The cytotoxic and antiproliferative properties of ruthenium nitrosyl complexes and their modulation effect on cytochrome P450 in the HepG2 cell line.

Ruthenium nitrosyl complexes are actively investigated as antitumor agents. Evaluation of potential interactions between cytochromes P450 (CYPs) with new compounds is carried out regularly during early drug development. In this study we have investigated the cytotoxic and antiproliferative activities of ruthenium nitrosyl complexes with methyl/ethyl esters of nicotinic and isonicotinic acids and γ-picoline against 2D and 3D cultures of human hepatocellular carcinoma HepG2 and non-cancer human lung fibroblasts MRC-5, assessed their photoinduced activity at λrad = 445 nm, and also evaluated their modulating effect on CYP3A4, CYP2C9, and CYP2C19. The study of cytotoxic and antiproliferative activities against 2D and 3D cell models was performed using phenotypic-based high content screening (HCS). The expression of CYP3A4, CYP2C9, and CYP2C19 mRNAs and CYP3A4 protein was examined using target-based HCS. The results of CYP3A4 mRNA expression were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The ruthenium nitrosyl complexes exhibited a dose-dependent cytotoxic effect against HepG2 and MRC-5 cells. The cytotoxic activity of complexes with ethyl isonicotinate (1) and nicotinate (3, 4) was significantly lower for MRC-5 than for HepG2, for a complex with methyl isonicotinate (2) it was higher for MRC-5 than for HepG2, for a complex with γ-picoline (5) it was comparable for both lines. The antiproliferative effect of complexes 2 and 5 was one order of magnitude higher for MRC-5; for complexes 1, 3, and 4 it was comparable for both lines. The cytotoxic activity of all compounds for 3D HepG2 was lower than for 2D HepG2, with the exception of 4. Photoactivation affected the activity of complex 1 only. Its cytotoxic activity decreased, while the antiproliferative activity increased. The ruthenium nitrosyl complexes 1-4 acted as inducers of CYP3A4 and CYP2C19, while the complex with γ-picoline (5) induced of CYP3A4. Among the studied ruthenium nitrosyl complexes, the most promising potential antitumor compound is the ruthenium compound with methyl nicotinate (4).

Serum miR-181a and miR-25 in patients with malignant and benign breast diseases.

Breast tumor diseases include a wide range of pathologies that require different approaches to their treatment. MicroRNA (miR) levels, reflecting regulation of the gene expression involved in tumorigenesis, can be diagnostic and prognostic markers of breast diseases. The levels of circulating miR-181a and miR-25 were measured in patients with benign breast diseases (BBD), patients with invasive carcinoma of a nonspecific type (ICNT) and also in conditionally healthy women. Expression of both miRs was higher in patients of both groups as compared to controls; at the same time, the content of serum miR-181a and miR-25 was higher in BBD patients than in ICNT patients. The detected changes may be of interest in the context of precancerous changes in BBD. It seems possible to use them in the future as markers of the pathological process as a part of a large diagnostic panel.

Stimulation of Diethylnitrosamine Metabolism Reduces Its General Toxic and Hepatocarcinogenic Effects.

The general toxic and hepatocarcinogenic effects of diethylnitrosamine after stimulation of its metabolism with 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) were studied. The hydroxylating activity of liver microsomes of C57Bl/6Mv mice towards p-nitrophenol increased more than 4-fold 3 days after injection of TCPOBOP. Injection of diethylnitrosamine 3 days after TCPOBOP caused a lesser body weight loss and decrease of food consumption in C57Bl/6Mv mice than in response to diethylnitrosamine without preinduction. Injection of diethylnitrosamine to suckling ICR mice after TCPOBOP induction of cytochrome P450 2e1 activity led to development of 2-fold lesser number of tumors and pretumorous nodes in the liver in comparison with animals injected with diethylnitrosamine without induction. These data indicated that metabolism stimulation reduced the general toxic and hepatocarcinogenic effects of diethylnitrosamine.

Iron metabolism after application of modified magnetite nanoparticles in rats.

The influence of modified nanosized magnetite (NSM) particles (magnetic microspheres coated with chitosan and magnetoliposomes) after a single intravenous infusion of their suspensions on iron metabolism in rats has been studied. Modern physical and chemical methods (X-ray fluorescence, dynamic light scattering, transmission electron microscopy) were used for standardization of the modified NSM particles (their size, structure, ζ-potential, and concentration were determined). Atomic emission spectroscopy was used to reveal the dynamics of iron content in rat liver, spleen, lungs, and kidneys during 120 days. Colorimetric and immunoturbidimetric methods were used to determine the concentrations of plasma iron and the proteins involved in its metabolism - ceruloplasmin, transferrin, and ferritin. Their dynamics throughout the experiments were studied.

The Mitochondria-Targeted Antioxidant SkQ1 Downregulates Aryl Hydrocarbon Receptor-Dependent Genes in the Retina of OXYS Rats with AMD-Like Retinopathy.

The mitochondria-targeted antioxidant SkQ1 is a novel drug thought to retard development of age-related diseases. It has been shown that SkQ1 reduces clinical signs of retinopathy in senescence-accelerated OXYS rats, which are a known animal model of human age-related macular degeneration (AMD). The aim of this work was to test whether SkQ1 affects transcriptional activity of AhR (aryl hydrocarbon receptor) and Nrf2 (nuclear factor erythroid 2-related factor 2), which are considered as AMD-associated genes in the retina of OXYS and Wistar rats. Our results showed that only AhR and AhR-dependent genes were sensitive to SkQ1. Dietary supplementation with SkQ1 decreased the AhR mRNA level in both OXYS and Wistar rats. At baseline, the retinal Cyp1a1 mRNA level was lower in OXYS rats. SkQ1 supplementation decreased the Cyp1a1 mRNA level in Wistar rats, but this level remained unchanged in OXYS rats. Baseline Cyp1a2 and Cyp1b1 mRNA expression was stronger in OXYS than in Wistar rats. In the OXYS strain, Cyp1a2 and Cyp1b1 mRNA levels decreased as a result of SkQ1 supplementation. These data suggest that the Cyp1a2 and Cyp1b1 enzymes are involved in the pathogenesis of AMD-like retinopathy of OXYS rats and are possible therapeutic targets of SkQ1.

Expression of genes for AhR and Nrf2 signal pathways in the retina of OXYS rats during the development of retinopathy and melatonin-induced changes in this process.

Modulation of oxidative stress is one of the experimental approaches to the therapy of age-related macular degeneration. Melatonin holds much promise in this respect. It was hypothesized that the efficiency of melatonin in age-related macular degeneration is associated with its ability to modulate gene expression for the AhR and Nrf2 signal pathways. Experiments were performed on premature aging OXYS rats, which serve as a reliable model of age-related macular degeneration in humans. We studied the effect of melatonin on gene mRNA for the AhR and Nrf2 signal pathways. Melatonin was shown to decrease the level of mRNA for AhR-dependent genes of CYP1A2 and CYP1B1 cytochromes in the retina, but had no effect on the content of mRNA for Nrf2-dependent genes in OXYS rats.

Blood microvesicles during chronic lymphoproliferative diseases.

The levels of CD19 (+) and CD20(+) microvesicles were estimated in the blood of patients with B-cell chronic lymphocytic leukemia and indolent non-Hodgkin lymphoma by flow cytometry method. It was found that the number of B cell microvesicles is several times higher in patients than in volunteers. The level of CD20 (+) microvesicles directly correlated with the number of CD20(+) lymphocytes in patients with chronic lymphoproliferative diseases. Extramedullary tumors cells can be a source of CD19 (+) microvesicles.

Role of xenobiotic metabolism enzyme gene polymorphism in the formation of chemotherapy resistance in patients with chronic lymphoproliferative diseases.

Enzymes of the cytochrome P450 and GSTP1 families play a pivotal role in the metabolism of a wide variety of antitumor drugs and polymorphisms of genes encoding for metabolizing enzymes can affect drug efficacy and toxicity. We studied the associations between functionally significant gene polymorphisms CYP2C8, CYP2C9, CYP2C19, CYP3A5, and GSTP1 and clinical response to chemotherapy in patients with chronic lymphoproliferative diseases. Significant correlations with chemotherapy resistance were observed for CYP2C8 3 (OR=7.05; CI 95%=1.76-29.55) and CYP2C9 2 polymorphisms (OR=4.1; CI 95%=1.03-16.81). No significant association between chemotherapy resistance and other examined polymorphisms were found.

Cold-induced activities of cytochromes P450 1A1 and 1A2 in rat liver: putative role of endogenous compounds in induction mechanism.

Adaptation to cold includes adaptive changes at the organism and molecular levels. One of the interesting facts is induction of cytochromes P450 subfamily 1A (CYP1A) in the liver of rats, inducible enzymes participating in biotransformation of procarcinogenic xenobiotics, under the effect of moderate cold exposure. Cold activation of CYP1A can be mediated by adaptive changes and the resultant redistribution or intensification of the synthesis of mediator compounds. This hypothesis is verified in the present study. The role of bilirubin, tocopherol, and corticosterone as mediators of cold induction of CYP1A in the rat liver was evaluated. The results indicate that these compounds can be involved in cold induction of CYP1A, but none of them is the only mediator in this process.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

Effect of cold stress on expression of genes for the AhR-dependent pathway of CYP1 regulation in rat liver.

The level of mRNA for cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1) and CYP1 regulatory proteins (heat shock protein, aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and aryl hydrocarbon receptor nuclear translocator) was measured in the liver of rats after cold stress (4 degrees C). The CYP1A1 mRNA level increased and remained high for 10 days after 5-day cold exposure. The level of mRNA for CYP1A2, heat shock protein, and aryl hydrocarbon receptor nuclear translocator decreased by the 10th day. The level of mRNA for CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator remained unchanged over this period.

Activity of 20S proteosomes and content of oxidized proteins in rat liver after long-term cold exposure.

We studied the effect of long-term (5 and 10 days) cold exposure (4 degrees C) on oxidative damage to proteins and proteosomal activity in the liver of Wistar rats. It was shown that core temperature on the 10th day of cold exposure decreased by 1.1 degrees C. The content of oxidized proteins increased at this term. Chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities increased, while trypsin-like activity decreased during cold exposure.

Possible role of P-glycoprotein in cyclophosphamide resistance of transplanted mouse RLS lymphosarcoma.

The causes of different sensitivity of mouse LS lymphosarcoma and its resistant RLS variant to cyclophosphamide were studied. Division of LS and RLS cells stops in the G2/M phase 24 h after cyclophosphamide treatment, but this stop lasts for more than 48 h in LS cells and less than 24 h in RLS cells. DNA fragmentation, a marker of apoptosis, is observed only in LS cells starting from 24 h after cyclophosphamide treatment. LS and RLS strains do not differ by the expression of bcl-2, bcl-6, bax, bad, mdr1a, mdr1b genes and P-glycoprotein protein. The strains differ by transport activity of P-glycoprotein, tested by SYTO 16 substrate release from cells: activity of P-glycoprotein in RLS cells was 2-fold higher than in LS cells. Presumably, the resistance of RLS tumor to cyclophosphamide-induced apoptosis is a result of inhibition of the apoptotic cascade by P-glycoprotein which is functionally more active in these cells than in LS cells.

Inhibitory effect of alpha-tocopherol on benzo(a)pyrene-induced CYPA1 activity in rat liver.

Combined treatment with benzo(a)pyrene (classic inductor of cytochromes P450 of subfamily 1A, CYPA1 and CYP1A2) and alpha-tocopherol decreased benzo(a)pyrene-induced CYP1A1 activity in rat liver. Activities of CYP1A2, NADPH-cytochrome P450 reductase, and glutathione S-transferase remained unchanged under these conditions. Addition of alpha-tocopherol to benzo(a)pyrene-induced microsomes in vitro decreased activity of CYP1A1. Immunoblotting of proteins in liver microsomes with antibodies against CYP1A1 did not reveal differences in CYP1A1 protein content in the liver of rats receiving benzo(a)pyrene alone or in combination with alpha-tocopherol. The in vivo decrease in benzo(a)pyrene-induced CYP1A1 activity did not result from free radical-produced damage to CYP1A1. The inhibition of benzo(a)pyrene-induced CYP1A1 activity with alpha-tocopherol is probably realized at the posttranslational level.

Dose- and time-dependent effects of menadione on enzymes of xenobiotic metabolism in rat liver.

We studied the effect of synthetic vitamin K analogue menadione on enzymes of xenobiotic metabolism in rat liver (total content of cytochrome P450 and catalytic activities of CYP1A1/2, CYP2B1, CYP2C, NADPH-cytochrome P450 reductase, and glutathione S-transferase). Menadione induced phase I and II enzymes for metabolism of xenobiotics, drugs, and procarcinogens. The effect of menadione depended on its dose and duration of treatment.

Transcriptional activation of cytochrome P450 1A1 with alpha-tocopherol.

Peroral administration of alpha-tocopherol in a daily dose of 150 mg/kg for 1, 4, 8, and 12 days leads to induction of cytochromes P450 1A in male rats. Activity of CYP1A1 and CYP1A2 increased most significantly one day after alpha-tocopherol administration (by 2.6 and 2.7 times, respectively). CYP1A1 was immunohistochemically detected in rat liver microsomes during this period. The content of CYP1A1 mRNA significantly increased in the liver. The amount of CYP1A2 mRNA and regulatory proteins for signal activation of CYP1A1 (AhR and Arnt) remained unchanged after treatment with alpha-tocopherol.

Effect of long-term cold exposure on activities of cytochrome P450-containing monooxygenases and glutathione S-transferase in rat liver microsomes.

Long-term cold exposure (5 degrees C) was followed by induction of rat liver monooxygenases. We revealed an increase in activity of NADPH-cytochrome C reductase, total content of cytochrome P450 (CYP), and activities of its molecular forms CYP1A1, 1A2, 2B1/B2, 2E1, and 3A1/A2 in microsomes. These indexes reached maximum by the 10th day, but decreased with lengthening of cold exposure. Glutathione S-transferase activity decreased under these conditions. Changes in enzyme activity could be related to the increase in blood corticosterone concentration.

MDR1 Gene C1236T and C6+139T polymorphisms in the Russian population: associations with predisposition to lymphoproliferative diseases and drug resistance.

Study of MDR1 polymorphism in intron 6 and exon 12 of healthy individuals and patients with chronic lymphoproliferative diseases showed that the presence of mutant 6+139T allele is a factor determining resistance to lymphoproliferative diseases. Comparison of genotyping results in 53 patients and the data on the efficiency of drug therapy showed no significant associations of C(6+139)T and C(1236)T genotypes with drug resistance.

Possible prediction of the efficiency of chemotherapy in patients with lymphoproliferative diseases based on MDR1 gene G2677T and C3435T polymorphisms.

Study of polymorphism in MDR1 gene exons 21 and 26 revealed that T2677T and T3435T alleles are not a factor predisposing to lymphoproliferative diseases, but they determine the efficiency chemotherapy. Individuals with T2677T and T3435T haplotypes are at highest risk of drug resistance. Association between genotypes G2677T and C3435T was detected in normal subjects and in patients with lymphoproliferative diseases.

Dose-dependent effect of alpha-tocopherol on activity of xenobiotic metabolizing enzymes in rat liver.

Oral treatment with alpha-tocopherol for 4 days dose-dependently increased the content of cytochrome P450 (CYP), catalytic activities of CYP1A1, CYP1A2, CYP2B1, CYP2C, and activity of NADPH-cytochrome-P450 reductase in the liver of male rats, but did not change activity of glutathione S-transferase. These results suggest that alpha-tocopherol induced the enzymes of phase I of xenobiotic metabolism, including CYP1 and CYP2 families involved in the metabolism of drugs and procarcinogenes.