Palladium and Platinum Complexes of the Antimetabolite Fludarabine with Vastly Enhanced Selectivity for Tumour over Non-Malignant Cells.

The purine derivative fludarabine is part of frontline therapy for chronic lymphocytic leukaemia (CLL). It has shown positive effects on solid tumours such as melanoma, breast, and colon carcinoma in clinical phase I studies. As the treatment of CLL cells with combinations of fludarabine and metal complexes of antitumoural natural products, e.g., illudin M ferrocene, has led to synergistically enhanced apoptosis, in this research study different complexes of fludarabine itself. Four complexes bearing a trans-[Br(PPh3)2]Pt/Pd fragment attached to atom C-8 via formal η1-sigma or η2-carbene bonds were synthesised in two or three steps without protecting polar groups on the arabinose or adenine. The platinum complexes were more cytotoxic than their palladium analogues, with low single-digit micromolar IC50 values against cells of various solid tumour entities, including cisplatin-resistant ones and certain B-cell lymphoma and CLL, presumably due to the ten-fold higher cellular uptake of the platinum complexes. However, the palladium complexes interacted more readily with isolated Calf thymus DNA. Interestingly, the platinum complexes showed vastly greater selectivity for cancer over non-malignant cells when compared with fludarabine.

SAM-Competitive EZH2-Inhibitors Induce Platinum Resistance by EZH2-Independent Induction of ABC-Transporters.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Multiple-stressor exposure of aquatic food webs: Nitrate and warming modulate the effect of pesticides.

Shallow lakes provide essential ecological and environmental services but are exposed to multiple stressors, including agricultural runoff (ARO) and climate warming, which may act on different target receptors disrupting their normal functioning. We performed a microcosm experiment to determine the individual and combined effects of three stressors-pesticides, nitrate and climate warming-on two trophic levels representative of communities found in shallow lakes. We used three submerged macrophyte species (Myriophyllum spicatum, Potamogeton perfoliatus, Elodea nuttallii), eight benthic or pelagic microalgal species and three primary consumer species (Daphnia magna, Lymnaea stagnalis, Dreissena polymorpha) with different feeding preferences for benthic and pelagic primary producers. Eight different treatments consisted of a control, only nitrate, a pesticide cocktail, and a combination of nitrate and pesticides representing ARO, each replicated at ambient temperature and +3.5°C, mimicking climate warming. Pesticides negatively affected all functional groups except phytoplankton, which increased. Warming and nitrate modified these effects. Strong but opposite pesticide and warming effects on Myriophyllum drove the response of the total macrophyte biomass. Nitrate significantly suppressed Myriophyllum final biomass, but not overall macrophyte and microalgal biomass. Nitrate and pesticides in combination caused a macrophyte decline, and the system tipped towards phytoplankton dominance. Strong synergistic or even reversed stressor interaction effects were observed for macrophytes or periphyton. We emphasize the need for more complex community- and ecosystem-level studies incorporating multiple stressor scenarios to define safe operating spaces.

Divergent Effects of EZH1 and EZH2 Protein Expression on the Prognosis of Patients with T-Cell Lymphomas.

T-cell lymphomas are highly heterogeneous and their prognosis is poor under the currently available therapies. Enhancers of zeste homologue 1 and 2 (EZH1/2) are histone H3 lysine-27 trimethyltransferases (H3K27me3). Despite the rapid development of new drugs inhibiting EZH2 and/or EZH1, the molecular interplay of these proteins and the impact on disease progression and prognosis of patients with T-cell lymphomas remains insufficiently understood. In this study, EZH1/2 mutation status was evaluated in 33 monomorphic epitheliotropic intestinal T-cell lymphomas by next generation sequencing and EZH1/2 and H3K27me3 protein expression levels were detected by immunohistochemistry in 46 T-cell lymphomas. Correlations with clinicopathologic features were analyzed and survival curves generated. No EZH1 mutations and one (3%) EZH2 missense mutation were identified. In univariable analysis, high EZH1 expression was associated with an improved overall survival (OS) and progression-free survival (PFS) whereas high EZH2 and H3K27me3 expression were associated with poorer OS and PFS. Multivariable analysis revealed EZH1 (hazard ratio (HR) = 0.183; 95% confidence interval (CI): 0.044-0.767; p = 0.020;) and EZH2 (HR = 8.245; 95% CI: 1.898-35.826; p = 0.005) to be independent, divergent prognostic markers for OS. In conclusion, EZH1/2 protein expression had opposing effects on the prognosis of T-cell lymphoma patients.

RBMX Protein Expression in T-Cell Lymphomas Predicts Chemotherapy Response and Prognosis.

T-cell non-Hodgkin's lymphomas (T-NHL) are a heterogeneous group of lymphomas with a mature T-cell phenotype. While in some hematological diseases the prognosis improved over the last decades, T-NHL cases often relapse early or present with an initially refractory course. Recently, it has been shown that RNA binding proteins have a crucial role for malignant tumor initiation, progression and treatment response while contributing to chemotherapy resistance. Therefore, we investigated the protein expression of the RNA binding protein X (RBMX), which has been shown to be of great relevance in disease initiation and progression in hematological diseases in 53 T-NHL cases using conventional immunohistochemistry. Low RBMX expression was associated with better response to anthracycline-containing first-line treatment. Furthermore, low RBMX expression predicted an improved overall survival and progression-free survival in univariate analysis. Multivariable Cox regression revealed RBMX as an independent prognostic marker for overall survival (p = 0.007; hazard ratio (HR) = 0.204; 95% confidence interval (CI): 0.064-0.646) and progression-free survival (p = 0.006; HR = 0.235; 95% CI: 0.083-0.666). The study identifies low RBMX expression to predict better chemotherapy response, overall survival and progression-free survival in patients with T-cell non-Hodgkin's lymphomas. These results suggest that RBMX protein expression levels might be a contributing factor towards chemotherapy resistance and thus affect prognosis. Hence, RBMX may be a potential therapeutic target and prognostic marker in T-cell lymphomas.

BRAF/EZH2 Signaling Represses miR-129-5p Inhibition of SOX4 Thereby Modulating BRAFi Resistance in Melanoma.

Many melanomas are associated with activating BRAF mutation. Targeted therapies by inhibitors of BRAF and MEK (BRAFi, MEKi) show marked antitumor response, but become limited by drug resistance. The mechanisms for this are not fully revealed, but include miRNA. Wishing to improve efficacy of BRAFi and knowing that certain miRNAs are linked to resistance to BRAFi, we wanted to focus on miRNAs exclusively associated with response to BRAFi. We found increased expression of miR-129-5p during BRAFi treatment of BRAF- mutant melanoma cells. Parallel to emergence of resistance we observed mir-129-5p expression to become suppressed by BRAF/EZH2 signaling. In functional analyses we revealed that miR-129-5p acts as a tumor suppressor as its overexpression decreased cell proliferation, improved treatment response and reduced viability of BRAFi resistant melanoma cells. By protein expression analyses and luciferase reporter assays we confirmed SOX4 as a direct target of mir-129-5p. Thus, modulation of the miR-129-5p-SOX4 axis could serve as a promising novel strategy to improve response to BRAFi in melanoma.

Litter chemistry prevails over litter consumers in mediating effects of past steel industry activities on leaf litter decomposition.

Soil pollution has adverse effects on the performance and life history traits of microorganisms, plants, and animals, yet evidence indicates that even the most polluted sites can support structurally-complex and dynamic ecosystems. The present study aims at determining whether and how litter decomposition, one of the most important soil ecological processes leaf, is affected in a highly trace-metal polluted site. We postulated that past steel mill activities resulting in soil pollution and associated changes in soil characteristics would influence the rate of litter decomposition through two non-exclusive pathways: altered litter chemistry and responses of decomposers to lethal and sub-lethal toxic stress. We carried out a litter-bag experiment using Populus tremula L. leaf litter collected at, and allowed to decompose in, a trace metal polluted site and in three unpolluted sites used as controls. We designed a fully-factorial transplant experimental design to assess effects of litter origin and exposure site on the rate of litter decomposition. We further determined initial litter chemistry, fungal biomass, mesofauna abundance in litter bags, and the soil macrofauna community. Irrespective of the site of litter exposure, litter originating from the polluted site had a two-fold faster decomposition than litter from the unpolluted sites. Litter chemistry, notably the lignin content, seemed most important in explaining the degradation rate of the leaf litter. Abundance of meso and macro-detritivores was higher at the polluted site than at the unpolluted sites. However, litter decomposition proceeded at similar rates in polluted and unpolluted sites. Our results show that trace metal pollution and associated soil and litter changes do not necessarily weaken consumer control on litter decomposition through lethal and sub-lethal toxic stress.

Induced defense mechanisms in an aquatic angiosperm to insect herbivory.

In terrestrial angiosperms, defense and resistance mechanisms against herbivores have been studied extensively; yet this topic is poorly understood in aquatic angiosperms. We investigated induced response mechanisms in Myriophyllum spicatum to the generalist insect herbivore Acentria ephemerella in three independent experiments. Various morphological and chemical response variables were examined in grazed apical shoots and compared to undamaged controls. We further estimated plant palatability of induced and non-induced apices in choice assays, and assessed the growth response of Acentria larvae in no-choice feeding assays. Leaves of induced apices were splayed out horizontally and changed in color from green to red. The dry matter content and thus plant toughness increased by up to 19%, but silica levels stayed constant. Induced apices exhibited a decline in chlorophyll content of up to 34%, reflected also by a 10% decrease in nitrogen levels, while nitrogen increased by 14% in lower parts of grazed shoots. Also, herbivore-deterring total phenolic compounds increased by up to 20% in apices. In choice trials, Acentria larvae strongly avoided grazed tips, and growth was reduced by 25% on induced apices. In total, we observed five different induced resistance and defensive traits in grazed apices: changes in appearance, increased plant toughness, delocalization of N-containing metabolites, increased polyphenols, and reduced nutritional value. The observed changes prevent herbivore damage and loss of apical tissue, which are most valuable for plant fitness. Our study presents the first evidence of multiple, parallel defense strategies including constitutive and induced defense mechanisms in a freshwater angiosperm.

Epiphytic bacterial community composition on two common submerged macrophytes in brackish water and freshwater.

BACKGROUND: Plants and their heterotrophic bacterial biofilm communities possibly strongly interact, especially in aquatic systems. We aimed to ascertain whether different macrophytes or their habitats determine bacterial community composition. We compared the composition of epiphytic bacteria on two common aquatic macrophytes, the macroalga Chara aspera Willd. and the angiosperm Myriophyllum spicatum L., in two habitats, freshwater (Lake Constance) and brackish water (Schaproder Bodden), using fluorescence in situ hybridization. The bacterial community composition was analysed based on habitat, plant species, and plant part. RESULTS: The bacterial abundance was higher on plants from brackish water [5.3 x 10(7) cells (g dry mass)-1] than on plants from freshwater [1.3 x 10(7) cells (g dry mass)-1], with older shoots having a higher abundance. The organic content of freshwater plants was lower than that of brackish water plants (35 vs. 58%), and lower in C. aspera than in M. spicatum (41 vs. 52%). The content of nutrients, chlorophyll, total phenolic compounds, and anthocyanin differed in the plants and habitats. Especially the content of total phenolic compounds and anthocyanin was higher in M. spicatum, and in general higher in the freshwater than in the brackish water habitat. Members of the Cytophaga-Flavobacteria-Bacteroidetes group were abundant in all samples (5-35% of the total cell counts) and were especially dominant in M. spicatum samples. Alphaproteobacteria were the second major group (3-17% of the total cell counts). Betaproteobacteria, gammaproteobacteria, and actinomycetes were present in all samples (5 or 10% of the total cell counts). Planctomycetes were almost absent on M. spicatum in freshwater, but present on C. aspera in freshwater and on both plants in brackish water. CONCLUSION: Bacterial biofilm communities on the surface of aquatic plants might be influenced by the host plant and environmental factors. Distinct plant species, plant part and habitat specific differences in total cell counts and two bacterial groups (CFB, planctomycetes) support the combined impact of substrate (plant) and habitat on epiphytic bacterial community composition. The presence of polyphenols might explain the distinct bacterial community on freshwater M. spicatum compared to that of M. spicatum in brackish water and of C. aspera in both habitats.

Gut pH, redox conditions and oxygen levels in an aquatic caterpillar: potential effects on the fate of ingested tannins.

Larvae of the freshwater moth Acentria ephemerella (Pyralidae, Lepidoptera) can fully develop on Myriophyllum spicatum, a submerged macrophyte containing 7-10% of its dry mass as tannins. We investigated the physicochemical gut parameters of larvae fed with M. spicatum or Potamogeton perfoliatus, a food plant lacking tannins, and the chemical fate of ingested polyphenols. Microelectrode studies revealed that larval midguts were slightly alkaline (pH 8) and had a positive redox potential. Whole guts were oxygen sinks owing to the oxygen demand of the gut contents. Oxygen penetrated the midgut up to 100 microm, but the centres of the foregut and midgut were always anoxic. The physicochemical parameters of the guts did not change with the food plant. The major tannin from M. spicatum, tellimagrandin II, was significantly depleted in the midgut and was not detected in faeces. In vitro studies indicated that tellimagrandin II is rapidly depleted mainly through oxidation, and hydrolysis might also occur. Our findings for A. ephemerella are compared with those for terrestrial Lepidoptera, and possible mechanisms for adaptations to tannin-rich food plants are discussed.

Polyphenolic allelochemicals from the aquatic angiosperm Myriophyllum spicatum inhibit photosystem II.

Myriophyllum spicatum (Haloragaceae) is a highly competitive freshwater macrophyte that produces and releases algicidal and cyanobactericidal polyphenols. Among them, beta-1,2,3-tri-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (tellimagrandin II) is the major active substance and is an effective inhibitor of microalgal exoenzymes. However, this mode of action does not fully explain the strong allelopathic activity observed in bioassays. Lipophilic extracts of M. spicatum inhibit photosynthetic oxygen evolution of intact cyanobacteria and other photoautotrophs. Fractionation of the extract provided evidence for tellimagrandin II as the active compound. Separate measurements of photosystem I and II activity with spinach (Spinacia oleracea) thylakoid membranes indicated that the site of inhibition is located at photosystem II (PSII). In thermoluminescence measurements with thylakoid membranes and PSII-enriched membrane fragments M. spicatum extracts shifted the maximum temperature of the B-band (S(2)Q(B)(-) recombination) to higher temperatures. Purified tellimagrandin II in concentrations as low as 3 microM caused a comparable shift of the B-band. This demonstrates that the target site of this inhibitor is different from the Q(B)-binding site, a common target of commercial herbicides like 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Measurements with electron paramagnetic resonance spectroscopy suggest a higher redox midpoint potential for the non-heme iron, located between the primary and the secondary quinone electron acceptors, Q(A) and Q(B). Thus, tellimagrandin II has at least two modes of action, inhibition of exoenzymes and inhibition of PSII. Multiple target sites are a common characteristic of many potent allelochemicals.

Impact of polyphenols on growth of the aquatic herbivore Acentria ephemerella.

Larvae of Acentria ephemerella live fully submerged, feeding on submersed aquatic angiosperms such as pondweeds (Potamogeton spp.) and Myriophyllum spicatum. Only the latter contains high concentrations of hydrolyzable tannins known to interfere with the growth of insect herbivores. We tested whether larvae grow faster on Potamogeton perfoliatus or M. spicatum and whether this is due to polyphenols in their food source. Larvae originating from the same egg clutch grew faster and larger on P. perfoliatus than on M. spicatum. The same growth response was observed with larvae that spent winter diapause on either P. perfoliatus or M. spicatum. These larvae were fed either with their host plant or the other macrophyte. No prior feeding effect was found, but growth of larvae reared on M. spicatum was less than when grown on P. perfoliatus. Larvae from another egg-clutch reared on M. spicatum, either from lake or cultivated in aquaria, exhibited reduced growth on the lake plants. P. perfoliatus contained less than 1% and M. spicatum (aquarium or field material) between 5 and 9% phenolic compounds. No differences in nitrogen content of leaves were found, but apical shoot sections of M. spicatum exhibited a significantly higher nitrogen content than P. perfoliatus. Our results indicate that hydrolyzable tannins are responsible for the reduced growth of Acentria when fed with M. spicatum.