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- Chatzakos, Vicky, 1977-, et al.
(författare)
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Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite
- 2012
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Ingår i: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839 .- 1873-2968. ; 84:5, s. 712-721
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Tidskriftsartikel (refereegranskat)abstract
- High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancercells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in thehepatocellular carcinoma cell line Huh7. Treatment with 20 mM of selenite reduced the viability of Huh7cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold.Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxiceffect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutralsphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophe-nyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 mM ofSK1-II prior to treatment with 10 mM of selenite caused induction of apoptosis and gave rise to a 2.5-foldincrease in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 mM of SK1-II combined with 10 mM ofselenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides.The formation of reactive oxygen species (ROS) after treatment with 10 mM of selenite was maximallyenhanced by 1 mM of SK1-II. Moreover, combined treatment with SK1-II and 10 mM of selenitesynergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell lineMIHA remained unaffected by the same treatment. These results raise the possibility that a combinationof selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant,at doses that are non-toxic to normal liver cells.
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| 2. |
- Nilsonne, Gustav, et al.
(författare)
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Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress
- 2006
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Ingår i: Free Radical Biology and Medicine. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 0891-5849.
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Tidskriftsartikel (refereegranskat)abstract
- Malignant mesothelioma cells differentiate into sarcomatoid or epithelioid phenotypes. The sarcomatoid cell type is more resistant to chemotherapy and yields a worse prognosis. We have investigated whether selenite alone and in combination with doxorubicin induced apoptosis in variously differentiated mesothelioma cells. Selenite in concentrations that could potentially be administered to patients strongly inhibited the growth of the sarcomatoid mesothelioma cells (IC 50 = 7.5 µM), whereas epithelioid cells were more sensitive to doxorubicin. Benign mesothelial cells remained largely unaffected. Selenite potentiates doxorubicin treatment. Apoptosis was the dominating mode of cell death. The toxicity of selenite was mediated by oxidative stress. Furthermore the activity of the thioredoxin system was directly dependent on the concentration of selenite. This offers a possible mechanism of action of selenite treatment. Our findings suggest that selenite is a promising new drug for the treatment of malignant mesothelioma.
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| 3. |
- Rundlöf, Anna-Klara
(författare)
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Expression of thioredoxin reductase 1 in mammalian cells with regulation by the core promoter and use of alternative splice variants
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Reactive oxygen species are generated in cells as by-products of aerobic metabolism. To protect from damaging effects the cells possess several antioxidant systems and one of the most important is the thioredoxin system. The thioredoxin system is found in all organisms and consists of thioredoxin (TXN), thioredoxin reductase (TXNRD) and NADPH and it participates in many different types of reactions. Mammalian TXNRD1 is a selenoprotein containing a redox active selenocysteine residue and displays a broad substrate specificity. Three mammalian TXNRD isoenzymes have been identified, one cytosolic (TXNRD1), one mitochondrial (TXNRD2), and one present mainly in testis (TGR). The aim of this thesis project has been to study the regulation of TXNRD1 expression at different levels and in different cells and tissues. The expression of TXNRD1 was analyzed with Northern blots revealing lowest mRNA levels in testis and highest in liver and kidney. To further analyze the expression in kidney in situ hybridization studies were performed. This showed a highly structured expression pattern with the mRNA prominently synthesized in the proximal tubules of the medullary rays. Using bioinformatic tools combined with 5'RACE PCR we found several TXNRD1-specific transcripts with different 5' ends. In total, 20 different splice variants were subsequently identified, arising from at least three separate promoters and encoding 5 different open reading frames, including one novel glutaredoxin motif and one alternative Nterminal domain likely to encompass a signal peptide for mitochondrial targeting. We have cloned and characterized a genomic region upstream of the most common 5'-UTR variant(s) of human TXNRD1. that showed promoter activity as detected with a luciferase reporter vector. Deletion constructs revealed that the promoter activity was maintained within the - 115 to +167 region in all cell lines tested and was considered the core promoter. This core promoter contained a POU motif, which bound the Oct-I transcription factor, and two sites in the promoter were found to bind Sp1 and Sp3. Mutants with only three altered key nucleotides prevented binding of Sp1/Sp3 and resulted in a marked decrease of transcriptional activity. The promoter displayed typical characteristics for a housekeeping gene. However, using RT-PCR analysis we found that oxidized LDL (oxLDL), but not native LDL, dose dependently increased TXNRD1 mRNA in human monocyte-derived macrophages (HMDM). and THP-1 cells or HMDM transiently transfected with TXNRD1 promoter fragments allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDL. Gel mobility shift analyses identified a 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDL, however without evident involvement of the Fos, Jun, Nrf2 or HoxA5 transcription factors. In the literature many reports show that TXNRD1 expression is regulated in response to diverse signaling events. With a housekeeping-type promoter for the most common transcripts, we believe that regulation in response to signaling mainly may be governed on other levels than an altered core promoter activity. TXNRD1 activity in cells hence seems to be modulated by an intricate interplay of mechanisms, involving a housekeeping-type promoter in combination with alternative splice variants and transcriptional start sites, post transcriptional regulation through AU-rich elements, inactivation by electrophilic agents and by the enzyme itself modulating the effects of several key signaling molecules,
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