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.

The role of UDP-glycosyltransferases in xenobioticresistance.

Uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) are an enzyme superfamily that catalyzes glycosyl residues transfer from activated nucleotide sugars to acceptor molecules. In addition to various endogenous compounds, numerous xenobiotics are substrates of UGTs. As the glycosides formed are generally less active/toxic and more hydrophilic than aglycones, UGTs effectively protect organisms from potentially harmful xenobiotics. Therefore, increased UGT expression and/or activity improve the protection of the organism and may contribute to the development of individuals that become more resistant to certain xenobiotics. While the function of UGTs in the resistance of human cancer cells to chemotherapy is now well known, other organisms and other xenobiotics have attracted much less attention. This review was designed to fill this knowledge gap by presenting complex information about the role of UGTs in xenobiotic-resistance in various organisms. This summarization and evaluation of the available information reveals that UGTs play an important role in defense against xenobiotics not only in humans, but in countless other organisms such as parasites, insects, and plants. Moreover, many recent studies clearly show the participation of UGTs in the resistance of nematodes to anthelmintics, insects to insecticides, weeds to herbicides as well as humans to various drugs (not only those used in cancer therapy but also in the treatment of epilepsy, psychiatric disorders, hypertension, hypercholesterolemia, and HIV infection). Nevertheless, although the contribution of UGTs to xenobiotic resistance in diverse organisms has become obvious, many pieces of information remain missing, for example with regard to the mechanisms of UGT regulation.

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.

The Modulation of Phase II Drug-Metabolizing Enzymes in Proliferating and Differentiated CaCo-2 Cells by Hop-Derived Prenylflavonoids.

Prenylflavonoids in the human organism exhibit various health-beneficial activities, although they may interfere with drugs via the modulation of the expression and/or activity of drug-metabolizing enzymes. As intestinal cells are exposed to the highest concentrations of prenylflavonoids, we decided to study the cytotoxicity and modulatory effects of the four main hop-derived prenylflavonoids on the activities and mRNA expression of the main drug-conjugating enzymes in human CaCo-2 cells. Proliferating CaCo-2 cells were used for these purposes as a model of colorectal cancer cells, and differentiated CaCo-2 cells were used as an enterocyte-like model. All the tested prenylflavonoids inhibited the CaCo-2 cells proliferation, with xanthohumol proving the most effective (IC50 8.5 µM). The prenylflavonoids modulated the activities and expressions of the studied enzymes to a greater extent in the differentiated, as opposed to the proliferating, CaCo-2 cells. In the differentiated cells, all the prenylflavonoids caused a marked increase in glutathione S-transferase and catechol-O-methyltransferase activities, while the activity of sulfotransferase was significantly inhibited. Moreover, the prenylflavonoids upregulated the mRNA expression of uridine diphosphate (UDP)-glucuronosyl transferase 1A6 and downregulated that of glutathione S-transferase 1A1/2.

The Hepatotoxicity of Alantolactone and Germacrone: Their Influence on Cholesterol and Lipid Metabolism in Differentiated HepaRG Cells.

The sesquiterpenes alantolactone (ATL) and germacrone (GER) are potential anticancer agents of natural origin. Their toxicity and biological activity have been evaluated using the differentiated HepaRG (dHepaRG) cells, a hepatocyte-like model. The half-maximal inhibitory concentrations of cell viability after 24-h treatment of dHepaRG cells are approximately 60 µM for ATL and 250 µM for GER. However, both sesquiterpenes induce reactive oxygen species (ROS) formation in non-toxic concentrations and significantly dysregulate the mRNA expression of several functional markers of mature hepatocytes. They similarly decrease the protein level of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and their transcription target, intercellular adhesion molecule 1 (ICAM-1). Based on the results of a BATMAN-TCM analysis, the effects of sesquiterpenes on cholesterol and lipid metabolism were studied. Sesquiterpene-mediated dysregulation of both cholesterol and lipid metabolism was observed, during which these compounds influenced the protein expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol regulatory element-binding protein 2 (SREBP-2), as well as the mRNA expression of HMGCR, CYP19A1, PLIN2, FASN, SCD, ACACB, and GPAM genes. In conclusion, the two sesquiterpenes caused ROS induction at non-toxic concentrations and alterations in cholesterol and lipid metabolism at slightly toxic and toxic concentrations, suggesting a risk of liver damage if administered to humans.

Sesquiterpenes Are Agonists of the Pregnane X Receptor but Do Not Induce the Expression of Phase I Drug-Metabolizing Enzymes in the Human Liver.

Sesquiterpenes, the main components of plant essential oils, are bioactive compounds with numerous health-beneficial activities. Sesquiterpenes can interact with concomitantly administered drugs due to the modulation of drug-metabolizing enzymes (DMEs). The aim of this study was to evaluate the modulatory effects of six sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, ß-caryophyllene, and caryophyllene oxide) on the expression of four phase I DMEs (cytochrome P450 3A4 and 2C, carbonyl reductase 1, and aldo-keto reductase 1C) at both the mRNA and protein levels. For this purpose, human precision-cut liver slices (PCLS) prepared from 10 patients and transfected HepG2 cells were used. Western blotting, quantitative real-time PCR and reporter gene assays were employed in the analyses. In the reporter gene assays, all sesquiterpenes significantly induced cytochrome P450 3A4 expression via pregnane X receptor interaction. However in PCLS, their effects on the expression of all the tested DMEs at the mRNA and protein levels were mild or none. High inter-individual variabilities in the basal levels as well as in modulatory efficacy of the tested sesquiterpenes were observed, indicating a high probability of marked differences in the effects of these compounds among the general population. Nevertheless, it seems unlikely that the studied sesquiterpenes would remarkably influence the bioavailability and efficacy of concomitantly administered drugs.

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.

Sulforaphane Alters ß-Naphthoflavone-Induced Changes in Activity and Expression of Drug-Metabolizing Enzymes in Rat Hepatocytes.

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, exerts many beneficial effects on human health such as antioxidant, anti-inflammatory, and anticancer effects. The effect of SFN alone on drug-metabolizing enzymes (DMEs) has been investigated in numerous in vitro and in vivo models, but little is known about the effect of SFN in combination with cytochrome P450 (CYP) inducer. The aim of our study was to evaluate the effect of SFN on the activity and gene expression of selected DMEs in primary cultures of rat hepatocytes treated or non-treated with ß-naphthoflavone (BNF), the model CYP1A inducer. In our study, SFN alone did not significantly alter the activity and expression of the studied DMEs, except for the glutathione S-transferase (GSTA1) mRNA level, which was significantly enhanced. Co-treatment of hepatocytes with SFN and BNF led to a substantial increase in sulfotransferase, aldoketoreductase 1C, carbonylreductase 1 and NAD(P)H:quinone oxidoreductase 1 activity and a marked decrease in cytochrome P450 (CYP) Cyp1a1, Cyp2b and Cyp3a4 expression in comparison to the treatment with BNF alone. Sulforaphane is able to modulate the activity and/or expression of DMEs, thus shifting the balance of carcinogen metabolism toward deactivation, which could represent an important mechanism of its chemopreventive activity.

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.

Influence of diet supplementation with green tea extract on drug-metabolizing enzymes in a mouse model of monosodium glutamate-induced obesity.

PURPOSE: Consumption of dietary supplements with green tea extract (GTE) is popular for weight management, but it may be accompanied by various side effects, including interactions with drugs. The aim of the present in vivo study was to evaluate the effect of defined GTE (Polyphenon 60) in three dosage schemes on insulin, leptin and drug-metabolizing enzymes in obese mice. METHODS: Experimental obesity was induced by repeated s.c. application of monosodium glutamate to newborn mice. Green tea extract was administered in three dosage schemes in chow diet. The plasmatic levels of insulin and leptin were assayed using enzyme-linked immunosorbent assay. Enzyme activities and mRNA expressions of drug-metabolizing enzymes (totally 13) were analyzed in liver and small intestine using spectrophotometric and HPLC assays and RT-PCR, respectively. RESULTS: GTE-treatment decreased insulin and leptin levels. Eleven enzymes were significantly affected by GTE-treatment. Long-term administration of 0.01% GTE caused increase in the activity and mRNA level of cytochrome P450 3A4 (CYP3A4) ortholog in the liver as well as in the small intestine. Interestingly, short-term overdose by GTE (0.1%) had more pronounced effects on enzyme activities and mRNA expressions than long-term overdose. CONCLUSIONS: GTE-mediated induction of CYP3A4 ortholog, the main drug-metabolizing enzyme, could result in decreased efficacy of simultaneously or subsequently administered drug in obese individuals.

The modulation of carbonyl reductase 1 by polyphenols.

Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1.

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.

Effect of oral administration of green tea extract in various dosage schemes on oxidative stress status of mice in vivo.

Green tea is a favorite beverage and its extracts are popular components of dietary supplements. The aim of the present in vivo study was to obtain detailed information about the effect of a standard green tea extract (Polyphenon, P), at different doses, on antioxidant enzymes and oxidative stress markers in murine blood, liver, small and large intestine. In all doses, P improved the oxidative stress status via an increased content of plasmatic SH-groups (by 21-67 %). Regarding antioxidant enzymes in tissues, the low dose of P had the best positive effect as it elevated the activity of NADPH quinone reductase in liver and small intestine, thioredoxin reductase in small intestine and hepatic superoxide dismutase. Based on these facts, consumption of green tea seems to be safe and beneficial, while consumption of dietary supplements containing high doses of catechins may disturb oxidative balance by lowering the activity of thioredoxin reductase, glutathione S-transferase, glutathione reductase and superoxide dismutase.

Antioxidant, pro-oxidant and other biological activities of sesquiterpenes.

Sesquiterpenes, 15-carbon compounds formed from 3 isoprenoid units, are secondary metabolites produced mainly in higher plants but also in fungi and invertebrates. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. Moreover, sesquiterpenes could become a rich reservoir of candidate compounds for drug discovery as several sesquiterpenes and their derivatives possess interesting biological activities. Recent efforts in the research and development of new drugs derived from natural products have led to the identification of a variety of sesquiterpenes that possess promising anti-inflammatory, antiparasitic and anti-carcinogenic activities. On the other hand, some sesquiterpenes can cause serious toxicity and other adverse effects. Therefore, more and more attention has been paid to the investigation of the mechanisms of biological activities of sesquiterpenes in vitro as well as in vivo. The data collected in this review show that many of sesquiterpenes biological activities are based on antioxidant or pro-oxidant actions of sesquiterpenes. Structure, concentration, metabolism as well as type of cells determine if sesquiterpene acts as anti-oxidant or pro-oxidant. Therefore, detailed research of sesquiterpenes is very important for evaluation of their efficacy and for their safe use.

Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.

Effect of selected catechins on doxorubicin antiproliferative efficacy and hepatotoxicity in vitro.

Catechins may influence both desirable and undesirable effects of many drugs. In this study, the in vitro effect of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epigallocatechin gallate (EGCG) on the efficacy of anticancer drug doxorubicin (DOX) was studied in HCT-8 cancer cells. Rat hepatocytes were used to study the influence of EGCG on DOX hepatotoxicity. Cell proliferation and viability were studied by 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide and neutral red uptake test assays. Formation of reactive oxygen species (ROS) was determined using the dichlorofluorescein assay. All of the studied catechins (1-25 µmol L(-1)) had no effect on the proliferation of intestinal cancer cells and did not affect the antiproliferative effect of DOX (1-8 µmol L(-1)) in these cells. Moreover, EGCG at 25 µmol L(-1) increased the viability of isolated hepatocytes and significantly protected these cells against DOX-induced toxicity and ROS production. Consumption of EGCG during DOX therapy seems to be safe and beneficial, since EGCG does not decrease DOX anticancer efficacy and could ameliorate DOX hepatotoxicity.

In vitro anti-proliferative and anti-inflammatory activity of leaf and fruit extracts from Vaccinium bracteatum Thunb.

The aim of this study was to evaluate in vitro anti-proliferative (tested on MCF-7, MDA-MB-231, and MCF-10A cell lines) and anti-inflammatory (evaluated as inhibition of prostaglandin E2 synthesis catalyzed by cyclooxygenase-2) effect of various extracts from Vaccinium bracteatum leaves and fruits. The highest anti-proliferative effect possessed leaf dichloromethane extract with IC50 values ranging from 93 to 198 µg/mL. In the case of cyclooxygenase-2 inhibition, n-hexane, dichloromethane, and ethanol fruit extracts showed the best activity with IC50 values = 2.0, 5.4, and 12.7 µg/mL, respectively. These results indicate that V. bracteatum leaves and fruits could be useful source of anti-cancer and anti-inflammatory compounds.

In vivo effect of oracin on doxorubicin reduction, biodistribution and efficacy in Ehrlich tumor bearing mice.

BACKGROUND: The limitation of carbonyl reduction represents one possible way to increase the effectiveness of anthracycline doxorubicin (DOX) in cancer cells and decrease its toxicity in normal cells. In vitro, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased the antiproliferative effect of DOX in MCF-7 breast cancer cells. Moreover, ORC significantly decreases DOX toxicity in non-cancerous MCF-10A breast cells and in hepatocytes. The present study was designed to test in mice the in vivo effect of ORC on plasma and tissue concentrations of DOX and its main metabolite DOXOL. The effect of ORC on DOX efficacy in mice bearing solid Ehrlich tumors (EST) was also studied. METHODS: DOX and DOX + ORC combinations were iv administered to healthy mice. Blood samples, livers and hearts were collected during the following 48 h. DOX and DOXOL concentrations were assayed using HPLC. The mice with inoculated EST cells were treated repeatedly iv with DOX and DOX + ORC combinations, and the growth of tumors was monitored. RESULTS: ORC in combination with DOX significantly decreased DOXOL plasma concentrations during four hours after administration, but this significantly affected neither DOX plasma concentrations nor DOX or DOXOL concentrations in the liver and heart at any of intervals tested. In EST bearing mice, ORC did not significantly affect DOX efficacy on tumor growth. However, EST was shown to be an improper model for the testing of ORC efficacy in vivo, as ORC did not inhibit DOXOL formation in EST. CONCLUSIONS: In vivo, ORC was able to retard DOXOL formation but was not able to improve DOX efficacy in EST-bearing mice.

Possibilities to increase the effectiveness of doxorubicin in cancer cells killing.

Anthracycline antibiotic doxorubicin (DOX) belongs among the most important antineoplastics used in cancer therapy. Unfortunately, its cytostatic effect in therapeutic doses is frequently insufficient; but the use of higher DOX doses is limited by the development of systemic toxicity, especially cardiotoxicity. Therefore, a searching for some possibilities of how to increase DOX efficacy in cancer cells, and minimizing associated toxicities to noncancerous tissues, is in the forefront of scientific research. Many approaches are based on altered DOX metabolism. The classical strategies include an enhancing of DOX uptake by cancer cells and/or an activation of DOX prodrug within cancer cells via liposomal encapsulation or conjugation with antibodies, peptides, or synthetic polymers. The diminishing of DOX deactivation, restriction of DOX efflux from cancer cells, decreased antioxidant defense of cancer cells, changes in cell cycle, or modulation of signaling pathways represent newer approaches in increasing DOX toxicity in tumors. Each way has certain advantages and limitations. The aim of this review was not to collect all reported results, but to bring an overview of various approaches and a summary of their principles. Possible advantages, disadvantages, and further perspectives are discussed and evaluated.

Oxidative stress parameters in different systemic rheumatic diseases.

The involvement of oxidative stress in the pathogenesis of rheumatic disorders, such as systemic sclerosis (SSc) and chronic polyarthritides, has been suggested yet not thoroughly verified experimentally. We analysed 4 plasmatic parameters of oxidative stress in patients with SSc (n = 17), psoriatic arthritis (PsA) (n = 10) and rheumatoid arthritis (RA) (n = 9) compared with healthy subjects (n = 22). The biomarkers were: total antioxidant capacity (TAC) measured by ferric reducing antioxidant power (FRAP) method, hydroperoxides determined by ferrous ion oxidation in presence of xylenol orange (FOX) method and sulfhydryl and carbonyl groups assessed by spectrophotometric assays. The results showed significantly increased hydroperoxides in SSc, PsA and RA (3.97 +/- 2.25, 4.87 +/- 2.18 and 5.13 +/- 2.36 micromol L(-1), respectively) compared with the control group (2.31 +/- 1.40 micromol L(-1); P < 0.05). Sulfhydryls were significantly lower in SSc (0.466 +/- 0.081 mmol L(-1)), PsA (0.477 +/- 0.059 mmol L(-1)) and RA (0.439 +/- 0.065 mmol L(-1)) compared with the control group (0.547 +/- 0.066 mmol L(-1); P < 0.05). TAC in all three diseases showed no difference in comparison with controls. Carbonyls were significantly higher in RA than in the control group (32.1 +/- 42 vs 2.21 +/- 1.0 nmol (mg protein)(-1); P < 0.05). The obtained data indicate augmented free radical-mediated injury in these rheumatic diseases and suggest a role for the use of antioxidants in prevention and treatment of these pathologies.