Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney.

BACKGROUND: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. RESULTS: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. CONCLUSIONS: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.

Novel propargylamine-based inhibitors of cholinesterases and monoamine oxidases: Synthesis, biological evaluation and docking study.

A combination of several pharmacophores in one molecule has been successfully used for multi-target-directed ligands (MTDL) design. New propargylamine substituted derivatives combined with salicylic and cinnamic scaffolds were designed and synthesized as potential cholinesterases and monoamine oxidases (MAOs) inhibitors. They were evaluated invitro for inhibition of acetyl- (AChE) and butyrylcholinesterase (BuChE) using Ellman's method. All the compounds act as dual inhibitors. Most of the derivatives are stronger inhibitors of AChE, the best activity showed 5-bromo-N-(prop-2-yn-1-yl)salicylamide 1e (IC50 = 8.05 µM). Carbamates (4-bromo-2-[(prop-2-yn-1-yl)carbamoyl]phenyl ethyl(methyl)carbamate 2d and 2,4-dibromo-6-[(prop-2-yn-1-yl)carbamoyl]phenyl ethyl(methyl)carbamate 2e were selective and the most active for BuChE (25.10 and 26.09 µM). 4-Bromo-2-[(prop-2-yn-1-ylimino)methyl]phenol 4a was the most potent inhibitor of MAOs (IC50 of 3.95 and ≈10 µM for MAO-B and MAO-A, respectively) along with a balanced inhibition of both cholinesterases being a real MTDL. The mechanism of action was proposed, and binding modes of the hits were studied by molecular docking on human enzymes. Some of the derivatives also exhibited antioxidant properties. Insilico prediction of physicochemical parameters affirm that the molecules would be active after oral administration and able to reach brain tissue.

Hepatotoxicity of monoterpenes and sesquiterpenes.

Public interest in natural therapies has increased significantly over past decades. Herbs and herbal products are extensively consumed worldwide and they are generally considered as safe. However, this may not always be true as many cases of herb-induced liver injury are reported every year. The liver is a frequent target tissue of toxicity from all classes of toxicants as liver structure and function predispose it to high sensitivity to xenobiotics. The present review is focused on the hepatotoxic properties of monoterpenes and sesquiterpenes, plant secondary metabolites that represent the major components of essential oils wildly used in folk medicines, pharmaceutical industry and cosmetics. Most of these terpenes easily enter the human body by oral absorption, penetration through the skin, or inhalation leading to measurable blood concentrations. Several studies showed that some monoterpenes (e.g., pulegone, menthofuran, camphor, and limonene) and sesquiterpenes (e.g., zederone, germacrone) exhibited liver toxicity, which is mainly based on reactive metabolites formation, increased concentration of reactive oxygen species and impaired antioxidant defense. There is a high probability that many other terpenes, without sufficiently known metabolism and effects in human liver, could also exert hepatotoxicity. Especially terpenes, that are important components of essential oils with proved hepatotoxicity, should deserve more attention. Intensive research in terpenes metabolism and toxicity represent the only way to reduce the risk of liver injury induced by essential oils and other terpenes-containing products.

The impact of sesquiterpenes ß-caryophyllene oxide and trans-nerolidol on xenobiotic-metabolizing enzymes in mice in vivo.

1. Sesquiterpenes, constituents of plant essential oil, are popular bioactive compounds due to the positive effect on human health, but their potential toxicity and possible herb-drug interactions are often omitted. In our in vivo study, we followed up the effect of p.o. administration of two sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) on various xenobiotic-metabolizing enzymes in mice liver and small intestine. 2. To spot the early effect of studied compounds, enzymatic activity and mRNA levels were assessed 6 and 24 h after single dose. 3. CAO and NER markedly increased cytochromes P450 (CYP2B, 3A, 2C) activity and mRNA levels in both tissues. Liver also showed elevated activity of aldo-ketoreductase 1C and carbonyl reductase after treatment. Contrary, sesquiterpenes decreased NAD(P)H:quinone oxidoreductase 1 activity in small intestine. Among conjugation enzymes, only liver sulfotransferase activity was increased by sesquiterpenes. 4. Our results document that single dose of sesquiterpenes modulate activities and expression of several xenobiotic-metabolizing enzymes.

The Effects of Selected Sesquiterpenes from Myrica rubra Essential Oil on the Efficacy of Doxorubicin in Sensitive and Resistant Cancer Cell Lines.

ß-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER) and valencene (VAL) are constituents of the essential oil of Myrica rubra (MEO), which has significant antiproliferative effect in various cancer cell lines. In the present study, we compared the antiproliferative effect of these sesquiterpenes alone and in combination with the cytostatic drug doxorubicin (DOX) in cancer cell lines with different sensitivity to DOX. Two ovarian cancer cell lines (sensitive A2780 and partly resistant SKOV3) and two lymphoblast cancer cell lines (sensitive CCRF/CEM and completely resistant CEM/ADR) were used. The observed effects varied among sesquiterpenes and also differed in individual cell lines, with only VAL being effective in all the cell lines. A strong synergism of DOX with NER was found in the A2780 cells, while DOX acted synergistically with HUM and CAO in the SKOV3 cells. In the CCRF/CEM cells, a synergism of DOX with CAO and NER was observed. In resistant CEM/ADR cells, sesquiterpenes did not increase DOX efficacy, although they significantly increased accumulation of DOX (up to 10-times) and rhodamine-123 (substrate of efflux transporter ABCB1) within cancer cells. In conclusion, the tested sesquiterpenes were able to improve DOX efficacy in the sensitive and partly resistant cancer cells, but not in cells completely resistant to DOX.

Nerolidol and Farnesol Inhibit Some Cytochrome P450 Activities but Did Not Affect Other Xenobiotic-Metabolizing Enzymes in Rat and Human Hepatic Subcellular Fractions.

Sesquiterpenes, 15-carbon compounds formed from three isoprenoid units, are the main components of plant essential oils. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. The aim of our study was to test and compare the potential inhibitory effect of acyclic sesquiterpenes, trans-nerolidol, cis-nerolidol and farnesol, on the activities of the main xenobiotic-metabolizing enzymes in rat and human liver in vitro. Rat and human subcellular fractions, relatively specific substrates, corresponding coenzymes and HPLC, spectrophotometric or spectrofluorometric analysis of product formation were used. The results showed significant inhibition of cytochromes P450 (namely CYP1A, CYP2B and CYP3A subfamilies) activities by all tested sesquiterpenes in rat as well as in human hepatic microsomes. On the other hand, all tested sesquiterpenes did not significantly affect the activities of carbonyl-reducing enzymes and conjugation enzymes. The results indicate that acyclic sesquiterpenes might affect CYP1A, CYP2B and CYP3A mediated metabolism of concurrently administered drugs and other xenobiotics. The possible drug-sesquiterpene interactions should be verified in in vivo experiments.

Essential Oil from Myrica rubra Leaves Potentiated Antiproliferative and Prooxidative Effect of Doxorubicin and its Accumulation in Intestinal Cancer Cells.

Essential oil from the leaves of Myrica rubra, a subtropical Asian fruit tree traditionally used in folk medicines, has a significant antiproliferative effect in several intestinal cancer cell lines. Doxorubicin belongs to the most important cytostatics used in cancer therapy. The present study was designed to evaluate the effects of defined essential oil from M. rubra leaves on efficacy, prooxidative effect, and accumulation of doxorubicin in cancer cell lines and in non-cancerous cells. For this purpose, intestinal adenocarcinoma CaCo2 cells were used. Human fibroblasts (periodontal ligament) and a primary culture of rat hepatocytes served as models of non-cancerous cells. The results showed that the sole essential oil from M. rubra has a strong prooxidative effect in cancer cells while it acts as a mild antioxidant in hepatocytes. Combined with doxorubicin, the essential oil enhanced the antiproliferative and prooxidative effects of doxorubicin in cancer cells. At higher concentrations, synergism of doxorubicin and essential oil from M. rubra was proved. In non-cancerous cells, the essential oil did not affect the toxicity of doxorubicin and the doxorubicin-mediated reactive oxygen species formation. The essential oil increased the intracellular concentration of doxorubicin and enhanced selectively the doxorubicin accumulation in nuclei of cancer cells. Taken together, essential oil from M. rubra leaves could be able to improve the doxorubicin efficacy in cancer cells due to an increased reactive oxygen species production, and the doxorubicin accumulation in nuclei of cancer cells.

The Influence of Sesquiterpenes from Myrica rubra on the Antiproliferative and Pro-Oxidative Effects of Doxorubicin and Its Accumulation in Cancer Cells.

The sesquiterpenes ß-caryophyllene, ß-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER), and valencene (VAL) are substantial components of the essential oil from Myrica rubra leaves which has exhibited significant antiproliferative effects in several intestinal cancer cell lines, with CaCo-2 cells being the most sensitive. The present study was designed to evaluate the effects of these sesquiterpenes on the efficacy and toxicity of the anticancer drug doxorubicin (DOX) in CaCo-2 cancer cells and in primary culture of rat hepatocytes. Our results showed that HUM, NER, VAL and CAO inhibited proliferation of CaCo-2 cancer cells but they did not affect the viability of hepatocytes. CAO, NER and VAL synergistically potentiated the efficacy of DOX in cancer cells killing. All sesquiterpenes exhibited the ability to selectively increase DOX accumulation in cancer cells and did not affect DOX concentration in hepatocytes. Additionally, CAO and VAL were able to increase the pro-oxidative effect of DOX in CaCo-2 cells. Moreover, CAO mildly ameliorated DOX toxicity in hepatocytes. Based on all results, CAO seems to be the most promising compound for further testing.

The induction and inhibition of UDP-glycosyltransferases in Haemonchus contortus and their role in the metabolism of albendazole.

Albendazole (ABZ) is an anthelmintic frequently used to treat haemonchosis, a common parasitosis of ruminants caused by the gastrointestinal nematode Haemonchus contortus. This parasite is able to protect itself against ABZ via the formation of inactive ABZ-glycosides. The present study was designed to deepen the knowledge about the role of UDP-glycosyltransferases (UGTs) in ABZ glycosylation in H. contortus. The induction effect of phenobarbital, a classical inducer of UGTs, as well as ABZ and ABZ-sulphoxide (ABZSO, the main active metabolite of ABZ) on UGTs expression and UGT activity toward ABZ was studied ex vivo in isolated adult nematodes. The effect of three potential UGT inhibitors (5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine and sulfinpyrazone) on ABZ glycosylation was tested. Pre-incubation of nematodes with ABZ and ABZSO led to increased expression of several UGTs as well as ABZ-glycosides formation in subsequent treatment. Phenobarbital also induced UGTs expression, but did not affect ABZ biotransformation. In the nematode's subcellular fraction, sulfinpyrazone inhibited UGT activity toward ABZ, although no effect of other inhibitors was observed. The inhibitory potential of sulfinpyrazone on the formation of ABZ-glycosides was also proved ex vivo in living nematodes. The obtained results confirmed the role of UGTs in ABZ biotransformation in H. contortus adults and revealed sulfinpyrazone as a potent inhibitor of ABZ glycosylation in this parasite. The possible use of sulfinpyrazone with ABZ in combination therapy merits further research.

Exploring Natura 2000 habitats by satellite image segmentation combined with phytosociological data: a case study from the Cierny Balog area (Central Slovakia).

Natura 2000 is a network of protected areas covering Europe's most valuable and threatened species and habitats. Recently, biota belonging to these networks have been threatened by both climate change and various human impacts. Regular monitoring is needed to ensure effective protection and proper management measures in these sites and habitats, but conventional field approaches are often time-consuming and inaccurate. New approaches and studies with different focuses and results are being developed. Our approach includes point data from field research and phytosociological databases as starting points for automatic segmentation, which has been developed just recently as a novel method that could help to connect ground-based and remote sensing data. Our case study is located in Central Slovakia, in the mountains around the village of Cierny Balog. The main aim of our case study is to apply advanced remote sensing techniques to map the area and condition of vegetation units. We focus on forest habitats belonging mainly to the Natura 2000 network. We concentrated on the verification of the possibilities of differentiation of various habitats using only multispectral Sentinel-2 satellite data. Our software NaturaSat created by our team was used to reach our objectives. After collecting data in the field using phytosociological approach and segmenting the explored areas in the program NaturaSat, spectral characteristics were calculated within identified habitats using software tools, which were subsequently processed and tested statistically. We obtained significant differences between forest habitat types. Also, segmentation accuracy was tested by comparing closed planar curves of ground based filed data and software results. This provided promising results and validation of the methods used. The results of this study have the potential to be used in a wider area to map the occurrence and quality of Natura 2000 habitats.

Environmental circulation of the anthelmintic drug albendazole affects expression and activity of resistance-related genes in the parasitic nematode Haemonchus contortus.

Veterinary anthelmintics excreted from treated animals pass to soil, subsequently to plants and then to their consumers. This circulation might have various consequences, including drug-resistance promotion in helminths. The present study was designed to follow the effect of the environmental circulation of the common anthelmintic drug albendazole (ABZ) in real farm conditions on the parasitic nematode Haemonchus contortus in vivo. Two fields with fodder plants (clover and alfalfa) were fertilized, the first with dung from ABZ-treated sheep (at the recommended dosage), the second with dung from non-treated sheep (controls). After a 10-week growth period, the fresh fodder from both fields was used to feed two groups of sheep, which were infected with H. contortus. Eggs and adult nematodes from the animals of both groups were isolated, and various parameters were compared. No significant changes in the eggs' sensitivity to ABZ and thiabendazole were observed. However, significantly increased expression of several cytochromes P450 and UDP-glycosyl transferases as well as increased oxidation and glycosylation of ABZ and ABZ-sulfoxide (ABZ-SO) was found in the exposed nematodes. These results show that ABZ environmental circulation improves the ability of the helminths to deactivate ABZ.

MicroRNAs mediated regulation of glutathione peroxidase 7 expression and its changes during adipogenesis.

Glutathione peroxidase 7 (GPx7) acts as an intracellular stress sensor/transmitter and plays an important role in adipocyte differentiation and the prevention of obesity related pathologies. For this reason, finding the regulatory mechanisms that control GPx7 expression is of great importance. As microRNAs (miRNAs) could participate in the regulation of GPx7 expression, we studied the inhibition of GPx7 expression by four selected miRNAs with relation to obesity and adipogenesis. The effect of the transfection of selected miRNAs mimics on GPx7 expression was tested in three cell models (HEK293, SW480, AT-MSC). The interaction of selected miRNAs with the 3'UTR of GPx7 was followed up on using a luciferase gene reporter assay. In addition, the levels of GPx7 and selected miRNAs in adipose tissue mesenchymal stem cells (AT-MSC) and mature adipocytes from four human donors were compared, with the changes in these levels during adipogenesis analyzed. Our results show for the first time that miR-137 and miR-29b bind to the 3'UTR region of GPx7 and inhibit the expression of this enzyme at the mRNA and protein level in all the human cells tested. However, no negative correlation between miR-137 nor miR-29b level and GPx7 was observed during adipogenesis. Despite the confirmed inhibition of GPx7 expression by miR-137 and miR-29b, the action of these two molecules in adipogenesis and mature adipocytes must be accompanied by other regulators.

Proof of the environmental circulation of veterinary drug albendazole in real farm conditions.

Anthelmintics, drugs against parasitic worms, are frequently used in livestock and might act as danger environmental microcontaminants. The present study was designed to monitor the possible circulation of common anthelmintic drug albendazole (ABZ) and its metabolites in the real agriculture conditions. The sheep were treated with the recommended dose of ABZ. Collected faeces were used for the fertilization of a field with fodder plants (alfalfa and clover) which served as feed for sheep from a different farm. The selective ultrasensitive mass spectrometry revealed surprisingly high concentrations of active ABZ metabolite (ABZ-sulphoxide) in all samples (dung, plants, ovine plasma, rumen content and faeces). Our results prove for the first time an undesirable permeation of ABZ metabolites from sheep excrement into plants (used as fodder) and subsequently to other sheep in real agricultural conditions. This circulation causes the permanent exposition of the ecosystems and food-chain to the drug and can promote the development of drug resistance in helminths.

Antiproliferative Effects of Hop-derived Prenylflavonoids and Their Influence on the Efficacy of Oxaliplatine, 5-fluorouracil and Irinotecan in Human ColorectalC Cells.

Beer, the most popular beverage containing hops, is also frequently consumed by cancer patients. Moreover, non-alcoholic beer, owing to its nutritional value and high content of biological active compounds, is sometimes recommended to patients by oncologists. However, the potential benefits and negatives have to date not been sufficiently evaluated. The present study was designed to examine the effects of four main hop-derived prenylflavonoids on the viability, reactive oxygen species (ROS) formation, activity of caspases, and efficiency of the chemotherapeutics 5-fluorouracil (5-FU), oxaliplatin (OxPt) and irinotecan (IRI) in colorectal cancer cell lines SW480, SW620 and CaCo-2. All the prenylflavonoids exerted substantial antiproliferative effects in all cell lines, with xanthohumol being the most effective (IC50 ranging from 3.6 to 7.3 µM). Isoxanthohumol increased ROS formation and the activity of caspases-3/7, but 6-prenylnaringenin and 8-prenylnaringenin exerted antioxidant properties. As 6-prenylnaringenin acted synergistically with IRI, its potential in combination therapy deserves further study. However, other prenylflavonoids acted antagonistically with all chemotherapeutics at least in one cell line. Therefore, consumption of beer during chemotherapy with 5-FU, OxPt and IRI should be avoided, as the prenylflavonoids in beer could decrease the efficacy of the treatment.

The Selection and Validation of Reference Genes for mRNA and microRNA Expression Studies in Human Liver Slices Using RT-qPCR.

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with ß-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and ß-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

High-fructose drinks affect microRNAs expression differently in lean and obese mice.

High fructose intake from soft drinks and sweets is assumed to have a negative impact on human health. Yet in spite of intensive research, the molecular mechanisms of these effects have not been fully elucidated yet, for example, the effect of high fructose intake could be different in normal and obese individuals. Four groups of mice were used in this study: control groups of lean mice and mice with obesity induced by a high-fat diet, then both of these groups with or without fructose administration in drinks. In plasma of each group, triacylglycerol, cholesterol, free fatty acids, alanine aminotransferase, insulin and adiponectin were measured. The expression levels of selected microRNAs (miRNAs) in plasma, the liver, white adipose tissue, brown adipose tissue and subcutaneous adipose tissue were quantified. In both lean and obese mice, high fructose intake increased cholesterol amount in the liver, up-regulated hepatic miR-27a, down-regulated miR-33a in white adipose tissue and increased plasmatic level of miR-21. The effect of high fructose intake on other miRNAs in the liver, plasma and adipose tissues differed in normal and obese mice. Fructose intake led to hepatic hypercholesterolemia and aberrant expression of several miRNAs participating in lipid metabolism, adipocytes differentiation and nonalcoholic fatty liver disease promotion. The effect of fructose on miRNAs expression differed in normal and obese mice. Nevertheless, plasmatic miR-21, which was induced by fructose in both lean and obese mice, may be considered as a potential biomarker of excessive fructose intake.

Sesquiterpenes α-humulene and ß-caryophyllene oxide enhance the efficacy of 5-fluorouracil and oxaliplatin in colon cancer cells.

The present study is designed to find out if sesquiterpenes, α-humulene (HUM), valencene (VAL), ß-caryphyllene-oxide (CAO) and trans-nerolidol (NER), are able to improve the antiproliferative effect of classical cytostatic drugs, 5-fluorouracil (FU) and oxaliplatin (1,2-diaminocyclohexaneoxalato-platinum, OxPt), in colon cancer cell lines Caco-2 and SW-620. In addition, the possible mechanisms of sesquiterpene action are studied. The results show significant ability of HUM and especially of CAO to enhance the anti-proliferative effects of FU and OxPt in cancer cell lines Caco-2 and SW-620. On the other hand, VAL and NER are ineffective. The action of CAO could be partly based on its ability to disrupt the mitochondrial membrane potential and to activate initiator caspases, but other mechanisms are probably also involved. Based on these results, CAO seems to have the potential for combination therapy of colon cancers and deserves further study.

Profiling microRNAs through development of the parasitic nematode Haemonchus identifies nematode-specific miRNAs that suppress larval development.

Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.

The metabolism of flubendazole in human liver and cancer cell lines.

Flubendazole (FLU), a benzimidazole anthelmintic drug widely used in veterinary medicine, has been approved for the treatment of gut-residing nematodes in humans. In addition, FLU is now considered a promising anti-cancer agent. Despite this, information about biotransformation of this compound in human is lacking. Moreover, there is no information regarding whether cancer cells are able to metabolize FLU in order to deactivate it. For these reasons, the present study was designed to identify all metabolites of Phase I and Phase II of FLU in human liver and in various cancer cells using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Precision-cut human liver slices and 9 cell lines of different origin (breast, colon, oral cavity) were used as in vitro model systems. Our study showed that FLU with a reduced carbonyl group (FLUR) is the only FLU metabolite formed in the human liver. All human cancer cell lines were able to form FLUR. In addition, methylated FLUR was detected in breast cells MCF7 and intestinal SW480 cells. The accumulation of FLU and its reduction to FLUR markedly differed among cells. The extent of FLU reduction was in a good correlation with the detected expression level of carbonyl reductase 1. In most cases, FLU entered in a higher amount and was reduced to a lesser extent in proliferating (metastatic) cells than in differentiated (non-cancerous, non-metastatic) ones. These results support the promising potential of FLU in anti-cancer therapy.

The effects of ß-caryophyllene oxide and trans-nerolidol on the efficacy of doxorubicin in breast cancer cells and breast tumor-bearing mice.

BACKGROUND: One approach to improve effect of chemotherapy is combination of classical cytostatic drugs with natural compounds, e. g. sesquiterpenes. In our previous study, sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) improved the anti-proliferative effect of doxorubicin (DOX) in intestinal cancer cell lines. PURPOSE: The present study was designed to evaluate the effect of CAO and NER on DOX efficacy, focusing on cell proliferation, migration, apoptosis and DOX accumulation in breast cancer cells MDA-MB-231 and MCF7 in vitro and in mice bearing solid Ehrlich tumors (EST) in vivo. METHODS: The impact of cytotoxic effect was assessed by the neutral red uptake test. The ability to migrate was tested using real-time measurement in x-CELLigence system. Expressions of molecules were examined using western blot analysis. The accumulation of DOX inside the cells using time lapse microscopy was observed. The mice with inoculated EST cells were treated repeatedly with DOX and DOX+CAO or DOX+NER and the growth of tumors were monitored. DOX concentrations in plasma and tumor were assayed using HPLC. RESULTS: In MDA-MB-231, combination of DOX with CAO enhanced anti-proliferative effect and acted strongly synergistic. NER increased accumulation of DOX inside the cells; moreover combination DOX with NER suppressed migration ability in vitro. In vivo, apoptosis was activated especially in group treated with DOX and CAO. However, none of tested sesquiterpenes was able to improve DOX accumulation in tumors and DOX-mediated inhibition of tumor growth. CONCLUSION: In conclusion, sesquiterpenes CAO and NER increased the efficacy of DOX in breast cancer cells in vitro, but did not improve its effect in vivo, in Ehrlich solid tumor bearing mice.