OBJECTIVETo explore the effect of nuclear factor erythroid-2 related factor 2 (Nrf2) and thioredoxin reductase (TrxR) gene on proliferation of chronic myeloid leukemia (CML) line cells and its mechanism.

METHODSFour interfering sequences of Nrf2 and one negative control sequence were designed and synthesised based on the principle of target sequence of siRNA, then constructed lentivirus vectors, which were transfected into K562 cell lines. The transfection effect was observed by laser scanning confocal microscope (LSCM) and flow cytometer (FCM); The depressing effect of siRNA was analyzed by real-time PCR. The cell proliferation inhibiting rate was measured with CCK-8 assay, the apoptotic rate by Annexin V-PE/PI with FCM and the apoptotic morphology of cells by LSCM.

RESULTSThe transfection efficiency of lentivirus was 65%. One cell line K562-C3 which significantly inhibited Nrf2 mRNA was obtained by real-time PCR, Nrf2 relative quantitation (RQ) expressions were 1.003±0.093 and 0.344±0.032 in the control group and K562-C3 respectively; TrxR expression also decreased with RQ as 1.090±0.549 and 0.395±0.029 respectively. The cellular proliferation inhibition rates of K562-C3 were (4.74±0.39)%, (6.13±1.78)% and (25.36±3.77)%, respectively at 24, 48 and 72 h. The apoptotic rate induced by K562-C3 (29.9%) at 72 hours was obviously higher than in the control group (7.9%). The Annexin V-PE positive K562-C3 cells presented the following apoptotic characteristics, such as karyopyknosis, nuclear fragmentation and apoptotic bodies observed by LSCM.

CONCLUSIONNrf2 specific siRNA could repress its expression at the cellular level and down-regulate the expression of its downstream antioxidant enzyme, such as TrxR, which lead to increased apoptotic rate and decreased cell proliferation.

Apoptosis ; Cell Proliferation ; Down-Regulation ; Genetic Vectors ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; NF-E2-Related Factor 2 ; metabolism ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Thioredoxin-Disulfide Reductase ; metabolism

PURPOSE: Thioredoxin is an endogenous antioxidant which directly scavenges reactive oxygen species(ROS) and regenerates oxidatively damaged protein by reducing potential at the redox active disulfide(-Cys-Gly-Pro-Cys-) site. Under oxidative stress, thiredoxin plays a protective and adaptative role by inducing expressions. The aim of this study was to clarify the role of thioredoxin on oxidative neuronal cell injury. We investigated the protective effects of E. coli thioredoxin, also acting as a substrate for mammalian thioredoxin reductase, against oxidative neuronal cell injury under oxidative stresses such as hydrogen peroxide and diamide. METHODS: PC 12 cells were cultured in RPMI 1640 media containing 10% fetal calf serum and subcultured in 96-well plates. Each well contained 30,000 cells. Cells were treated with hydrogen peroxide or diamide 30 minutes after thioredoxin treatment and then incubated for 24 hours. Cytotoxicity and cellular viability were assessed by measuring of lactate dehydrogenase(LDH) release and MTT reduction. RESULTS: Thioredoxin not only decreased the cytotoxicity of PC 12 cell treated with hydrogen peroxide by decreasing LDH release and preventing the decrease of MTT reduction but also thioredoxin showed greater protective effects when simultaneously treated with hydrogen peroxide. Also, thioredoxin decreased cytotoxicity by decreasing LDH release from PC 12 cells damaged by diamide. Thioredoxin did not prevent the decrease of MTT reduction on PC 12 cells damaged by diamide. CONCLUSION: Thioredoxin protected PC 12 cells under oxidative stresses by directly scavenging and inhibiting oxidants such as hydrogen peroxide and diamide.
Diamide ; Hydrogen Peroxide ; Lactic Acid ; Neurons* ; Oxidants ; Oxidation-Reduction ; Oxidative Stress ; Oxygen ; Thioredoxin-Disulfide Reductase ; Thioredoxins*

Carcinoma, Hepatocellular ; enzymology ; pathology ; Glutathione Peroxidase ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Thioredoxin-Disulfide Reductase

Human thioredoxin reductase (TrxR) system is associated with cancer cell growth and anti-apoptosis process. Effects of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane (BBSKE), a novel TrxR inhibitor, were investigated on human leukemia cell lines HL-60 and K562. BBSKE treatment induced cell growth inhibition and apoptosis in both cell lines. Apoptosis induced by BBSKE is through Bcl-2/Bax and caspase-3 pathways. Ehrlich's ascites carcinoma-bearing mice were used to investigate the anti-tumor effect of BBSKE in vivo. Tumor-bearing mice treated with BBSKE showed an increase of life span with a comparable effect to cyclophosphamide (CTX). These results suggest a potential usage of BBSKE as a therapeutic agent against non-solid tumors.
Apoptosis ; drug effects ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Proliferation ; drug effects ; Enzyme Inhibitors ; pharmacology ; HL-60 Cells ; Humans ; K562 Cells ; Organoselenium Compounds ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; physiology ; Thioredoxin-Disulfide Reductase ; antagonists & inhibitors ; bcl-2-Associated X Protein ; physiology

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.
Animals ; Antioxidants/*isolation & purification/*metabolism ; Cell Fractionation ; Cytosol/enzymology ; Erythrocytes/parasitology ; Glutathione Reductase/*isolation & purification/*metabolism ; Mice ; Plasmodium berghei/*enzymology ; Thioredoxin-Disulfide Reductase/*isolation & purification/*metabolism

BACKGROUND: Peroxiredoxin(Prx) is a novel peroxidase family to remove hydrogen peroxide using thioredoxin system, which is consisted of thioredoxin, thioredoxin reductase and NADPH. Several enzymatic antioxidants, such as superoxide dismutase, catalase and peroxidases are known to be present in the normal skin. But very little is known on the expression of Prx in the normal skin. OBJECTIVE: The aim of this study was to evaluate the expression of Prx isotypes in the normal human and rat skin, and thus to understand the role of Prx in the skin. MATERIALS AND METHODS: In this study, expression of 5 isotypes of Prx was evaluated in the primary cultures of keratinocytes and fibroblasts from human and rat, HaCaT cells and A431 cells by Western blot analysis. Also, immunohistochemical study for Prx I-IV expression was performed in the rat fibroblasts. RESULTS: Western blot analysis provided strong signals for Prx I, II and III with the cell extracts of cultured cells from the human and rat. The signals for Prx IV were weakly positive in hK, A431, hF and rF. The signals for Prx VI were positive in human cells, but were negative in rat cells. The finding were also identified in the intact skin. From immunocytochemical study for Prx I-IV, they were stained positively as a reticulated pattern in the cytoplasm of rF without isotype-specific difference. The positive reaction was strong in perinuclear cytoplasm. CONCLUSIONS: This study shows that Prx is ubiquitously expressed in the normal human and rat skin with an isotype-specific expression by species and cell types.
Animals ; Antioxidants ; Blotting, Western ; Catalase ; Cell Extracts ; Cells, Cultured* ; Cytoplasm ; Fibroblasts ; Humans* ; Hydrogen Peroxide ; Keratinocytes ; NADP ; Peroxidase ; Peroxidases ; Peroxiredoxins* ; Rats* ; Skin* ; Superoxide Dismutase ; Thioredoxin-Disulfide Reductase ; Thioredoxins

BACKGROUND: Although the pathogenesis of vitiligo isn't fully understood, a recent study demonstrates that oxidative stress plays an important role to induce vitiligo. Peroxiredoxin (Prx) is a novel peroxidase family to remove hydrogen peroxide using thioredoxin system, which is consisted of thioredoxin, thioredoxin reductase, and NADPH. OBJECTIVE: This study aimed to investigate the change of expression of Prx I to elucidate the role of oxidative stress in the pathogenesis of vitiligo. METHODS: Sample specimens were obtained from the lesional skin of vitiligo patients, and non-depigmented skin was obtained from the perilesional area as control samples. The skin samples were immediately frozen using liquid nitrogen, and then section samples were prepared to perform immunohistochemical staining with antibodies for Prx I. Some of the skin biopsy samples were used for primary culture of keratinocytes. Protein extracts from the expanded keratinocytes were prepared for Western blot analysis of Prx I. RESULTS: In vitiligo, the ubiquitous expression of Prx I in all layers of epidermis, which was also observed in the normal perilesional skin, was reduced in the depigmented lesion of vitiligo patients. The reduction of Prx I was remarkable from the lesions which were exposed to sunlight. Consistently, Prx I expression from the lesional keratinocytes were noticeably reduced in comparison with that from perilesional keratinocytes. CONCLUSION: Our results showing that Prx I is impaired in the epidermis of depigmented lesions of vitiligo patients suggest that oxidative stress is an important factor to induce vitiligo.
Antibodies ; Biopsy ; Blotting, Western ; Epidermis ; Humans ; Hydrogen Peroxide ; Keratinocytes ; Nitrogen ; Oxidative Stress ; Peroxidase ; Peroxiredoxins ; Skin ; Sunlight ; Thioredoxin-Disulfide Reductase ; Thioredoxins ; Vitiligo

OBJECTIVETo establish gemcitabine-resistant pancreatic cancer cell strain and study the role of thioredoxin reductase (TrxR) in drug-resistant process.

METHODSGemcitabine-resistant pancreatic cancer cell strain SW1990/GZ was induced by increasing drug dosage intermittently, then the changes of its biological features and the activity of TrxR were examined.

RESULTSStable drug-resistant SW1990/GZ cell strain was established by culturing with gemcitabine for 9 months. The morphology and growth characteristics of the cell strain changed remarkably. The cells shrunk and became rounder; its endoplasm expanded; granular substances increased; and the doubling-time was prolonged. Resistance of the cell line to gemcitabine, fluorouracil, adriamycin, and mitomycin significantly increased. The TrxR activity of the drug-resistant cells was increased markedly.

CONCLUSIONSW1990/GZ has certain multidrug resistance to some chemotherapy drugs, and TrxR plays a role in the drug-resistant process.

Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Pancreatic Neoplasms ; enzymology ; pathology ; Thioredoxin-Disulfide Reductase ; drug effects ; metabolism

BACKGROUND: Continuous growth stimulation by various factors, as well as chronic oxidative stress, may co-exist in many solid tumors, such as lung cancer. A new family of antioxidant proteins, the peroxiredoxins (Prxs), have been implicated in the regulation of many cellular processes, including cell proliferation, differentiation and apoptosis. However, a real pathophysiological significance of Prx proteins, especially in lung disease, has not been sufficiently defined. Therefore, this study was conducted to investigate the distribution and expression of various Prx isoforms in lung cancer and other pulmonary conditions. METHOD: Patients diagnosed with lung cancer, and who underwent surgery at the Ajou Medical Center, were enrolled. The expressions of Prxs, Thioredoxin (Trx) and Thioredoxin reductase (TR) were analyzed using proteomic techniques and the subcellular localization of Prx proteins was studied using immunohistochemistry on normal mouse lung tissue. RESULT: Immunohistochemical staining has shown the isoforms of Prx I, II, III and V are predominantly expressed in bronchial and alveolar lining epithelia, as well as in the alveolar macrophages of the normal mouse lung. The isoforms of Prx I and III, and thioredoxin were also found to be over-expressed in the lung cancer tissues compared to their paired normal lung controls. There was also an increased amount of the oxidized form of Prx I, as well as a putative truncated form of Prx III, in the lung cancer samples when analyzed using 2-dimensional electrophoresis. In addition, a 43 kDa intermediate molecular weight protein band, and other high molecular weight bands of over 20 kDa, recognized by the anti-Prx I antibody, were present in the tissue extracts of lung cancer patients on 1-Dimensional electrophoresis, which require further investigation. CONCLUSION: The over-expressions of Prx I and III, and Trx in human lung cancer tissue, as well as their possible chaperoning function, may represent an attempt by tumor cells to adjust to their microenvironment in a manner advantageous to their survival and proliferation, while maintaining their malignant potential.
Animals ; Apoptosis ; Cell Proliferation ; Electrophoresis ; Humans* ; Immunohistochemistry ; Lung Diseases ; Lung Neoplasms* ; Lung* ; Macrophages, Alveolar ; Mice ; Molecular Weight ; Oxidative Stress ; Peroxiredoxins* ; Protein Isoforms ; Thioredoxin-Disulfide Reductase ; Thioredoxins* ; Tissue Extracts

Space flight is known to produce a number of neurological disturbances. The etiology is unknown, but it may involve increased oxidative stress. A line of experimental evidence indicates that space flight may disrupt antioxidant defense system and result in increased oxidative stress. In vitro studies found that abundant of NO was produced in rat pheochromocytoma (PC12) cells, SHSY5Y neuroblastoma cells, and protein nitration was increased in PC12 cells within a simulated microgravity rotating wall bioreactor high aspect ratio vessel system or clinostat system. In the present study, we observed the change of redox status in SH-SY5Y cells after parabolic flight, and studied the effects of key redox molecule, thioredoxin (TRX), during the altered gravity. SH-SY5Y cells were divided into four groups: control cells, control cells transfected with TRX, flight cells and flight cells transfected with TRX. The expression levels of 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), TRX and thioredoxin reductase (TRXR) were observed by immunocytochemical method. It was shown that after parabolic flight, the staining of 3-NT and TRX were enhanced, while the expression level of TRXR was down-regulated compared with control. As for flight cells transfected with TRX, the staining of 3-NT and iNOS were weakened compared with flight cells. These results obtained suggest that altered gravity may increase protein nitration, down-regulate TRXR and elicit oxidative stress in SH-SY5Y cells, while TRX transfection could partly protect cells against oxidative stress induced by parabolic flight.
Animals ; Antioxidants ; Cell Line, Tumor ; Humans ; Hypogravity ; Nitric Oxide Synthase Type II ; physiology ; Oxidative Stress ; PC12 Cells ; Rats ; Space Flight ; Thioredoxin-Disulfide Reductase ; physiology ; Thioredoxins ; physiology ; Transfection ; Tyrosine ; analogs & derivatives ; physiology