Pyridine Derivative of Succinic Acid Hydroxylamide Enhances the Cytotoxic Effect of Cisplatin and Actinomycin D.

We studied the possibility of inhibition of histone deacetylases (HDAC) in the nuclear extract of HeLa cells by N1-hydroxy-N4-(pyridin-4-yl)succinamide (compound 1). Compound 1 inhibits HDAC and showed low toxicity for A-172, HepG2, HeLa, MCF-7, and Vero cells. HeLa cells were most sensitive to the compound. Increasing the interval between administration of compound 1 and the chemotherapeutic agent to 8 h led to an increase in the cytotoxic effect of cisplatin (actinomycin D) on HeLa cells. The combination of compound 1 with cisplatin (actinomycin D) reduced the cytotoxic effect of these drugs for non-tumor Vero cells.

Cationic dinitrosyl iron complexes with thiourea exhibit selective toxicity to brain tumor cells in vitro.

The cytotoxic activity of a series of dinitrosyl iron complexes (DNICs) with thioureas against cells of different origin has been studied in this work. The cytotoxicity of the studied DNICs proved to be substantially different depending on the structure of the complexes and cell line. Complexes with thiourea and 1,3-dimethylthiourea were found to induce notable cell death in different cell lines of both cancerous and non-cancerous origin, while the N-ethylthiourea-bearing complex induced cell death in cells derived from brain tumors. The studied DNICs effectively release NO while decomposing in solutions, as follows from the electrochemical analysis. It was found that the cytotoxic effects of the studied DNICs did not correlate with their NO-donating ability, hence suggesting that their cytotoxic activity is, in a big part, defined by the long-lived nitrosyl iron-sulfur intermediates formed during the decomposition of the complexes. The structures of the products formed upon hydrolytic decomposition of all studied DNICs have been studied by electrospray ionization mass spectrometry. Stable high-molecular cluster ions containing NO groups namely [Fe4S3(NO)7]- (Roussin's "black salt" anion), [Fe4S3(NO)5]-, [Fe4S4(NO)4]-, [Fe4S3(NO)4]- and [Fe4S3(NO)6]- have been detected in the solution of the N-ethylthiourea-bearing complex. The mechanism of Roussin's "black salt" anion formation in a solution of DNIC with N'-ethylthiourea was studied using density functional theory. This moved us near understanding the reasons for the formation of biologically active intermediates upon the decomposition of the complex with N'-ethylthiourea, which are apparently responsible for the unique antiglioma activity of the complex.

Nrf2-Dependent Expression of Glutathione Antioxidant System Genes and Redox Status in Cells of In Vivo Drug-Resistant Murine P388 Leukemia Strains.

We measured the content of ROS and malondialdehyde in cells of in vivo drug-resistant murine P388 leukemia strains. It was found that the strains did not differ by malondialdehyde concentration, but intracellular concentration of ROS in cells of the cyclophosphamide-resistant strain (P388/CP) was higher than in cells of the original (P388) and other studied strains (P388/Rub, P388/cPt). Nuclear localization of the transcription factor Nrf2 in cells of strain P388/CP attested to its constitutive activation. Enhanced relative expression of the GCLM gene was found in all studied drug-resistant strains; the expression of the GSR and GPX1 genes was increased only in cells of the cyclophosphamide-resistant strain. These findings suggest that the mechanism of resistance of strain P388/CP is associated with increased activity of glutathione metabolism that developed as a result of activation of the antioxidant response transcription factor Nrf2 against the background of high intracellular concentration of ROS.

Influence of Tumor Suppressor p53 Functioning on the Expression of Antioxidant System Genes under the Action of Cytotoxic Compounds.

The effect of inhibition of the tumor suppressor p53 on the antioxidant system genes expression under the influence of cytotoxic compounds of the platinum group was studied. It was found that the action of platinum(II) and platinum(IV) complexes induced accumulation of p53 protein with a maximum in 12 h, which was confirmed by an increase in the expression of the P21 gene, the target gene of the p53 protein. It was shown that the action of platinum complexes activated the expression of catalase and superoxide dismutase 2 genes. Suppression of p53 protein functions with specific inhibitor α-piphitrin under the action of platinum complexes reduced the expression of catalase and superoxide dismutase 2 genes and the target gene P21, which attested to the p53-dependent regulation of these genes.

Activity of Antioxidant Enzymes and Content of Reduced Glutathione in Cells of Drug-Resistant Murine Leukemia P388 Strains.

Activities of superoxide dismutase and catalase and content of reduced glutathione in cells of drug-resistant murine leukemia P388 strains were studied without or after administration of antitumor compounds. In the absence of chemotherapeutic agents, no significant differences in activities of the studied enzymes in cells of the initial strain and strains resistant to cyclophosphamide, cisplatin, and rubomycin were observed. Compounds to which resistance was developed did not significantly affect activity of enzymes in cells of drug-resistant strains, while the use of compounds that were not resistance inductors was accompanied by a significant decrease in enzyme activity in cells resistant to cisplatin and rubomycin. In cells of strains resistant to cisplatin and cyclophosphamide, the content of reduced glutathione significantly differed from that in the initial strain. In addition, the concentration of reduced glutathione in cells of cyclophosphamide-resistant strain considerably decreased upon addition of the drug producing a therapeutic effect. Our findings suggest that the mechanism of resistance of in vivo derived cyclophosphamide resistant cell strain is related to increased level of reduced glutathione and activity of its metabolism.

Effect of Chitosan-(Poly)Nitroxides on Normal and Tumor Cells under Conditions of Induced Oxidative Stress.

The cytotoxicity and antioxidant effects of chitosan-(poly)nitoxides of different molecular weights containing a nitroxide radical of the piperidine structure were studied on tumor (HeLa, A172, and HepG2) and normal (Vero) cell lines. The chitosan-(poly)nitroxides exhibited low cytotoxicity. Under conditions of oxidative stress induced with tert-butyl hydroperoxide, the most pronounced decrease in ROS levels in the presence of chitosan-(poly)nitroxides was observed in normal cells. In cell homogenates, the decrease in malondialdehyde levels was observed only in the presence of low-molecular-weight chitosan-(poly)nitroxide irrespective of the cell line. Our data demonstrate that the cell-specific antioxidant properties of chitosan-(poly)nitroxides are related to their penetration into cells and interaction with intracellular membranes.

Screening of Some Dioxaboreninopyridine and Aniline Derivatives for Carcinogenic Properties Using a Model Cell-Free System of Regenerating Rat Liver.

We studied the effects of some aniline and dioxaborininopyridine derivatives on the rate of oxidative deamination of putrescine and polyamines in a tissue with high mitotic index. These effects were evaluated quantitatively by measuring diamine oxidase and polyamine oxidase activities in a model cell-free test system of regenerating rat liver tissue. Aniline derivatives exhibited mainly antiproliferative effects and promoted oxidative degradation of putrescine, spermidine, and spermine. Dioxaborininopyridine derivatives inhibited this process, thus exhibiting carcinogenic properties.

Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

[Lymphocyte apoptosis enhancement by the synthetic peptide E in experimental ulcer].

The influence of regulatory peptide type of AFP on the course of experimental ulcer was investigated. It has been shown that activation of apoptosis enhances local necrotic inflammatory reaction, on the one hand, and enables the development of adhesions with on the other.

Dysfunction of membrane-receptor system of blood cells and kidney tissue in experimental diabetes mellitus.

The effect of streptozotocin-induced diabetes mellitus on some parameters of energy metabolism and functional status of cell membranes was studied in experiments on rats. It was found that the development of diabetes mellitus is associated with dramatic changes in the metabolism of blood cells and kidney tissue: inhibition of aerobic ATP synthesis, accumulation of lactate, uncoupling of oxidative phosphorylation, and development of lactic acidosis. Diabetes mellitus leads to restructuring of membrane lipids, changes in microviscosity, and suppression of insulin receptors and membrane-bound Na(+), K(+)-ATPase, and Ca(2+)-ATPase. Sharply increased levels of LPO products and lactic acidosis during DM indicate an imbalance in the LPO-antioxidant system and development of oxidative stress.

Serum IgG-like glycoferroprotein: identification of its final dissociation form of thermostable protein coupled with albumin.

Human serum IgG-like glycoferroprotein identical to ascitic IgG-like glycoferroprotein that binds labeled monoclonal antibodies to CA125 is a complex consisting of three proteins: IgG, human serum albumin, and unidentified thermostable protein. Final dissociation form of serum IgG-like glycoferroprotein also appears as a complex of three nonidentical polypeptides with a molecular weight of 55 kDa (PC55) migrating in the albumin zone of thermostable protein coupled with albumin and structures chemically identical to human serum albumin and IgG heavy chains. Under denaturing conditions of electrophoresis in polyacrylamide gel, IgG-like glycoferroprotein and PC55 have the same molecular weight (about 55 kDa), while under reducing conditions their weight is about 75 kDa. Transition form (form the lower to the higher molecular weight) appears as an oblique (at about ≈ 30°) protein band creating a ladder string effect. Ladder string effect was reproduced with thermostable protein coupled with albumin, PC55, IgG-like glycoferroprotein, with all commercially available human and bovine albumins, rat albumin as well as with heated and renatured albumins and can serve as electrophoretic identification sign for thermostable protein coupled with albumin. Renatured after boiling (100°C for 15 min) bovine albumin under reducing conditions appeared as bow string twisted in helix, that raises molecule in 2.5 turns from ≈ 2 to ≈ 75 kDa. These data attest to the existence of an albumin double and to its possible double structure.

[Disfunction of membrane-receptor system of blood cells in children with diabetes mellitus of type I and II].

When metabolic failure in children and adolescents with diabetes, are violations of the structural and functional properties of membrane - the receptor apparatus of cells, accompanied by a decrease in ATP levels, inhibition of activity of membrane-bound enzyme Na+, K(+)-ATPase, a sharp decrease in insulin binding receptor activity and decrease glucose uptake by cells that indicates a decline in cell sensitivity to insulin. Diabetes in children and adolescents occurs with lipid disorders, activation of the processes of lipid peroxidation, manifested increasing concentrations of both primary and secondary products of lipid peroxidation, changes in structural and functional properties of erythrocyte membranes, as well as disturbances in the antioxidant defense system. Changes in the studied indexes depend on the type of diabetes and duration of the disease. Imbalance in the system LPO-AOD in the background shows the development of dyslipidemia, oxidative stress, particularly pronounced in type 2 diabetes.

Effect of nitric oxide donor, a modulator of tumor drug resistance, on cell death and p53 protein expression.

Exogenous NO donor 3,3-bis-(nitroxymethyl)oxetane (NMO) was synthesized at the Institute for Problems of Chemical Physics (Russian Academy of Sciences). This compound was shown to inhibit cell death (apoptosis and necrosis) in cyclophosphamide-sensitive and cyclophosphamide-resistant P388 murine tumor. p53 protein was expressed in both lines of tumor cells. NO donor NMO had little effect on p53 protein expression in cells of both stains. Our results suggest that the proapoptotic effect of NMO is mediated by the p53-independent molecular mechanisms.

[Relationship between structure and function of alpha-fetoprotein: conformational status and biological activity].

Alpha-fetoprotein (AFP) is the major mammalian fetal protein and the recognized tumor marker. This review summarizes data on structure and function of AFP with emphasis on human AFP, which is intensively investigated. During the last decade multiple functionally important sites of human AFP have been revealed or predicted by searching of similarity between primary structures of AFP and other proteins or their DNA sequences. A number of peptides derived from human AFP have been studied by different teams of investigators. These peptides were obtained by limited proteolysis of AFP or synthesized using solid phase chemistry. Study of biological (physiological) activities of these peptides allows determining biologically active sites of alpha-fetoprotein and constructing its structural and functional map. Biomodulating properties of these peptides make them a potential basis for design of drugs for different purposes including using in anticancer therapy. Conformational changes in AFP molecule have been intensively studied for the last few years and sufficient conformational mobility of AFP with the ability to form molten globule form (MGF) despite its stability in solution has been demonstrated. Native molecule of AFP may contain cryptic biologically active sites, which are not available for ligand binding. These sites become open and available for interaction after changes in conformation of AFP molecule. Study of conformational changes of AFP under different conditions allows understanding molecular mechanisms of its functioning. This review describes and analyses data obtained, mainly, during the last few years on study of conformational states of alpha-fetoprotein and relationship between conformational changes of AFP and its biological activity. Biochemical, biophysical and functional characterislics of some well-studied peptide fragments of AFP and their structural and functional mapping are presented.

[Change of the endocrine function of the pancreas in patients with chronic pancreatitis]. / Izmenenie éndokrinnoi funktsii podzheludochnoi zhelezy u bol'nykh khronicheskim pankreatitom.

There was a study of 19 chronic pancreatitis patients (10 male and 9 female), 11 chronic pancreatitis and pancreatogenic diabetes mellitus patients (8 male and 3 female) and 12 type 2 diabetes mellitus patients (4 male and 8 female) at the age of 30-60 as well as 15 control group subjects at the same age range. The content of the C-peptide and such peptides as INCINE, PAMG-cine and PAMG-tin in the blood serum was subjected to the immunoradiometric assay. It was discovered that there is a trend to the increased C-peptide level in CP patients while the C-peptide level in CP patients with diabetes mellitus was smaller than that in the control group; the C-peptide level in CP patients with type 2 diabetes mellitus was higher as compared to that in the control group. It was shown that erythrocytes of CP patients are less sensitive to insulin action and do not respond to the presence of insulinomimetic peptides under examination during the glucose uptake test. CP patients with diabetes mellitus and type 2 diabetes mellitus patients are more sensitive to the action of insulin and peptides applied. Synthetic insulinomimetic peptides can serve as a means for discovering the functional cell deficiency under the glucose uptake test.

Dual effects of endogenous DNases on transcriptionally active and inactive chromatin.

Experiments on resting hepatocytes with inactive c-fos gene and active albumin gene. We revealed that DNA of the transcribed gene is less susceptible to the influence of endogenous Ca2+/Mg(2+)-dependent DNases in matrix-associated and highly soluble chromatin fractions. In the fraction of low soluble chromatin active gene was more accessible for DNases. Our results indicate that activity of endogenous DNases can change in the transcribed gene locus.

Functional association of immediate early gene c-fos with nuclear matrix.

Accumulation of c-fos gene locus DNA in the nuclear matrix of hepatocyte nuclei was observed during induction of c-fos with cycloheximide. No enhanced association with the nuclear matrix was detected for inactive immunoglobulin gene locus. The use of endogenous DNAses allows isolation of nuclear matrix preparations enriched with transcribing chromatin.

[Endogenous DNases as a tool for isolation of nuclear matrix: critical parameters of nucleolysis]. / Endogennye DNKazy kak instrument polucheniia preparatov iadernogo matriksa: kriticheskie parametry nukleoliza.

Degree of nucleolysis has critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences as demonstrated at the example of inactive (c-fos, c-myc, and Ck) and active (p53, albumin, and 28S rRNA) genes in resting hepatocytes. Optimal degree of nucleolysis features degradation of loop domains of chromatin with preserved relatively uniform molecular weight distribution of DNA. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix.