibrinogen concentrations in CRF rats, compared with pair-fed and control animals. Liver Aα-chain fibrinogen and HNF1α mRNAs levels correlated positively with (a) liver and plasma fibrinogen levels and (b) liver HNF1α protein levels. The positive correlation between (a) liver Aα-chain fibrinogen mRNA level, (b) liver Aα-chain fibrinogen level, and (c) serum markers of renal function suggest that fibrinogen gene transcription is closely related to the progression of kidney disease. Knockdown of Hnfα in the HepG2 cell line by small interfering RNA (siRNA) led to a decrease in fibrinogen mRNA levels. Clofibrate, an anti-lipidemic drug that reduces plasma fibrinogen concentration in humans, decreased both HNF1α and Aα-chain fibrinogen mRNAs levels in (a) the liver of CRF rats and (b) HepG2 cells. The obtained results suggest that (a) an elevated level of liver HNF1α can play an important role in the upregulation of fibrinogen gene expression in the liver of CRF rats, leading to an elevated concentration of plasma fibrinogen, a protein related to the risk of cardiovascular disease in CKD patients, and (b) fibrates can decrease plasma fibrinogen concentration through inhibition of HNF1α gene expression.

Vascular inflammation is an important factor in the pathophysiology of cardiovascular diseases, such as atherosclerosis. Changes in the extracellular nucleotide and in particular adenosine catabolism may alter a chronic inflammation and endothelial activation. This study aimed to evaluate the relation between vascular ecto-adenosine deaminase (eADA) activity and endothelial activation in humans and to analyze the effects of LPS-mediated inflammation on this activity as well as mechanisms of its increase. Moreover, we investigated a therapeutic potential of ADA inhibition by deoxycofromycin (dCF) for endothelial activation. We demonstrated a positive correlation of vascular eADA activity and ADA1 mRNA expression with endothelial activation parameters in humans with atherosclerosis. The activation of vascular eADA was also observed under LPS stimulation in vivo along with endothelial activation, an increase in markers of inflammation and alterations in the lipid profile of a rat model. Ex vivo and in vitro studies on human specimen demonstrated that at an early stage of vascular pathology, eADA activity originated from activated endothelial cells, while at later stages also from an inflammatory infiltrate. We proposed that LPS-stimulated increase in endothelial adenosine deaminase activity could be a result of IL-6/JAK/STAT pathway activation, since the lack of IL-6 in mice was associated with lower vascular and plasma eADA activities. Furthermore, the inhibitors of JAK/STAT pathway decreased LPS-stimulated adenosine deaminase activity in endothelial cells. We demonstrated that cell surface eADA activity could be additionally regulated by transcytosis pathways, as exocytosis inhibitors including lipid raft inhibitor, methyl-β-cyclodextrin decreased LPS-induced eADA activity. This suggests that cholesterol-dependent protein externalization mediated by lipid rafts could be an important factor in the eADA increase. Moreover, endocytosis inhibitors and exocytosis activators increased this activity on the cell surface. Furthermore, the inhibition of adenosine deaminase in endothelial cells in vitro attenuated LPS-mediated IL-6 release and soluble ICAM-1 and VCAM-1 concentration in the incubation medium through the restoration of the extracellular adenosine pool and adenosine receptor-dependent pathways. This study demonstrated that the vascular endothelial eADA activity remains under control of inflammatory mediators acting through JAK/STAT pathway that could be further modified by dyslipidemic-dependent exocytosis and transcytosis pathways. Inhibition of eADA blocked endothelial activation suggesting a crucial role of this enzyme in the control of vascular inflammation. This supports the concept of eADA targeted vascular protection therapy.

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis. Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5'-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC. The activity of ecto5'-nucleotidase differ in hearts of LDLR-/- and ApoE-/- mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models. This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.

Inflammation related to chronic kidney disease (CKD) is an important clinical problem. We recently determined that hepatocyte nuclear factor 1α (HNF1α) was upregulated in the livers of chronic renal failure (CRF) rats—experimental model of CKD. Considering that the promoter region of gene encoding C-reactive protein (CRP) contains binding sites for HNF1α and that the loss-of-function mutation in the Hnfs1α leads to significant reduction in circulating CRP levels, we hypothesized that HNF1α can activate the Crp in CRF rats. Here, we found coordinated upregulation of genes encoding CRP, interleukin-6 (IL-6), HNF1α, and HNF4α in the livers and white adipose tissue (WAT) of CRF rats, as compared to the pair-fed and control animals. This was accompanied by elevated serum levels of CRP and IL-6. CRP and HNFs’ mRNA levels correlated positively with CRP and HNFs’ protein levels in the liver and WAT. Similar upregulation of the Crp, Il-6, and Hnfs in the liver and WAT and increased serum CRP and IL-6 concentrations were found in lipopolysaccharide (LPS)-induced systemic inflammation in rats. Moreover, silencing HNF1α in HepG2 cells by small interfering RNA led to decrease in CRP mRNA levels. Our results suggests that (a) HNFs act in concert with IL-6 in the upregulation of CRP production by the liver and WAT, leading to an increase in circulating CRP concentration in CRF rats and (b) CRF-related inflammation plays an important role in the upregulation of genes that encode HNFs and CRP in the liver and WAT of CRF rats. Electronic supplementary material The online version of this article (10.1007/s11010-018-3268-1) contains supplementary material, which is available to authorized users.

Little is known about the effects of coffee that are not related to the presence of caffeine. The aim of the study was to analyse changes in kidney function and nucleotide metabolism related to high intake of decaffeinated coffee. Mice consumed decaffeinated coffee extract for two weeks. Activities of AMP deaminase, ecto5′-nucleotidase, adenosine deaminase, purine nucleoside phosphorylase were measured in kidney cortex and medulla by analysis of conversion of substrates into products using HPLC. Concentration of nucleotides in kidney cortex, kidney medulla and serum were estimated by HPLC. Activity of ecto5′-nucleotidase increased from 0.032 ± 0.006 to 0.049 ± 0.014 nmol/mg tissue/min in kidney cortex of mice administered high-dose decaffeinated coffee (HDC) together with increase in cortex adenosine concentration and decrease in plasma creatinine concentration. HDC leads to increased activity of ecto5′-nucleotidase in kidney cortex that translates to increase in concentration of adenosine. Surprisingly this caused improved kidney excretion function.

BACKGROUND/AIM Pancreatic cancer is a disease with very poor prognosis, and none of currently available pharmacotherapies have proven to be efficient in this indication. The aim of this study was to analyze the expression of fatty acid synthase (FASN) gene as a potential therapeutic target in proliferating human pancreatic cancer cells (PANC-1), and verify if orlistat, originally developed as an anti-obesity drug, inhibits PANC-1 proliferation. MATERIALS AND METHODS The effects of orlistat on gene expression, lipogenesis, proliferation and apoptosis was studied in PANC-1 cell culture. RESULTS Expression of FASN increased during proliferation of PANC-1. Inhibition of FASN by orlistat resulted in a significant reduction of PANC-1 proliferation and enhanced apoptosis of these cells. CONCLUSION This study showed, to our knowledge for the first time, that orlistat exhibits significant antitumor activity against PANC-1 cells. This implies that orlistat analogs with good oral bioavailability may find application in pharmacotherapy of pancreatic cancer.

Introduction Human placenta mitochondria produces huge amounts of progesterone necessary for maintaining the pregnancy. Lipid peroxidation in human placental mitochondria inhibits progesterone synthesis and that inhibition can be reversed by superoxide dismutase and other antioxidants. Paraquat (PQ) a highly toxic herbicide generates superoxide radical inside cells and induces lipid peroxidation. Hence, it is supposed to stimulate lipid peroxidation in human placental mitochondria and in consequence to inhibit a placental mitochondrial steroidogenesis. Methods Placentas were obtained from normal pregnancies. All experiments were done using isolated human placental mitochondria. Mitochondrial lipid peroxidation was determined as tiobarbituric acid reactive substances (TBARS). A conversion of cholesterol to pregnenolone or pregnenolone to progesterone was measured using radiolabeled steroids and thin layer chromatography. Results PQ enhanced the iron-dependent lipid peroxidation as also PQ heightened the inhibitory action of this process on progesterone synthesis in isolated human placental mitochondria. Paradoxically, a superoxide dismutase (SOD) reversed the inhibition of progesterone synthesis only minimally although it strongly inhibited PQ stimulated iron-dependent lipid peroxidation. When iron was absent, PQ stimulated only negligible lipid peroxidation but strongly inhibited progesterone synthesis. SOD had no effect on inhibition of progesterone synthesis by PQ. PQ strongly inhibited of the conversion of cholesterol to pregnenolone but had not got any influence on the enzymatic activity of mitochondrial 3β-hydroxysteroid dehydrogenase. PQ strongly decreased the efficiency of NADPH-dependent cytochrome P450 reduction as well as it promoted the rapid oxidation of the pre-reduced mitochondrial cytochrome P450. However PQ has not inhibited combined activity of adrenodoxin reductase and adrenodoxin. Discussion We conclude that the most important reason of the inhibition of progesterone synthesis by PQ is the escape of electrons from cytochrome P450scc to that compound what leads to cytochrome oxidation and, in consequence the inhibition of the reaction catalyzed by it. The action of PQ described here should be considered as potentially harmful for pregnancy and fetal development.

Neuroblastoma is one of the most common childhood malignancies and the primary cause of death from pediatric cancer. Derivatives of 17β-estradiol, 2-methoxyestradiol, as well as selective estrogen receptor modulators, such as fulvestrant, are novel potentially active anticancer agents. In particular, 2-methoxyestradiol is effective in treatment of numerous malignancies, including breast and prostate cancer, Ewing sarcoma, and osteosarcoma. Herein, we treated neuroblastoma SH-SY5Y cells with physiologically and pharmacologically relevant concentrations of 2-methoxyestradiol. We used flow cytometry in order to determine cell viability, cell death, level of nitric oxide and mitochondrial membrane potential. We demonstrated that at pharmacologically relevant concentrations, 2-methoxyestradiol results in induction of apoptosis of neuroblastoma SH-SY5Y cells via nitric oxide generation and reduction of mitochondrial membrane potential. Based on the obtained data, we propose that 2-methoxyestradiol may be a natural modulator of cancer cell death and survival through nitro-oxidative stress-dependent mechanisms. Moreover, the results confirm the efficiency of 2-methoxyestradiol in treatment of neuroblastoma.

During pregnancy the isoform composition of human placental AMP-deaminase changes. This may reflect the adaptation of enzyme to changing metabolic requirements of the growing fetus. In this paper kinetic and regulatory properties of AMP-deaminase purified from human preterm (∼25 week of gestation) placenta were described and compared with these of the enzyme purified from term placenta. AMP-deaminase from preterm placenta was less sensitive to pH changes and in contrast to the enzyme from the term organ, at low range of substrate concentrations was not inhibited but activated by physiological concentrations of orthophosphate. This may significantly improve the catalytic efficiency of enzyme at early phase of the pregnancy.

In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe2+ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) - a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and alpha-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM.

The effects of ascorbate and a-tocopherol as antioxidants and as co-operative factors against NADPH-dependent lipid peroxidation in human placental mitochondria have been studied. The addition of ascorbate at low concentration (up to 50 μM) to the NADPH-generating system resulted in increasing lipid peroxidation and Fe3+ to Fe2+ reduction. High concentration of ascorbate (150 μM), which produced maximal rate of ascorbate-dependent lipid peroxidation, was found to inhibit almost completely NADPH-dependent lipid peroxidation by maintaining too much iron in its reduced form. Either stimulatory or inhibitory effect of ascorbate on NADPH-dependent lipid peroxidation depends on the appropriate Fe3+/Fe2+ ratio. α-Tocopherol caused a decrease of NADPH-dependent lipid peroxidation, inhibiting completely this process at 150 μM concentration. The inhibitory effect of α-tocopherol increased rapidly with the increasing ascorbate concentration, almost complete inhibition of NADPH-dependent lipid peroxidation being obtained at 25 μM α-tocopherol and 50 μM ascorbate. This strong inhibitory combined effect of α-tocopherol and ascorbate was independent of the Fe3+/Fe2+ ratio, as a-tocopherol is not able to reduce Fe3+ to Fe2+ under the conditions employed. These findings suggest that antioxidant effects of ascorbate in placental mitochondria are mediated by recycling of a-tocopherol rather than by strong reduction of Fe3+ to Fe2+. On the basis of the results obtained, we assume that adequate concentrations of a-tocopherol and ascorbate in placental tissue may prevent the release of lipid peroxide from placental mitochondria and therefore could be protective against the development of preeclampsia.

A C34T mutation in the AMPD1 gene is proposed to cause local or systemic augmentations in blood adenosine level and improvement of prognoses in heart diseases like congestive heart failure or heart ischemic disease. This study examines some physico-chemical properties of AMP-deaminase isolated from cardiac muscle of a 10-year-old boy heterozygote for this mutation.

Human placental mitochondria might be a significant source of NADPH- and iron-dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH- and iron-dependent lipid peroxidation of human placental mitochondria in a concentration-dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal-chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and alpha-tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 mum ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 mum ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro-oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and alpha-tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.

Human placental mitochondria might be a significant source of NADPH- and iron-dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH- and iron-dependent lipid peroxidation of human placental mitochondria in a concentration-dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal-chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and alpha-tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 mum ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 mum ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro-oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and alpha-tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.

During pregnancy the activity of AMP-deaminase in developing human placenta gradually decreases, being in homogenates of mature, term placenta (approximately 40 week of gestation) one fourth to one third of that in homogenates of immature (approximately 25 week of gestation) organ. The gradual decrease of activity correlates inversely with the increasing presence of the form of enzyme predominating in homogenates of the mature placenta. The discrepancy observed indicate that isozymic pattern of AMP-deaminase in developing human placenta changes.

Estrogens control a large range pivotal life functions as reproductive development and fertility, bone growth and sexual behavior. Aromatase is a key enzyme of estrogen biosynthesis. The property, structure and reaction mechanism of aromatase as well as detailed structure of human aromatase cytochrome P450 gene (CYP19) was discussed in this article. It was pointed that unique human CYP19 gene expression results from presence of many tissue specific promoters and alternative splicing. The molecular mechanism of control aromatase cytochrome P450 gene expression in various species ovaries, testes and human adipose tissue and placenta was discussed in details. Because of a very important role of estrogen in breast cancer a molecular base of aberrant expression CYP19 gene in breast tumor and adipose tissue proximal to breast tumor and potential possibility of pharmacological silencing of this gene expression was discussed in the article.

Mitochondrial dysfunction plays a crucial role in cell types that exhibit necrosis-like death after activation of their death program. Tumour necrosis factor (TNF) induces abnormal, perinuclear clustering of mitochondria from an evenly spread distribution throughout the cytoplasm. The mitochondria withdraw from the cell periphery and aggregate in a unipolar perinuclear cluster. TNF-induced mitochondrial clustering is caused by impaired kinesin-mediated transportation of mitochondria. In this report, we describe a novel activity of menadione (MEN), namely the induction of an altered spatial distribution of mitochondria in the choriocarcinoma JAR cells. Strikingly, 2 hours of cell exposition to menadione did not disrupt the integrity of the plasma membrane, while the intracellular ATP level significantly decreased. Control (untreated) cells displayed a typically scattered distribution of filamentary mitochondria inside the cell. After 2 hours of MEN treatment the spatial distribution of the mitochondria was markedly altered to an asymmetric perinuclear clustered distribution. Menadione-stressed cells displayed a highly asymmetrical perinuclear clustered distribution of the mitochondria. The exposure of cells to MEN also results in a change in shape of the mitochondria into a population of enlarged granular structures. The results of our study demonstrate that in JAR cells menadione causes mitochondria to translocate from the cell periphery into the perinuclear region several hours before disruption of cell membrane integrity and cell death.