Activation of Transcription Factor Nrf2 in HeLa Cells under the Action of Nitrosyl Iron Complex with N-Ethylthiourea.

We studied the effect of an NO donor, nitrosyl iron complex with N-ethylthiourea, on Nrf2-dependent antioxidant system activation of tumor cells in vitro. The complex increased intracellular accumulation of Nrf2 transcription factor and induced its nuclear translocation. It was shown that both heme oxygenase-1 gene and protein expression increased significantly under the influence of the complex. Nrf2 activation was accompanied by a decrease in the intracellular accumulation of proinflammatory transcription factor NF-κB p65 subunit and expression of its target genes. The cytotoxic effect of N-ethylthiourea leads to induction of Nrf2/HO-1 antioxidant response and suppression of NF-κB-dependent processes in tumor cells.

Effect of Pyridoxine Derivative B6NO on Transcription Factor Nrf2 Activity and Cytotoxic Properties of Doxorubicin In Vitro.

The effect of a new pyridoxine derivative B6NO on doxorubicin cytotoxicity and Nrf2-dependent cellular processes in vitro was studied. Antioxidant B6NO enhances the cytotoxic effect of doxorubicin on tumor cells, which is associated with G2/M cell division arrest and an increase in activity of proapoptotic enzyme caspase-3. The antioxidant promotes intracellular accumulation and nuclear translocation of Nrf2 transcription factor in non-tumor and tumor cells. In non-tumor cells, B6NO increases the expression of antioxidant system proteins and reduces ROS generation in the presence of doxorubicin. In tumor cells, no activation of Nrf2-dependent processes occurs under the action of the antioxidant. Our findings demonstrate the prospect of further studies of pyridoxine derivatives as antioxidants to reduce adverse reactions during chemotherapy.

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.

Effect of Cytotoxic Compounds on Activity of Antioxidant Enzyme System in MCF-7 and H1299 Cells.

We studied the function of the antioxidant system in tumor cell lines MCF-7 and H1299 that differ by the state of tumor suppressor gene p53. Exposure to different classes of cytotoxic compounds induced several types of antioxidant system responses that depend on the type of cell line. The effects of platinum(II) and platinum(IV) complexes on activity of antioxidant enzymes vary, which can be explained by differences in their accumulation and biotransformation in tumor cells. Triazole and oxazolidinone derivatives had little effect on activity of superoxide dismutase and catalase in H1299 cells, but increased superoxide dismutase activity in MCF-7 cells.