Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells.

BACKGROUND INFORMATION: TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. RESULTS: Stably transfected HEK-293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK-TrxR15 and HEK-TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK-TrxR15 and HEK-TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F-actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCdelta by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCdelta, and PKCdelta siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. CONCLUSIONS: The overexpression of TrxR1 inhibits migration of HEK-293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCdelta activation.

The role of thioredoxin reductase activity in selenium-induced cytotoxicity.

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.

Drug-resistant human lung cancer cells are more sensitive to selenium cytotoxicity. Effects on thioredoxin reductase and glutathione reductase.

The human U-1285 and GLC(4) cell lines, both derived from small cell carcinoma of the lung, are present in doxorubicin-sensitive (U-1285 and GLC(4)) and doxorubicin-resistant MRP-expressing (U-1285dox and GLC(4)/ADR) variants. These sublines were examined here with respect to their susceptibilities to the toxic effects of selenite and compared to the toxic effects of selenite on the promyelocytic leukemia cell line HL-60 and its doxorubicin-resistant P-glycoprotein expressing variant. The drug-resistant U-1285dox and GLC(4)/ADR sublines proved to be 3- and 4-fold, respectively, more sensitive to the cytotoxicity of selenite than the drug-sensitive U-1285 and GLC(4) sublines, whereas no difference was observed between the HL-60 sublines. The presence of doxorubicin at a concentration equal to the IC(10) did not significantly potentiate the toxic effects of selenite. The presence of selenite did not significantly affect the expression of the multi-drug resistant proteins (MRP1, LRP and topoisomerase IIalpha) in the drug-resistant cells. The activities of thioredoxin reductase (TrxR) were higher (50 and 25%, respectively) in the drug resistant cell sublines U-1285dox and GLC(4)/ADR compared to the drug-sensitive parental lines. The activity of glutathione reductase (GR) was essentially the same in the drug-sensitive and -resistant cell lines. Exposure to selenite resulted in a 4-fold increase in both TrxR and GR activities in U-1285 cells, an effect, which was less pronounced in the presence of doxorubicin. Under similar conditions the increase in the TrxR activity in the resistant U-1285dox cell line, was only 30% and the activity of GR was unaltered. Different responses in the activity of the key enzymes in selenium metabolism are one possible mechanism explaining the differential cytotoxicity of selenium in these cells.

Animal models for treatment of unresectable liver tumours: a histopathologic and ultra-structural study of cellular toxic changes after electrochemical treatment in rat and dog liver.

INTRODUCTION: Electrochemical treatment (EChT) has been taken under serious consideration as being one of several techniques for local treatment of malignancies. The advantage of EChT is the minimal invasive approach and the absence of serious side effects. Macroscopic, histopathological and ultra-structural findings in liver following a four-electrode configuration (dog) and a two-electrode EChT design (dog and rat) were studied. MATERIALS AND METHODS: 30 female Sprague-Dawley rats and four female beagle dogs were studied with EChT using Platinum:Iridium electrodes and the delivered dose was 5, 10 or 90 C (As). After EChT, the animals were euthanized. RESULTS: The distribution of the lesions was predictable, irrespective of dose and electrode configuration. Destruction volumes were found to fit into a logarithmic curve (dose-response). Histopathological examination confirmed a spherical (rat) and cylindrical/ellipsoidal (dog) lesion. The type of necrosis differed due to electrode polarity. Ultra-structural analysis showed distinct features of cell damage depending on the distance from the electrode. Histopathological and ultra-structural examination demonstrated that the liver tissue close to the border of the lesion displayed a normal morphology. CONCLUSIONS: The in vivo dose-planning model is reliable, even in species with larger tissue mass such as dogs. A multi-electrode EChT-design could obtain predictable lesions. The cellular toxicity following EChT is clearly identified and varies with the distance from the electrode and polarity. The distinct border between the lesion and normal tissue suggests that EChT in a clinical setting for the treatment of liver tumours can give a reliable destruction margin.

Cellular toxicity induced by different pH levels on the R3230AC rat mammary tumour cell line. An in vitro model for investigation of the tumour destructive properties of electrochemical treatment of tumours.

INTRODUCTION: The aim of this study was to evaluate the cellular toxicity of different pH levels on the R3230AC mammary tumour cell line (clone-D) in vitro and to determine in what way the pH affects the tumour cells. The results could be used to interpret the cell damaging effects seen in electrochemical treatment of tumours (EChT), where pH alteration in tissue is the major event. METHODS: Tumour cells were treated with pH 3.5, 5, 7, 9, 10 and 11 for 10, 20 or 30 min, respectively, followed by studies with the viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (methyltetrazolium (MTT)), morphological observation in phase contrast microscope (PCM) and light microscope, nucleotide analogue incorporation (BrdU; 5-Brdmo-2'-deoxyuridine), Caspase-3 activity measurement and detection of DNA fragmentation by an agarose gel electrophoresis. RESULTS: In the viability assay, it was found that different pH levels had cytotoxic effects; these effects were dependent on the pH value and on the time of exposure at a given pH. Morphologically, cells in pH 3.5 and 5 had shrunk, were rounded and had condensed chromatin, whereas prominent cell swelling and nuclear expansion were seen in the pH 9- and 10-treated cells. Gross cytolysis was found in pH 11. A BrdU incorporation assay indicated that proliferation rate is inhibited markedly both with decreasing and increasing pH. Significant Caspase-3 activity was found in pH 3.5 and 5 groups. Caspase-3 levels for the alkaline exposure were equal or below the normal control. DNA ladder formation, a characteristic of apoptosis, was only visualised in the treatment of pH 3.5 for 30 min. CONCLUSIONS: pH changes inhibit cell proliferation and decrease cell viability. The pathway of killing tumour cell in low pH probably has at least two directions: apoptosis and cell necrosis, whereas high pH results in only cell necrosis. The study suggests that low pH environment can induce apoptosis in unphysiological condition comparable with tissue pH at EChT. In addition, it seems that R3230AC mammary tumour cells are more tolerant to high pH than to acidic changes. This supports the theory that anodic EChT should be more efficient than cathodic.

Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase.

Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.

Adriamycin cytotoxicity may stimulate growth of hepatocellular tumours in an experimental model for adjuvant systemic chemotherapy in liver transplantation.

Adjuvant treatment with adriamycin has been suggested to improve results after liver transplantation for hepatocellular cancer. Here we have applied an animal model for evaluation of treatment with adriamycin and/or cyclosporine A on liver tumour growth. Three chemically induced rat liver tumours with various degree of differentiation were transferred to the spleens of syngenic rats. Each recipient group was divided into four subgroups, treated with adriamycin and/or cyclosporine A or none of the drugs. When the tumour was well differentiated no proliferation was found in any of the subgroups. When the tumour exhibited a more pronounced dysplasia, adriamycin stimulated tumour growth. This effect was further increased by cyclosporine. In the animals transplanted with the most aggressive tumour, adriamycin inhibited tumour growth. When given together with cyclosporine this inhibition was counteracted. These data suggest that adriamycin, especially when given together with cyclosporine, may have a stimulatory effect on liver tumour cell growth.

Lovastatin stimulates up-regulation of alpha7 nicotinic receptors in cultured neurons without cholesterol dependency, a mechanism involving production of the alpha-form of secreted amyloid precursor protein.

The cholesterol-lowering drug lovastatin enhances the secretion of the alpha-secretase cleavage product of amyloid precursor protein (APP). To investigate whether this effect is mediated via activation of alpha7 nicotinic acetylcholine receptors (nAChRs), we treated SH-SY5Y cells and PC12 cells with lovastatin and measured the levels of alpha7 nAChRs, the alpha-form of secreted APP (alphaAPPs), and lovastatin-related lipids, including cholesterol and ubiquinone. The results showed that low concentrations of lovastatin significantly induced up-regulation of alpha7 nAChRs. No effects of lovastatin were observed on alpha3-containing nAChRs, muscarinic receptors, or N-methyl-D-aspartate receptors. alphaAPPs levels increased in the culture medium of cells treated with lovastatin, whereas no change in whole APP was observed. The increase in alphaAPPs was inhibited by prior exposure of these cells to alpha-bungarotoxin, an antagonist of alpha7 nAChRs. The concentrations of lovastatin used in the study did not change the cholesterol content, but high doses can decrease the levels of ubiquinone and cell viability. These results indicate that lovastatin may play a neuronal role that is cholesterol independent. We also show that the up-regulation of alpha7 nAChRs stimulated by lovastatin is involved in a mechanism that enhances production of alphaAPPs during APP processing.

Ubiquinone biosynthesis in rat liver peroxisomes.

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans-prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans-Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn(2+). Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress.

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.

Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation.

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.