Apoptotic Efficacy of Etomoxir in Human Acute Myeloid Leukemia Cells. Cooperation with Arsenic Trioxide and Glycolytic Inhibitors,and Regulation by Oxidative Stress and Protein Kinase Activities

Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 µM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs (etoposide, cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25–200 µM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress, caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic efficacy of ATO and (with some limitations) 2-deoxy-D-glucose which, although clinically important anti-tumour agents, exhibit low efficacy in monotherapy.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Growth enhancement of Citrus reshni after inoculation with Glomus intraradices and Trichoderma aureoviride and associated effects on microbial populations and enzyme activity in potting mixes

There have been some scientific reports suggesting that dual inoculations with arbuscular mycorrhizal (AM) and saprophytic soil fungi may cause an additive or synergistic growth enhancement of the inoculated host plant. Some Trichoderma spp. have shown antagonistic potential against pathogenic fungi and a beneficial effect on plant growth. Joint inoculations of the mycorrhizal fungus Glomus intraradices Schenck and Smith, isolated from a citrus nursery (Tarragona, Spain) and a strain of Trichoderma aureoviride Rifai, isolated from an organic compost, were tested on a citrus rootstock, Citrus reshni Hort. ex Tan. The interactions between both microorganisms and their influence on mycorrhizal root colonization and plant growth enhancement, the changes produced in the soil microbial activity, like esterase, trehalase, phosphatase and chitinase activities, and on microbial populations were evaluated in three organic substrates: (1) sphagnum peat and autoclaved sandy soil (1/1, v/v), (2) sphagnum peat, quartz sand and perlite (1/1/1, v/v) and (3) pine bark compost (BVU, Prodeasa Product). Substrate characteristics were more important than the AM inoculation treatment in the determination of enzyme activity. In bark compost, the number of bacterial colonies obtained on soil-dilution plates was significantly higher than in peat and sand mixtures. Inoculation with T. aureoviride alone produced no significant effect on growth enhancement of C. reshni. However, dual inoculation with both, T. aureoviride and G. intraradices significantly increased plant growth in two of the substrates used and was the best treatment in pine bark amended compost. The inoculation with T. aureoviride did not affect the development of mycorrhizal root colonization. These results show a synergistic effect of G. intraradices and T. aureoviride on the growth of C. reshni in organic substrates and indicate the potential benefits of using combined inoculations.     

Investigation of the Listeria monocytogenes Scott A acid tolerance response and associated physiological and phenotypic features via whole proteome analysis

The global proteomic responses of the foodborne pathogen Listeria monocytogenes strain Scott A, during active growth and transition to the stationary growth phase under progressively more acidic conditions, created by addition of lactic acid and HCl, were investigated using label-free liquid chromatography/tandem mass spectrometry. Approximately 56% of the Scott A proteome was quantitatively assessable, and the data provides insight into its acquired acid tolerance response (ATR) as well as the relation of the ATR to the growth phase transition. Alterations in protein abundance due to acid stress were focused in proteins belonging to the L. monocytogenes common genome, with few strain-dependent proteins involved. However, one of the two complete prophage genomes appeared to enter lysogeny. During progressive acidification, the growth rate and yield were reduced 55% and 98%, respectively, in comparison to nonacidified control cultures. The maintenance of the growth rate was determined to be connected to activation of cytoplasmic pH homeostatic mechanisms while cellular reproductive-related and cell component turnover proteins were markedly more abundant in acid stressed cultures. Cell biomass accumulation was impeded predominantly due to repression of phosphodonor-linked enzymes involved with sugar phosphotransfer, glycolysis, and cell wall polymer biosynthesis. Acidification caused a shift from heterofermentation to an oxidatively stressed state in which ATP appears to be generated mainly through the pyruvate dehydrogenase/pyruvate oxidase/phosphotransacetylase/acetate kinase and branched chain acid dehydrogenase pathways. Analysis of regulons indicated energy conservation occurs due to repression by the GTP/isoleucine sensor CodY and also the RelA mediated stringent response. Whole proteome analysis proved to be an effective way to highlight proteins involved with the acquisition of the ATR.     

Isolation of a soluble and membrane-associated Fe(III) reductase from the thermophile, Thermus scotoductus (SA-01)

The Fe(III) reductase activity was studied in the South African Fe(III)-reducing bacterium, Thermus scotoductus (SA-01). Fractionation studies revealed that the membrane as well as the soluble fraction contained NAD(P)H-dependent Fe(III) reductase activity. The membrane-associated enzyme was solubilized by KCl treatment and purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A combination of ion-exchange and gel filtration chromatography was used to purify the soluble enzyme to apparent homogeneity. The molecular mass of the membrane-associated Fe(III) reductase was estimated to be 49 kDa, whereas the soluble Fe(III) reductase had an apparent molecular mass of 37 kDa. Optimum activity for the membrane-associated enzyme was observed at around 75 degrees C, whereas the soluble enzyme exhibited a temperature optimum at 60 degrees C.