Chemo-sensitizing effects of EP4 receptor-induced inactivation of nuclear factor-κB.

The EP4 receptor conveys growth-inhibitory effects in mature and immature B cells via NF-κB. Herein, the EP4 receptor was evaluated as a potential therapeutic target for leukemia and lymphoma, whose survival depends on the constitutive activity of NF-κB. Utilizing a pharmacological approach, we proved that the EP4 receptor induces caspase-mediated apoptosis in malignantly transformed B cells, with the most prominent effect being on Burkitt׳s lymphoma cells. Since the increased activation of NF-κB underlies multi-drug resistance phenomena, we modulated this signaling pathway via EP4 receptor triggering. Pge1-OH, a specific EP4 receptor agonist, led to decreased NF-κB activity and a consequent decrease in levels of the antiapoptotic gene Bcl-xL in Ramos cells, resulting in an elevated sensitivity of cells towards bortezomib- and doxorubicin-induced chemotherapeutic effects. Our study identifies the as yet unrecognized potential of EP4 receptor agonists as chemo-sensitizing agents in B-cell lymphoma. The specific downregulation of NF-κB-dependent pathways in B-cell malignancies opens new possibilities for treatment and current therapy optimization using specific EP4 receptor agonists.

Fullerenol nanoparticles: toxicity and antioxidant activity.

Fullerenes are a relatively new group of compounds and represent a class of sphere-shaped molecules made exclusively of carbon atoms. Since their discovery in 1985, many aspects of both fullerene and its analogues have been intensively studied to reveal their physical and chemical reactivity, as well as potential use in biological systems. Both in vitro and in vivo studies have shown that polyhydroxylated fullerene derivatives, fullerenol nanoform (C60(OH) n , n = 2-72), can be potential antioxidative agents in biological systems. This chapter represents a review of published studies of fullerenes' biological activities with special accent on the most tested fullerenol nanoform C60(OH)24.

Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

Selective cytotoxicity of amidinopiperidine based compounds towards Burkitt's lymphoma cells involves proteasome inhibition.

Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-based compounds manifest cytotoxic activity, we have selected serine protease inhibitors designed and synthesized in-house with large hydrophobic naphthalene moiety for screening. The cytotoxic potential of screened molecules was correlated to modifications of R(1) residues. The most cytotoxic were compounds with greater basicity; amidinopiperidines, piperidines and benzamidines. Amidinopiperidine-based compounds exert cytotoxicity in low µM range, with IC(50) 18 µM and 22 µM for inhibitors 15 and 16 respectively. These compounds exhibited selective cytotoxicity towards the Burkitt's lymphoma cells Ramos and Daudi, and proved nontoxic to PMBC, Jurkat and U937. They induce caspase-dependent apoptotic cell death, as demonstrated by the use of a pan-caspase inihibitor, zVADfmk, which was able to rescue Ramos cells from compound(s)-induced apoptosis. We confirm a disruption of the pro-survival pathway in Burkitt's lymphoma through NFκB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IκB) molecules with no subsequent release of active NFκB implicated the involvement of proteasome. Indeed, we show that the amidinopiperidine-based compounds inhibit all three proteolytical activities of the human 20S proteasome, with the most prominent effect being on the trypsin-like activity. Consistently, treatment of Ramos cells with these compounds led to an increase in ubiquitinated proteins. The amidinopiperidine-based serine protease inhibitors presented are, as selective inducers of apoptosis in Burkitt's lymphoma cells, promising leads for the development of novel chemotherapeutics.

EP4 receptor signalling in immature B cells involves cAMP and NF-κB dependent pathways.

OBJECTIVES: Delineation of EP4 receptor signalling properties in immature B cells. METHODS: WEHI 231 cells were used as a model of immature B lymphocytes. The effects of PGE2, EP4 receptor antagonist, EP4 receptor agonist, forskolin and adenylate cyclase inhibitor on proliferation of WEHI 231 cells were examined by MTS assay. Cyclic adenosine monophosphate (cAMP) levels were examined by ELISA, whereas phosphorylation of vasodilator-stimulated phosphoprotein (VASP), kinase, extracellular signal-regulated kinase1/2, IκB-α and nuclear factor (NF)-κB subunit p105 were subjected to Western blot analysis. Translocation of NF-κB subunit p65 and EPRAP (EP4 receptor associated protein) was examined by fluorescence microscopy. Levels of early growth response factor (Egr)-1 mRNA were determined by quantitative PCR. KEY FINDINGS: We identified the EP4 receptor as the principal molecule mediating the growth-suppressive effect of prostaglandin E2 in WEHI 231 cells. EP4 receptor activation results in cAMP formation and the activation of protein kinase A, NF-κB1 p105 subunit stabilization and inhibition of IκBα phosphorylation, followed by the accumulation of NF-κB p65 subunit in the cell cytoplasm, whereas the activation of PI3K is not involved in EP4 receptor signalling. Elevation of cAMP and inhibition of NF-κB activation are two possible mechanisms by which the EP4 receptor inhibits the proliferation of immature B lymphocytes. CONCLUSIONS: Modulation of the EP4 receptor on immature B lymphocytes provides important insight into the observed action of PGE2 and opens new possibilities for the development of therapies for autoimmune diseases, leukaemia and lymphomas.

Prostaglandin EP4 receptor enhances BCR-induced apoptosis of immature B cells.

Prostaglandin E2 (PGE2) is emerging as an important co-modulator of B cell responses. Using a pharmacological approach, we aimed to delineate the role of PGE2 in B cell receptor (BCR) induced apoptosis of immature B cells. Gene and protein expression analyses showed that, of the four PGE2 receptors subtypes, only EP4 receptor is upregulated upon BCR cross-linking, leading to sensitization of WEHI 231 cells towards PGE2 mediated inhibitory effects. EP4 receptor antagonist ONO-AE3-208, was able to completely revert the observed effects of PGE2. The engagement of EP4 receptor promotes BCR-induced G0/G1 arrest of WEHI 231 cells, resulting in enhanced caspase mediated, BCR-induced apoptosis. We addressed, mechanistically, the interplay between BCR and EP4 receptor signaling components. Prostaglandin1-alcohol (Pge1-OH), a selective EP4 receptor agonist inhibits BCR-induced activation of NF-κB by suppression of BCR-induced IκBα phosphorylation. Disruption of prosurvival pathways is a possible mechanism by which PGE2 enhances BCR-induced apoptosis in immature B lymphocytes.

Potential hepatoprotective effects of fullerenol C60(OH)24 in doxorubicin-induced hepatotoxicity in rats with mammary carcinomas.

The aim of this study was to investigate the potential protective role of fullerenol C60(OH)24 on doxorubicin-induced liver toxicity using in vivo (female Sprague-Dawley rats) and in vitro (human hepatocellular carcinoma - HepG2; colorectal adenocarcinoma cell lines - Caco-2) approaches. The first (healthy control) and second (control with chemically induced mammary carcinomas) group received saline only. The third, fourth and fifth group (all with breast cancer) were injected (i.p.) with a single dose of doxorubicin (8mg/kg), doxorubicin/fullerenol (100mg/kg of fullerenol 30min before administration of 8mg/kg doxorubicin) and fullerenol (100mg/kg), respectively. Two days after treatment, the rats were sacrificed. Results showed that treatment with doxorubicin alone caused significant changes in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), as well as in the levels of malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), total antioxidant status (TAS), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) in the liver tissue. These effects were significantly reduced for all investigated parameters by pre-treatment with fullerenol but not for the MDA and GSH level. The HepG2 and Caco-2 cell lines were continuously treated with fullerenol for 12h, 24h, 48h and 96h at concentrations of 10microg/mL and 44microg/mL. With the aim of evaluating the modulating activity of fullerenol on doxorubicin-induced hepatotoxicity, the cell lines were simultaneously treated with doxorubicin (1microm; 5microm) and fullerenol (10microg/mL; 44microg/mL) in different combinations. When the cells are treated with 5microm doxorubicin along with the fullerenol, we can see a significant improvement of the cell capability during the entire time-line. We can conclude that fullerenol has cytotoxic effects on HepG2 by itself, but when the oxidative stress is too high the cytotoxic effects of fullerenol are overcome by its protective role as a strong antioxidant compound.

Determination of caffeine and associated compounds in food, beverages, natural products, pharmaceuticals, and cosmetics by micellar electrokinetic capillary chromatography.

A method is described for quantitating caffeine, theobromine, theophylline, paracetamol, propyphenazone, acetylsalicylic acid, salicylic acid, and codeine phosphate in corresponding real samples of food, beverages, natural products, pharmaceuticals, and cosmetic preparations by micellar electrokinetic capillary chromatography. The separation is carried out at 25 degrees C and 25 kV, using a 20 mM phosphate buffer (pH 9.0), 80 mM sodium dodecyl sulfate, and 7.5% (v/v) acetonitrile. UV detection is at 210 nm. The method is shown to be specific, accurate (recoveries over the range 98.9-101.2%), linear over the tested range (correlation coefficients>or=0.9993), and precise (relative standard deviation below 2.1%). The method is applied for the quantitative analysis of these compounds in different foods, beverages, natural products, pharmaceuticals, and cosmetic products.