Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-ß and VEGF Production and Metastatic Potential of Cells.

Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G1 stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor ß (TGF-ß) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.

Alpha ketoglutarate exerts a pro-osteogenic effect in osteoblast cell lines through activation of JNK and mTOR/S6K1/S6 signaling pathways.

Although numerous in vivo studies have suggested that alpha-ketoglutarate (AKG), i.e. the key intermediate in the Krebs cycle, may have an anabolic effect on bone tissue, the direct influence of AKG on osteoblasts and the underlying mechanism of its action have not been investigated so far. The aim of this study was to assess the impact of AKG (disodium salt dihydrate) on osteogenesis in vitro and identification of some signaling mechanisms involved in this activity. The human and mouse normal osteoblast cell lines hFOB 1.19 and MC3T3-E1 were used in this study. The results showed that AKG did not increase the proliferation of osteoblasts; however, it upregulated the expression of transcription factors RUNX2 and Osterix, the mRNA and protein levels of osteoblast differentiation markers (alkaline phosphatase, type I collagen, bone sialoprotein II, osteopontin, osteocalcin), and the mineralization levels in the hFOB 1.19 and MC3T3-E1 cell cultures. Moreover, AKG increased JNK, mTOR, S6K1, and S6 phosphorylation and decreased ERK1/2 phosphorylation in both osteoblast cell lines. The JNK inhibitor and rapamycin, but not the ERK inhibitor, abolished the AKG-promoted osteoblast differentiation. Using immunofluorescence staining, qRT-PCR, and Western blot analysis, we detected the presence of an AKG receptor GPR99 activated by alpha ketoglutaric acid in the tested osteoblast cell lines. However, AKG salt did not activate GPR99. Our findings suggest that AKG salt activates the JNK and mTOR/S6K1/S6 signaling pathways to promote differentiation of osteoblasts, independently of GPR99 activation. We can conclude that AKG salts might be promising candidates for bone anabolic drugs used for prevention or/and treatment of osteoporosis.

Neutral endopeptidase (NEP) inhibitors - thiorphan, sialorphin, and its derivatives exert anti-proliferative activity towards colorectal cancer cells in vitro.

Neutral endopeptidase (NEP) is an enzyme implicated in development of different tumors, e.g. colorectal cancer (CRC). In this study, the anti-cancer effects of NEP inhibitors, thiorphan (synthetic compound) and sialorphin (naturally occurring pentapeptide) on CRC cells were investigated. Moreover, we synthesized some derivatives of sialorphin (alanine scan analogues: AHNPR, QANPR, QHAPR, QHNAR; N-acetylated sialorphin; C-amidated sialorphin, and C-amidated alanine scan analogues) to examine the biological activity of these inhibitors on CRC cells. The cytotoxic activity of the NEP inhibitors against CRC cell lines (SW620 and LS180) and normal human fibroblasts (HSF) was evaluated. Additionally, the influence of NEP inhibitors on proliferation, cell cycle progression, induction of apoptosis, and the level of phosphorylation of MAP kinases and mTORC1 signaling pathway proteins in CRC cells were examined. The NEP inhibitors were non-cytotoxic to HSF cells; however, most of them slightly decreased the viability and inhibited proliferation of CRC cells. The N-acetylation or C-amidation of sialorphin or its alanine scan analogues resulted in decreased or abolished anti-proliferative activity of the NEP inhibitors towards the CRC cells. Additionally, thiorphan and sialorphin enhanced the anti-proliferative activity of other CRC-cell growth inhibitors (atrial natriuretic peptide-ANP and melphalan-MEL). The mechanisms involved in the anti-proliferative effects of the tested inhibitors were mediated via NEP and associated with induction of cell cycle arrest in the G0/G1 phase, increased activity of ERK1/2, and a reduced level of phosphorylation of mTOR (Ser2448), 4E-BP1, and p70S6K. However, the NEP inhibitors did not induce apoptosis in the CRC cells. These results have indicated that thiorphan and sialorphin or its derivatives AHNPR, QANPR, QHAPR, and QHNAR have the potential to be used as agents in treatment of patients with CRC.

Metformin inhibits both oleic acid-induced and CB1R receptor agonist-induced lipid accumulation in Hep3B cells: The preliminary report.

Fatty liver is characterized by excessive accumulation of triglycerides within hepatocytes. Recent findings indicate that natural history of nonalcoholic fatty liver is regulated, in part, by endogenous cannabinoids. Metformin is an oral hypoglycemic medication which inhibits gluconeogenesis and glycogenolysis in hepatocytes and limits lipid storage in the liver through the inhibition of free fatty acid formation via induction of activated protein kinase activity (AMPK). Both endocannabinoids and metformin may modulate hepatosteatosis; therefore, it was interesting to examine whether metformin may affect lipid accumulation in hepatocytes by acting on cannabinoid receptors, CB1 and CB2, in in vitro study. Hep3B cells were incubated with or without metformin (Met), phosphatidylcholine (PC), and oleic acid (OA). Cells without any of the examined substances served as negative control. Cells treated only with OA served as positive control. The quantity of intracellular lipids was assessed using Oil-Red-O staining. Selective CB1R agonist, arachidonyl-2-chloromethylamide (ACEA), and CB2R agonist, AM1241 (2-iodo-5-nitrophenyl)-[1-(methylpiperidin-2-ylmethyl)-1 H-indol-3-yl]methanone, were also used to treat Hep3B cells. In some experiments, antagonist for CB1R, AM6545, or SR144528 as selective antagonist of CB2R were used. In the study, Met decreased lipid accumulation in cells treated with OA and inhibited CB1R agonist-induced lipid accumulation in hepatocytes. The CB2R agonist-induced hepatic lipid accumulation was not inhibited by metformin. The results indicate that metformin may interact with endocannabinoid system in the liver by inhibiting CB1R agonist-stimulated fat accumulation in hepatocytes.

Colon cancer-derived conditioned medium induces differentiation of THP-1 monocytes into a mixed population of M1/M2 cells.

Macrophages play an important role in the immune response and in the maintenance of tissue homeostasis. It is well known that many tumors recruit monocytes from circulation and influence their differentiation, mainly into suppressive M2-like subsets. Since there are contradictory data concerning the importance of macrophages for colon cancer progression, we used in our experiments four colon cancer cell lines representing different stages of tumor development (HT29, LS180, SW948, SW620). An acute monocytic leukemia cell line THP-1 was used as a human model of monocytes. Our work revealed that conditioned medium from the tumor cell lines induced activation and differentiation of THP-1 cells. The changes involved increased expression of CD68, a macrophage differentiation marker. Moreover, we also observed increased expression of CD206 and CD163, which are widely considered as markers of tumor-associated macrophages. The tumor-derived conditioned medium decreased the proliferation of THP-1 cells and blocked their cell cycle at the G1 stage. The tumor-conditioned medium also upregulated the production of several cytokines and chemokines characteristic of both M1 and M2 subsets and induced the expression of important pro-angiogenic factors, vascular endothelial growth factor, and matrix metalloproteinase-9 in THP-1 cells. Moreover, the tumor-conditioned medium induced the expression of galectin-3, which is implicated in malignant transformation, and indoleamine 2,3-dioxygenase, that is, a key enzyme of the kynurenine pathway. Our data suggest that tumor cells can actively influence the phenotype of monocytes and switch their differentiation into a population of non-adherent mixed M1 and M2 cells. These preliminary studies suggest that colon cancer cells produce soluble factors that influence monocyte differentiation, most probably into suppressive subsets. These data provide a better understanding of the influence of colon cancer on polarization of monocytes.

Biologically and chemically important hydrazino-containing imidazolines as antioxidant agents.

Biologically and chemically useful hydrazinoimidazolines were evaluated as antioxidant and antihaemolytic agents. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH•), galvinoxyl radical (GOR), nitric oxide (NO) and hydrogen peroxide (H2O2) scavenging assays, ferric ions reducing power assay, and ex vivo model of rat erythrocytes exposed to 2,2'-azobis(2-methylpropionamidine)dihydrochloride (AAPH) or H2O2 were used. The most potent DPPH• scavengers proved to be hydrazinoimidazolines 3, 2, and 4, revealing excellent antiradical effects - superior or comparable to that of all antioxidant standards used. Moreover, these molecules showed strong NO neutralising potencies - better to that of ascorbic acid (AA) (3), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (3 and 2), butylated hydroxytoluene (BHT) (3 and 2), and butylated hydroxyanisole (BHA) (3, 2, and 4). Compound 4 was also effective in GOR scavenging. The excellent scavenger of GOR, NO, and H2O2 proved to be structure 5, with the potency superior or comparable to the majority of antioxidant standards used. In turn, compound 9 was effective in H2O2 and GOR neutralisation. All hydrazinoimidazolines revealed the reducing power that is higher than BHT. Moreover, the protective effects of most test compounds on oxidatively stressed erythrocytes were observed. Some structure-activity relationships were disclosed. A significance of the primary hydrazino group on antioxidant effects was confirmed. The most likely DPPH• and GOR scavenging mechanisms for test compounds were propound. Among all the investigated molecules, hydrazinoimidazolines 5, 3, 2, 4, and 9, due to their excellent or good antiradical activities, can represent promising antioxidant candidates with prospective utility for prevention of diseases related to reactive oxygen/nitrogen species.

Tumor-Associated Macrophages as Target for Antitumor Therapy.

It is well known that the microenvironment of solid tumors is rich in inflammatory cells that influence tumor growth and development. Macrophages, called tumor-associated macrophages (TAMs), are the most abundant immune cell population present in tumor tissue. Several studies have demonstrated that the density of TAMs is associated with a poor prognosis and positively correlates with tumor growth. Several studies have proved that TAMs may activate and protect tumor stem cells, stimulate their proliferation as well as promote angiogenesis and metastasis. Furthermore, TAMs-derived cytokines and other proteins, such as CCL-17, CCL-22, TGF-ß, IL-10, arginase 1, and galectin-3, make a significant contribution to immunosuppression. Since TAMs influence various aspects of cancer progression, there are many attempts to use them as a target for immunotherapy. The numerous studies have shown that the primary tumor growth and the number of metastatic sites can be significantly decreased by decreasing the population of macrophages in tumor tissue, for example, by blocking recruitment of monocytes or eliminating TAMs already present in the tumor tissue. Moreover, there are attempts at reprogramming TAMs into proinflammatory M1 macrophages or neutralizing the protumoral products of TAMs. Another approach uses TAMs for anticancer drug delivery into the tumor environment. In this review, we would like to summarize the clinical and preclinical trials that were focused on macrophages as a target for anticancer therapies.

8-methoxypsoralen reduces AKT phosphorylation, induces intrinsic and extrinsic apoptotic pathways, and suppresses cell growth of SK-N-AS neuroblastoma and SW620 metastatic colon cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: 8-methoxypsoralen (8-MOP) is a furanocoumarin and an active compound of a traditional Egyptian medicinal plant Ammi majus L, whose juice/fruit has been used for many years in folk phototherapy for the treatment of vitiligo or a hyperproliferative skin disorder, psoriasis. 8-MOP together with UVA light is also used as an anticancer drug for the treatment of cutaneous T-cell lymphoma. However, furanocoumarins exert anticancer activity even without UV irradiation. AIM OF THE STUDY: Evaluation UV-independent anticancer activity of 8-MOP in human cancer cell lines and identification of the mechanisms involved in this action. Results could provide new data about a potential role of 8-MOP in prevention and growth suppression in a broad spectrum of cancers. MATERIALS AND METHODS: 8-MOP (99%, HPLC/MS assay) was isolated from A. majus fruits by chromatographic methods. The effect of 8-MOP on cell viability was evaluated by the MTT test in several human cancer cell lines. Anti-proliferative activity of 8-MOP was evaluated by the BrdU assay in neuroblastoma (SK-N-AS) and metastatic colon cancer (SW620) cells. The Hoechst/PI staining was used for morphological analysis of cell death. An annexin V-FITC/PI double labelling and Cell Death Detection ELISA kit were used to detect apoptosis. The expression of apoptosis-associated proteins and the AKT activation status were determined by Western blot analysis. RESULTS: 8-MOP inhibited cell growth in several cancer cell lines. The SK-N-AS and SW620 cells were the most sensitive to the compound. 8-MOP reduced the phosphorylation of AKT308, decreased the expression of Bcl-2, increased the Bax protein level, and activated caspases -8, -9, and -3 in both cell lines. CONCLUSIONS: 8-MOP impairs the PI3K/AKT signalling pathway and, independently of photoactivation, can inhibit the growth of neuroblastoma and colon cancer cells by induction of apoptosis via intrinsic and extrinsic pathways.

Correlations of Kynurenic Acid, 3-Hydroxykynurenine, sIL-2R, IFN-α, and IL-4 with Clinical Symptoms During Acute Relapse of Schizophrenia.

Several lines of evidence suggest that up-regulation of immune response and alterations of kynurenine pathway function are involved in pathogenesis of schizophrenia. Correlations among clinical status (using PANNS, SANS and SAPS scales) and blood levels of kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK) and levels of selected immunoactive molecules, soluble interleukin-2 receptor (sIL-2R), interferon-α (IFN-α) and IL-4 were analyzed in 51 chronic schizophrenia patients during acute relapse, after four weeks of therapy and at remission. KYNA levels were significantly lower in comparison with controls (N=45) throughout the study, whereas 3-HK did not differ from controls at admission and during therapy, but increased at remission. The KYNA/3-HK ratio and IL-4 levels, but not sIL-2R and IFN-α levels, were consistently decreased in schizophrenia patients at all analyzed time points. KYNA level and KYNA/3-HK ratio measured at admission correlated negatively with the duration of illness, whereas 3-HK level correlated negatively with the improvement of SANS score at discharge. sIL-2R level before treatment was positively linked with number of relapses. In the subgroup of patients with poor response to pharmacotherapy, treated with clozapine later on, initial KYNA level and the ratio KYNA/3-HK correlated negatively with number of relapses. Positive association of sIL-2R level with number of relapses was also evident in this subgroup. Furthermore, among these patients, starting IFN-α level was negatively linked with the improvement of total PANSS score at discharge. Presented here data support the concept of disturbed kynurenine pathway function in schizophrenia and suggest that assessment of KYNA and 3-HK levels during acute relapse might be useful in prediction of response to antipsychotic therapy. Deficit of peripheral KYNA and higher 3-HK levels could be associated with more severe symptoms of schizophrenia. Further studies with larger samples size are needed to validate our results.

Alpha-Ketoglutarate as a Molecule with Pleiotropic Activity: Well-Known and Novel Possibilities of Therapeutic Use.

Alpha-ketoglutarate (AKG), an endogenous intermediary metabolite in the Krebs cycle, is a molecule involved in multiple metabolic and cellular pathways. It functions as an energy donor, a precursor in the amino acid biosynthesis, a signalling molecule, as well as a regulator of epigenetic processes and cellular signalling via protein binding. AKG is an obligatory co-substrate for 2-oxoglutarate-dependent dioxygenases, which catalyse hydroxylation reactions on various types of substrates. It regulates the activity of prolyl-4 hydroxylase, which controls the biosynthesis of collagen, a component of bone tissue. AKG also affects the functioning of prolyl hydroxylases, which, in turn, influences the function of the hypoxia-inducible factor, an important transcription factor in cancer development and progression. Additionally, it affects the functioning of enzymes that influence epigenetic modifications of chromatin: ten-eleven translocation hydroxylases involved in DNA demethylation and the Jumonji C domain containing lysine demethylases, which are the major histone demethylases. Thus, it regulates gene expression. The metabolic and extrametabolic function of AKG in cells and the organism open many different fields for therapeutic interventions for treatment of diseases. This review presents the results of studies conducted with the use of AKG in states of protein deficiency and oxidative stress conditions. It also discusses current knowledge about AKG as an immunomodulatory agent and a bone anabolic factor. Additionally, the regulatory role of AKG and its structural analogues in carcinogenesis as well as the results of studies of AKG as an anticancer agent are discussed.

Chemerin, retinol binding protein-4, cytokeratin-18 and transgelin-2 presence in sera of patients with non-alcoholic liver fatty disease.

 Background. Chemerin and retinol binding protein-4 (RBP-4) are adipokines which may play a role in the progression of NAFLD. It has been also suggested that cytokeratin-18 (CK-18) could be a marker of hepatocyte caspase-directed death while transgelin-2 production could reflect stage of liver fibrosis. The aim of this study was to evaluate the level of the above adipokines in sera of patients with NAFLD and determine the relation between the level of transgelin-2 and fibrosis-4 index (FIB-4). MATERIAL AND METHODS: Ninety-five subjects included initially to the study were divided into four groups: (I) prediabetics, obese with NAFLD and metabolic syndrome (MS), (II) lean with NAFLD and without MS, (III) obese without NAFLD and MS, and (IV) healthy individuals. We determined the levels of chemerin, RBP-4, transgelin-2 and CK-18 fragments in sera of patients with NAFLD. Moreover, we examined if the levels of CK-18 fragments and transgelin-2 correlates with FIB4 value. RESULTS: Chemerin and RBP-4 were highly expressed in sera of all NAFLD, especially in obese individuals. Chemerin level was also linked to MS. High level of serum CK-18 fragments and transgelin-2 did not correlate with obesity and MS, but seemed to correlate with progression of NAFLD to liver fibrosis. CONCLUSIONS: In conclusion, the production of the two adipokines, chemerin and RBP-4, is strongly associated with obesity in patients with NAFLD. Serum concentrations of CK-18 fragments and transgelin-2 correlate with the severity of NAFLD, but no with obesity.

[The kynurenic acid hypothesis - a new look at etiopathogenesis and treatment of schizophrenia]. / Koncepcja kynureninowa ­ nowe spojrzenie na etiopatogeneze i leczenie schizofrenii.

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan formed in the brain and in the periphery, known to block ionotropic glutamate receptors and α7 nicotinic receptors, and to act as a ligand of G protein-coupled GPR35 receptors and human aryl hydrocarbon (AHR) receptors. KYNA seems to modulate a number of mechanisms involved in the pathogenesis of schizophrenia including dopaminergic transmission in mesolimbic and mesocortical areas or glutamatemediated neurotransmission. The kynurenine hypothesis of schizophrenia links the occurrence of positive and negative symptoms of schizophrenia and cognitive impairments characteristic for the disease with the disturbances of kynurenine pathway function. Available data suggest that antipsychotic drugs may restore balance among kynurenine pathway metabolites, and that co-administration of glycine with antipsychotics may reduce extrapyramidal symptoms in patients suffering from schizophrenia. Central level of KYNA may increase in the course of inflammation, which is consistent with the inflammatory hypothesis of schizophrenia. Alterations of immune response and disturbed functioning of kynurenine pathway may lead to disproportion between neuroprotective and neurotoxic mechanisms in the brain. Currently, intense research efforts are focused on the role of kynurenine pathway metabolites in pathogenesis of schizophrenia, their association with the response to antipsychotic treatment, and search for novel medications modulating the function of kynurenine pathway.

Neutral endopeptidase (NEP) is differentially involved in biological activities and cell signaling of colon cancer cell lines derived from various stages of tumor development.

The presented studies were aimed at exploring the role of neutral endopeptidase (NEP) in the function of colon cancer cell lines LS 180 and SW 620, derived from different grades and stages of tumor development. NEP silencing by siRNA resulted in decreased viability and proliferation accompanied by increased apoptosis in both cell lines. Additionally, cell cycle arrest at the G2/M phase was observed, but only in LS 180 cells. Opposite to these results, serum-stimulated migration was increased in both cell lines. Furthermore, NEP silencing influenced the invasive activity of LS 180 and SW 620 cells in an opposite manner: while LS 180 cells showed an enhanced invasiveness, SW 620 cells exerted a reduced activity. An exploration of the activity of signaling molecules responsible for the function of tumor cells-Akt, PTEN, and FAK-after NEP silencing indicated that the endopeptidase is involved in their regulation. The increased phosphorylation level of Akt was accompanied by a decrease in PTEN in the presence of a high concentration of serum. A reduced concentration of serum did not change the phosphorylation status of Akt. Enhanced autophosphorylation of FAK was observed in LS 180 and SW 620 cells cultivated in a medium with a high concentration of serum. Taken together, these results confirm that NEP is implicated in the regulation of the survival, growth, and motile activity of colon cancer. This is also the first report which shows that NEP mediates cancer cell migration and invasiveness, but not growth and survival, through Akt/FAK signaling pathways.

Biological activity of terpene compounds produced by biotechnological methods.

CONTEXT: Biotransformation systems are profitable tools for structural modification of bioactive natural compounds into valuable biologically active terpenoids. OBJECTIVE: This study determines the biological effect of (R)-(+)-limonene and (-)-α-pinene, and their oxygenated derivatives, (a) perillyl alcohol and (S)-(+)- and (R)-(-)-carvone enantiomers and (b) linalool, trans-verbenol and verbenone, respectively, on human colon tumour cells and normal colonic epithelium. MATERIALS AND METHODS: Biotransformation procedures and in vitro cell culture tests were used in this work. Cells were incubated for 24 h with terpenes at concentrations of 5-500 µg/mL for NR, MTT, DPPH, and NO assays. IL-6 was determined by ELISA with/without 2 h pre-activation with 10 µg/mL LPS. RESULTS: trans-Verbenol and perillyl alcohol, obtained via biotransformation, produced in vitro effect against tumour cells at lower concentrations (IC50 value = 77.8 and 98.8 µg/mL, respectively) than their monoterpene precursors, (R)-(+)-limonene (IC50 value = 171.4 µg/mL) and (-)-α-pinene (IC50 value = 206.3 µg/mL). They also showed lower cytotoxicity against normal cells (IC50 > 500 and > 200 µg/mL, respectively). (S)-(+)-Carvone was 59.4% and 27.1% more toxic to tumour and normal cells, respectively, than the (R)-(-)-enantiomer. (R)-(+)-limonene derivatives decreased IL-6 production from normal cells in media with or without LPS (30.2% and 13.9%, respectively), while (-)-α-pinene derivatives induced IL-6 (verbenone had the strongest effect, 60.2% and 29.1% above control, respectively). None of the terpenes had antioxidative activity below 500 µg/mL. DISCUSSION AND CONCLUSIONS: Bioactivity against tumour cells decreased in the following order: alcohols > ketones > hydrocarbons. (R)-(+)-limonene, (-)-α-pinene, and their derivatives expressed diverse activity towards normal and tumour cells with noticeable enantiomeric differences.

Hyperreactivity of Blood Leukocytes in Patients with NAFLD to Ex Vivo Lipopolysaccharide Treatment Is Modulated by Metformin and Phosphatidylcholine but Not by Alpha Ketoglutarate.

INTRODUCTION AND AIMS: Toll-like receptor 4 and proinflammatory cytokines play a central role in the progression of nonalcoholic fatty liver disease. We investigated IL-1, IL-6 and TNFα production and toll-like receptor 4 in both--obese and lean patients with non-alcoholic fatty liver disease who met different sets of metabolic syndrome criteria and linked the results with the disease burden. MATERIALS AND METHODS: 95 subjects were divided into four groups depending on the following criteria: presence or absence of metabolic syndrome and/or non-alcoholic fatty liver disease, glucose tolerance (prediabetes or normoglycemia) and BMI value (obese or lean). We determined the levels of IL-1ß, IL-6, TNFα, and monocyte toll-like receptor 4 expression in fresh blood as well as in blood cultures treated with lipopolysaccharide with or without metformin, alphaketoglutarate or phosphatidylcholine supplementation. RESULTS: The blood leukocytes of patients with non-alcoholic fatty liver disease are hypersensitive to lipopolysaccharide treatment and produce elevated levels of pro-inflammatory cytokines in response to ex vivo treatment with lipopolysaccharide. Moreover, they overexpress toll-like receptor-4. Hyperreactivity was typical mainly for obese patients with non-alcoholic fatty liver disease together with metabolic syndrome and decreased with the severity of disease. Metformin was the most effective in attenuation of hyperreactivity in all groups of patients with non-alcoholic fatty liver disease, but in obese patients the effectiveness of metformin was weaker than in lean. The reduction of cytokine level by metformin was accompanied by the decrease in toll-like receptor-4 expression. phosphatidylcholine also attenuated hyperreactivity to lipopolysaccharide but mainly in obese patients. Alpha ketoglutarate did not modulate cytokines' level and toll-like receptor 4 expression in non-alcoholic fatty liver disease patients. CONCLUSIONS: Metformin and phosphatidylcholine attenuated lipopolysaccharide induced toll-like receptor 4 overexpression and overproduction of pro-inflammatory cytokines; however, their efficacy depended on combined presence of non-alcoholic fatty liver disease, metabolic syndrome and obesity.

[Umbilical cord blood as a source of nerve and stem cells]. / Krew pepowinowa jako zródlo komórek nerwowych i macierzystych.

OBJECTIVES: The aim of the study was to assess proliferative ability of the stem cells in the umbilical cord blood and their potential to differentiate in in vitro culture. MATERIAL AND METHODS: The material consisted of 14 samples of umbilical cord blood collected from the umbilical cord vein. Mononuclear cells were isolated using the method of density gradient medium. Next, CD34 cells were isolated from the interphase with the use of the VarioMACS sorter and anti-CD34 antibodies. Long-term cultures were conducted on Iscove's modified Dulbecco medium (IMDM) with addition of granulocyte-macrophage colony stimulating factor (GM-CSF) and nerve growth factor (NGF). Qualitative identification was performed using the May-Grunwald-Giemsy staining method, taking photographs with a confocal microscope, and with the immunoenzymatic method. RESULTS: In our research, CD34+ stem cells constituted 1.16% of the mononuclear cells, and after centrifugation in medium 0.37% of leukocytes in whole umbilical cord blood. Even after 60 days of culture without addition of the growth factors, CD34+ cells were present in the fraction of adherent cells. After stimulation with GM-CSF and NGF a part of the umbilical cord blood cells were transformed into nerve cells (presence of neuron-specific enolase was shown) and into cells morphologically similar to fibroblast and dendritic cells. CONCLUSIONS: After stimulation with GM-CSF and NGF cytokines, the umbilical cord blood cells proliferate in long-term medium, transform into nerve cells and into cells similar to fibroblast and dendritic cells.

Fungus Cerrena unicolor as an effective source of new antiviral, immunomodulatory, and anticancer compounds.

In the report, three bioactive fractions from Cerrena unicolor: laccase (LAC), endopolysaccharides (c-EPL), and low molecular weight (ex-LMS) were tested for the first time towards their antiviral, immunostimulatory, cytotoxic and antiproliferative effect. The immunomodulatory activity was studied by means of THP-1-derived macrophages able to synthesize and secrete IL-6 and TNF-α. We used cervical carcinoma cell lines SiHa (ATCC, HTB-35) and CaSki (ATCC, CRL 1550) to determine antitumor activity and human skin fibroblasts (HSF) as a control. SiHa and L929 cell lines were used in the antiviral activity assay to propagate HHV-1 and EMCV, respectively. LAC was the most active against HSV at an early stage of viral replication, whereas the activity of laccase against EMCV was evident after incubation of the virus with LAC before and after the adsorption step. Moreover, the investigations showed that the fungal c-EPL fraction stimulated the production and secretion of TNF-α and IL-6 by THP-1-derived macrophages up to a level of 2000 pg/ml and 400 pg/ml, respectively. It was indicated for the first time that the LAC and ex-LMS fractions exhibited anticancer activity. This resulted from their cytotoxic or antiproliferative action against the investigated tumor cells at concentrations above 250 µg/ml and 10 µg/ml, respectively.

Opioids, Neutral Endopeptidase, its Inhibitors and Cancer: Is There a Relationship among them?

The role of endogenous animal opioids in the biology of cancer is widely recognized but poorly understood. This is, among others, because of the short half-life of these peptides, which are quickly inactivated by endopeptidases, e.g., neutral endopeptidase (NEP, CD10). It has been established that NEP is engaged in the modulation of the tumor microenvironment, among others that of colon cancer, by exerting influence on cell growth factors, the extracellular matrix and other biologically active substances. Although there are some discrepancies among the findings on the role of both opioids and NEP in cancer development, authors agree that their role seems to depend on the origin, stage and grade of tumor, and even on the method of examination. Moreover, recently, natural inhibitors of NEP, such as sialorphin, opiorphin and spinorphin have been detected. Their analgesic activity has been established. It is interesting to ask whether there is a relationship among opioid peptides, tumor-associated NEP and its inhibitors.

Exopolysaccharide from Ganoderma applanatum as a promising bioactive compound with cytostatic and antibacterial properties.

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

Fluvastatin inhibits growth and alters the malignant phenotype of the C6 glioma cell line.

BACKGROUND: Fluvastatin is a member of the family of HMG-CoA reductase inhibitors (statins) extensively used in medical practice. Increasing evidence suggests that fluvastatin may be implicated in suppression of cancer growth and development. The aim of the present study was to investigate the anti-cancer potential of fluvastatin in C6 rat malignant glioma cells. METHODS: First, the effects of fluvastatin on cell viability (MTT assay), proliferation (BrdU assay), cell morphology, and cytoskeleton were examined. Subsequently, its effect on extracellular signal regulated kinase 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 1 and 2 (JNK 1/2) expression was estimated by Western blot. Finally, the influence of fluvastatin on cell migration and production of MMP-9 and VEGF was determined using a wound-healing assay and ELISA test, respectively. RESULTS: The results obtained showed that fluvastatin had a remarkable inhibitory and cytotoxic effect on tumor C6 cells (IC(50) = 8.6 µM, 48 h), but did not inhibit the growth of normal neuronal cells. The concentrations from 1 to 10 µM induced marked morphologic alterations typical for apoptosis including shrinkage of cytoplasm, chromatin condensation, and nucleus breakdown. CONCLUSION: The inhibitory effects of fluvastatin on cell proliferation seemed to be associated with decreased p-ERK1/2 expression, upregulation of p-JNK1/2, and reduction in the MMP-9 and VEGF concentrations in culture media. The high anticancer (antiproliferative, proapoptotic, antiinvasive) activity of fluvastatin and lack of its toxicity against normal cells indicate a potential use of this statin in the treatment of malignant glioma.