Marrubium vulgare ethanolic extract induces proliferation block, apoptosis, and cytoprotective autophagy in cancer cells in vitro.

Marrubium vulgare is a European medicinal plant with numerous beneficial effects on human health. The aim of the study was to isolate the plant ethanolic extract (MVE) and to investigate its anti-melanoma and anti-glioma effects. MVE was prepared by the modified pharmacopoeial percolation method and characterized by UHPLC-LTQ OrbiTrap MS. MVE dose-dependently reduced viability of melanoma (B16) and glioma (U251) cells, but not peripheral blood mononuclear cells. It arrested cell cycle in S+G2/M phase, which was associated with the activation of MAP kinase p38 and up-regulation of antiproliferative genes p53, p21 and p27. MVE induced oxidative stress, while antioxidants abrogated its antitumor effect. Furthermore, MVE induced mitochondrial depolarization, activation of caspase-9 and -3, Parp cleavage, phosphatidylserine exposure and DNA fragmentation. The mitochondrial apoptotic pathway was associated with the up-regulation of proapoptotic genes Pten, Bak1, Apaf1, and Puma and down-regulation of antiapoptotic genes survivin and Xiap. MVE also stimulated the expression of autophagy-related genes Atg5, Atg7, Atg12, Beclin-1, Gabarab and Sqstm1, as well as LC3-I conversion to the autophagosome associated LC3-II, while autophagy inhibitors exacerbated its cytotoxicity. Finally, the most abundant phenolic components of MVE, ferulic, p-hydroxybenzoic, caffeic and chlorogenic acids, did not exert a profound effect on viability of tumor cells, suggesting that other components individually or in concert are the mediators of the extracts' cytotoxicity. By demonstrating the ability of MVE to inhibit proliferation, induce apoptosis and cytoprotective autophagy, our results suggest that MVE, alone or combined with autophagy inhibitors, could be a good candidate for anti-melanoma and anti-glioma therapy.

AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells.

The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose- and time-dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up-regulation of autophagy-inducing protein beclin-1 and subsequent appearance of acridine orange-stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin-treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2. Autophagy induction in cisplatin-treated cells was preceded by activation of adenosine monophosphate-activated protein kinase (AMPK) and concomitant down-regulation of mammalian target of rapamycin (mTOR)-mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA-mediated AMPK down-regulation and AMPK inhibitor compound C increased cisplatin-induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

Increased activity of interleukin-23/interleukin-17 proinflammatory axis in obese women.

OBJECTIVE: To compare the concentrations of cytokines belonging to Th17 axis (interleukin (IL)-17 and IL-23) and Th1 axis (IL-12 and interferon (IFN)-gamma) in obese and lean women, and to investigate their relationships with the proinflammatory adipokine leptin, proinflammatory cytokine macrophage migration inhibitory factor (MIF) and anthropometric and metabolic parameters of obesity. DESIGN: Cross-sectional study. SUBJECTS: Twenty-six obese women (age 20-52 years, body mass index (BMI): 30-48 kg/m(2)) and 20 healthy lean women (age 23-46 years, BMI: 18-25 kg/m(2)). MEASUREMENTS: Plasma levels of cytokines and leptin, BMI, waist circumference (WC) and insulin resistance index HOMA (homeostatic model assessment). RESULTS: Blood concentrations of IL-17, IL-23, MIF and leptin, but not IL-12 or IFN-gamma, were higher in obese compared with lean women (P=0.002, 0.046, 0.006 and 0.002, respectively). There was a positive correlation between IL-17 and IL-23 (r(s)=0.530), which was at the border of statistical significance (P=0.065). Neither IL-17 nor IL-23 correlated with leptin or MIF, and there was no association between IL-17 and IL-23 levels with BMI, WC or HOMA index. CONCLUSION: Interleukin-23/IL-17 axis is stimulated in obese women independently of the increase in abdominal fat, insulin resistance, leptin and MIF levels.

A novel method for the functionalization of gamma-irradiated single wall carbon nanotubes with DNA.

In this work we describe a novel method for highly efficient functionalization of single wall carbon nanotubes (SWCNTs) by DNA wrapping. Exposure of SWCNTs to gamma-irradiation (50 kGy) has lowered by one order of magnitude the amount of single stranded deoxyribonucleic acid (ssDNA) required for SWCNT modification. The resulting hybrids of gamma-irradiated SWCNTs and ssDNA were characterized by optical absorbance spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy was used to investigate the morphology of hybrids. While gamma-irradiation in three different media has significantly improved the process of SWCNT dispersion, irradiation in ammonia was the most efficient. The gamma-irradiated SWCNTs functionalized with ssDNA were stabilized by electrostatic forces. This preliminary study suggests that gamma-irradiation can significantly improve the functionalization of SWCNTs with DNA.

Antiproliferative effect of vitamin A and D analogues on adult human keratinocytes in vitro.

BACKGROUND: Vitamin A and D analogues play an important role in epidermal homeostasis and are used in the treatment of various skin diseases. The failure of retinoid and vitamin D treatments is sometimes difficult to explain. METHODS: We analyzed the effect of all-trans retinoic acid (all-trans RA), 13-cis retinoic acid (13-cis RA), ergocalciferol and cholecalciferol in keratinocyte cultures established from adult donors, on the cell proliferation by means of [(3)H]thymidine incorporation and apoptosis after fluorescein diacetate/trypan blue staining. RESULTS: All tested agents exerted a dose-dependent inhibition of keratinocyte proliferation in the concentration range of 1.25-5 microM. Based on IC(50) values, the antiproliferative efficiency was as follows: cholecalciferol > ergocalciferol = all-trans RA > 13-cis RA. The observed effect of cholecalciferol and ergocalciferol, but not retinoids, involved the induction of apoptotic cell death. Combining vitamins A and D did not further increase the proliferation block and even displayed an antagonistic effect. CONCLUSION: The susceptibility of keratinocytes to the antiproliferative action of vitamins A and D was markedly different in cell cultures derived from different donors, indicating a possible predictive value of the in vitro testing for the efficiency of the clinical response to these agents.

Modulation of inducible nitric oxide synthase activation by immunosuppressive drugs.

The activation of inducible form of nitric oxide (NO) synthase (iNOS, type II, or macrophage NOS) and subsequent production of free radical gas NO is an important anti-infectious and anti-tumor mechanism of innate immunity. On the other hand, high amounts of iNOS-derived NO have been implicated in self-tissue destruction during autoimmune diseases, allograft rejection, sepsis, and other disorders accompanied by excessive activation of the immune system. It is generally accepted that beneficial effects of some recently designed immunosuppressive agents primarily stem froin their ability to interfere with the function of T and/or B cells, thus preventing deleterious consequences of specific immunity-innate immunity positive feedback, with high NO production being one of them. However, it has been recently observed that drugs like cyclosporin A, FK506, leflunomide, mycophenolate mofetil, pentoxifylline, and linomide can directly modulate cytokine and/or LPS-induced NO production in various cell types in vitro, probably by interfering with iNOS gene transcription or catalytic activity of iNOS enzyme. Interestingly, some of these drugs exhibited cell-specific pattern of iNOS modulation, thus indirectly revealing distinct requirements for iNOS induction in different cell types. Possible impact of this direct and cell-selective interference with iNOS activation on the therapeutic effectiveness of immunosuppressive drugs is discussed.

Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes.

The effect of interleukin-17 (IL-17) on production of nitric oxide (NO) in rodent astrocytes was investigated. While IL-17 by itself did not induce NO production, it caused a dose-dependent enhancement of IFN-gamma-triggered NO synthesis in both mouse and rat primary astrocytes. In contrast, IL-17 was unable to stimulate NO synthesis in either murine or rat macrophages. IFN-gamma-triggered expression of mRNA for iNOS, but not for its transcription factor interferon regulatory factor-1 (IRF-1), was markedly elevated in IL-17-treated astrocytes. The induction of iNOS mRNA by IL-17 in IFN-gamma-pretreated astrocytes was abolished by antagonists of nuclear factor-kappaB (NF-kappaB) activation--a proteasome inhibitor MG132 and an antioxidant agent PDTC, as well as with specific p38 MAP kinase inhibitor SB203580. While IL-17 stimulated both IL-1beta and IL-6 production in astrocytes, only IL-1 was partly responsible for IL-17-induced NO release. Finally, IL-17 synergized with exogenous IL-1beta and TNF-alpha for astrocyte NO production. Having in mind a well-known neurotoxic action of NO, these results suggest a possible role for IL-17 in the inflammatory diseases of the CNS.

Regulation of inducible nitric oxide synthase by cAMP-elevating phospho-diesterase inhibitors.

Among the numerous genes controlled by cyclic adenosine monophosphate (cAMP)/protein kinase A signalling machinery is the gene encoding the inducible nitric oxide synthase (iNOS), an enzyme catalyzing the synthesis of a highly reactive free radical nitric oxide (NO). While being a major microbicidal and tumoricidal molecule, iNOS-derived NO has also been implicated in tissue destruction, as well as in regulation of inflammatory/immune cell function in various disorders associated with excessive inflammation. A feasible way for cAMP-dependent therapeutic control of inflammation, including iNOS-mediated NO synthesis, could involve the administration of drugs that block the enzymatic activity of cAMP-degrading phosphodiesterases (PDE). Indeed, cAMP-elevating PDE inhibitors can influence iNOS activation in different cell types in vitro, and their potent anti-inflammatory effects in experimental disease models and clinical studies were frequently accompanied with profound modulation of NO production. A set of conflicting data has been generated over the years, ranging from strong suppression to marked enhancement of NO release by cAMP-increasing PDE inhibitors, depending on cell-type, iNOS stimuli, and/or the agents used. The present review summarizes the data on iNOS modulation by cAMP-elevating PDE inhibitors and possible mechanisms behind it, speculating on its contribution to the therapeutic effects of these drugs.

Differential regulation of nitric oxide production by increase of intracellular cAMP in murine primary fibroblasts and L929 fibrosarcoma cell line.

The effect of intracellular cAMP rise on nitric oxide (NO) production was compared in murine primary fibroblasts isolated from the spleens of CBA mice, and L929 fibrosarcoma cell line. Treatment of confluent L929 cells with cAMP analogues -dibutyryl-cAMP (db-cAMP) or 8-Cl-cAMP caused dose-dependent augmentation of inducible NO synthase (iNOS)-mediated NO production, which has been abrogated by inhibition of protein synthesis with cycloheximide or addition of selective iNOS inhibitor aminoguanidine. In contrast, under the same cultivating conditions, cAMP analogues were not able to upregulate NO synthesis in primary fibroblasts. Treatment with cAMP analogues or non-selective phosphodiesterase (PDE) inhibitor pentoxifylline affected IFNgamma-induced NO synthesis in both cell types, but in the opposite manner-enhancing in L929 cells and suppressive in primary fibroblasts. The induction of iNOS, but not its catalytic activity, was impaired in cAMP-treated primary fibroblasts. Finally, PDE type IV inhibitor rolipram enhanced IFN-gamma-triggered NO synthesis in L929 cells, but was unable to mimic cAMP analogue or PTX-mediated suppression of NO synthesis in spleen fibroblasts. These results suggest that, in contrast to L929 fibrosarcoma cell line, intracellular cAMP rise might have a role in downregulation of NO production in murine primary fibroblasts.

Inducible nitric oxide synthase inhibition by mycophenolic acid.

The focus of this review is the influence of an immunosuppressive xenobiotic drug mycophenolic acid on the induction of nitric oxide production in various cell types. The potential therapeutic significance of the cell-specific fine-tuning of nitric oxide release by mycophenolic acid, as well as the mechanisms behind the drug action are discussed.

Possible virulence factors of Staphylococcus sciuri.

Staphylococcus sciuri is an opportunistic pathogen of controversial clinical significance. The factors that contribute to colonization and/or infection caused by this bacterium have not been studied intensively so far. The present research was carried out in order to study the presence of potential virulence factors in 121 human and animal isolates of this bacterium. Isolates were examined for biofilm formation, hemagglutination, presence of clumping factor, production of spreading factors and exotoxins, cytotoxicity and capacity to stimulate nitric oxide production. The results showed that S. sciuri is highly capable of biofilm production, that it displays strong proteolytic and DNase activities, produces hemolysins and stimulates nitric oxide production by rat macrophages. Although the present study showed existence of a wide spectrum of possible virulence determinants of S. sciuri, their exact contribution to virulence of this bacterium in vivo remains to be determined.

Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line.

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-gamma caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-gamma-induced expression of iNOS, but not interferon regulatory factor-1 (IRF-1) mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-gamma and any further induction was blocked by protein synthesis inhibitor cycloheximide (CHX). FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action.

5-Aza-2'-deoxycytidine stimulates inducible nitric oxide synthase induction in C6 astrocytoma cells.

The influence of a nucleoside analog 5-aza-2'-deoxycytidine (5-AzadC) on inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) production in various rat cell types was investigated. In C6 astrocytoma cell line and primary astrocytes, 5-AzadC enhanced proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-1)-triggered NO synthesis in a time- and dose-dependent manner. In contrast, 5-AzadC did not potentiate NO production in IFN-gamma-stimulated macrophages, fibroblasts, or endothelial cells. Blockade of transcription or translation in C6 cells abolished the observed effect, suggesting the iNOS gene expression, rather than its catalytic activity, as a target for the drug action. Accordingly, 5-AzadC upregulated IFN-gamma-induced expression of iNOS mRNA in C6 astrocytes. The effect of 5-AzadC on astrocyte NO release was blocked by the inhibitor of p44/42 mitogen activated protein kinase-dependent signaling. Finally, the observed stimulatory effect of 5-AzadC on iNOS expression was apparently independent of DNA demethylation, as DNA digestion with methylation-sensitive restriction enzyme HpaII showed that 5-AzadC failed to demethylate cellular DNA in conditions used for iNOS induction.

Muramyl dipeptide potentiates cytokine-induced activation of inducible nitric oxide synthase in rat astrocytes.

We investigated the influence of muramyl dipeptide (MDP), a cell wall component of Gram-positive bacteria, on cytokine-induced nitric oxide (NO) production in rat primary astrocytes. MDP alone did not induce NO release in astrocyte cultures. However, MDP increased astrocyte NO production and subsequent nitrite accumulation triggered by IFN-gamma. IFN-gamma-activated expression of mRNA for inducible NO synthase (iNOS) and iNOS transcription factor interferon regulatory factor-1 (IRF-1) was markedly enhanced in astrocytes treated with MDP. The potentiating effect of MDP on IFN-gamma-induced NO production in astrocytes was completely blocked with protein tyrosine kinase (PTK) inhibitor genistein or mitogen activated protein kinase (MAPK) inhibitor PD98059. In contrast, protein kinase C (PKC) inhibitor calphostin C did not affect ability of MDP to augment IFN-gamma-triggered astrocyte NO synthesis. These results suggest that MDP synergizes with IFN-gamma in the induction of iNOS gene in astrocytes through mechanisms involving PTK and MAPK, but not PKC activation. Finally, MDP also augmented NO production and iNOS mRNA expression in astrocytes treated with IL-1beta.

Leflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes.

Highly reactive gaseous free radical nitric oxide (NO), generated by astrocytes and infiltrating macrophages is implicated in inflammatory destruction of brain tissue, including that occurring in multiple sclerosis. Therefore, the influence of immunosuppressive drug leflunomide on inducible nitric oxide synthase (iNOS)-dependent NO production in rat astrocytes and macrophages was investigated. Under the same cultivating conditions, leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-gamma+LPS-triggered expression of iNOS mRNA in astrocytes. In the presence of transcription inhibitor actinomycin D, A77 1726 failed to inhibit astrocyte NO production, suggesting transcriptional regulation of iNOS by leflunomide. This assumption was further supported by the ability of A77 1726 to inhibit IFN-gamma+LPS-induced expression of mRNA for an important iNOS transcription factor IRF-1. PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), but not genistein, an unselective protein tyrosine kinase inhibitor, completely mimicked cell type-specific inhibition of NO synthesis by A77 1726. Therefore, previously described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for A77 1726-mediated suppression of iNOS activation in astrocytes. Accordingly to results obtained with primary astrocytes, both A77 1726 and PD98059 significantly reduced IFN-gamma+LPS-induced NO synthesis in the cultures of rat astrocytoma cell line C6. The ability to suppress iNOS induction in astrocytes supports potential use of leflunomide in the treatment of multiple sclerosis and other NO-dependent inflammatory brain disorders.

Tumor cell-specific inhibition of inducible nitric oxide synthase activation by tiazofurin.

The effects of tiazofurin (TR) on proliferation and cytokine-induced nitric oxide (NO) production in the L929 fibrosarcoma cell line and murine embryonic fibroblasts were investigated. Treatment with TR inhibited the growth of nonconfluent L929 cells in a dose-dependent manner. TR, at concentrations not affecting cell viability or proliferation, markedly decreased IFN-gamma + LPS-induced expression of inducible NO synthase (iNOS) mRNA and, subsequently, NO production in confluent L929 cultures. However, TR did not interfere with the IFN-gamma-triggered expression of mRNA for IRF-1, an important iNOS transcription factor, implying that TR interferes with some other intracellular pathway involved in iNOS induction triggered by IFN-gamma + LPS. In contrast to the results obtained in L929 cells, iNOS mRNA expression induced by IFN-gamma + LPS in murine embryonic fibroblasts was resistant to TR, indicating a tumor-selective action of this agent.

Relationship between activity of silica thin films and density of cells occupation.

The SiO2 thin films (STFs) were deposited on the surfaces of stainless steel tapes and their activity was particularly investigated from the aspect of the number density of hydroxyl groups on their surfaces. The calculation procedure of density of active OH groups includes determination of average length of silica chains that constitute silica sol particles with almost uniform size, on the base of thermogravimetric analysis. The size of SiO2 particles is analyzed by transmission electron microscopy and dynamic light scattering method. Fibroblast (L929) cell densities on the surfaces of these films were investigated using phase contrast microcopy. It was shown that there is a relationship between OH group densities and density of attached cells. Besides, the cytotoxicity effect was studied and compared for various thermally treated STFs.

A new approach to the drug release kinetics of a discrete system: SiO2 system obtained by ultrasonic dry spraying.

Mesoporous silica materials have already proved to be non-toxic and biocompatible, and also to have large pore volume and very high specific surface area suitable for loading of small molecules. Having this in mind and the fact that silicon dioxide (SiO(2)) powders can be so designed to obtain particle structures organized at multi levels, SiO(2) was chosen as a potential carrier for metronidazole, an antibiotic drug. SiO(2) powder was synthesized in two stages: first silica sol was prepared by hydrothermal synthesis and second the sol was converted into powder by dry spraying with simultaneous incorporation of the antibiotic into its structure. Scanning and transmission electron microscopy study revealed very complex structure and sub-structure of SiO(2) particles. Cell viability tests were used for estimation of cytotoxicity of so synthesized SiO(2). The drug release data showed that the system can provide drug release for a long time. Also, the device behavior is fully predictable, according to our theoretical model of multilevel structure design, and gives many opportunities for model investigations of drug release and its kinetics. The pore sizes and their distribution were observed as a limiting factor of drug release kinetics. Therefore, as the pore sizes are given as a set of discrete values, the kinetics of drug release might also be given as a set of corresponding discrete values.

Cell-type dependent response of melanoma cells to aloe emodin.

Intrinsic characteristics of melanoma cells such as expression of inducible nitric oxide synthase (iNOS), redox status, and activity of signaling pathways involved in proliferation, differentiation and cell death define the response of the cells to the diverse treatments. In this context we compared the effectiveness of herbal antaquinone aloe emodin (AE) against mouse B16 melanoma and human A375, different in initial activity of ERK1/2, constitutive iNOS expression and basal level of reactive oxygen species (ROS). Both cell lines are sensitive to AE treatment. However, while the agent induces differentiation of B16 cells toward melanocytes, in A375 cells promoted massive apoptosis. Differentiation of B16 cells, characterized by enhanced melanin production and tyrosinase activity, was mediated by H(2)O(2) production synchronized with rapid p53 accumulation and enhanced expression of cyclins D1 and D3. Caspase mediated apoptosis triggered in A375 cells was accompanied with Bcl-2 but not iNOS down-regulation. In addition, opposite regulation of Akt-ERK1/2 axis in AE treated B16 and A375 cells correlated with different outcome of the treatment. However, AE in a dose-dependent manner rescued both B16 and A375 cells from doxorubicin- or paclitaxel-induced killing. These data indicate that caution is warranted when AE is administrated to the patients with conventional chemotherapy.