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      Hyperglycemia regulates thioredoxin-ROS activity through induction of thioredoxin-interacting protein (TXNIP) in metastatic breast cancer-derived cells MDA-MB-231

      , 1 , 2 , 1 , 2 , 3

      BMC Cancer

      BioMed Central

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          We studied the RNA expression of the genes in response to glucose from 5 mM (condition of normoglycemia) to 20 mM (condition of hyperglycemia/diabetes) by microarray analysis in breast cancer derived cell line MDA-MB-231. We identified the thioredoxin-interacting protein (TXNIP), whose RNA level increased as a gene product particularly sensitive to the variation of the level of glucose in culture media. We investigated the kinesis of the TXNIP RNA and protein in response to glucose and the relationship between this protein and the related thioredoxin (TRX) in regulating the level of reactive oxygen species (ROS) in MDA-MB-231 cells.


          MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Cells were analyzed with Affymetrix Human U133A microarray chip and gene expression profile was obtained. Semi-quantitative RT-PCR and Western blot was used to validate the expression of TXNIP RNA and protein in response to glucose, respectively. ROS were detected by CM-H2DCFDA (5–6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) and measured for mean fluorescence intensity with flow cytometry. TRX activity was assayed by the insulin disulfide reducing assay.


          We found that the regulation of TXNIP gene expression by glucose in MDA-MB-231 cells occurs rapidly within 6 h of its increased level (20 mM glucose) and persists through the duration of the conditions of hyperglycemia. The increased level of TXNIP RNA is followed by increased level of protein that is associated with increasing levels of ROS and reduced TRX activity. The inhibition of the glucose transporter GLUT1 by phloretin notably reduces TXNIP RNA level and the inhibition of the p38 MAP kinase activity by SB203580 reverses the effects of TXNIP on ROS-TRX activity.


          In this study we show that TXNIP is an oxidative stress responsive gene and its expression is exquisitely regulated by glucose level in highly metastatic MDA-MB-231 cells.

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          Most cited references 24

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          Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function.

           E Junn,  E. Cho,  Ivana K. Kim (2000)
          As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.
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            Hyperglycemia promotes oxidative stress through inhibition of thioredoxin function by thioredoxin-interacting protein.

            Increased intracellular reactive oxygen species (ROS) contribute to vascular disease and pro-atherosclerotic effects of diabetes mellitus may be mediated by oxidative stress. Several ROS-scavenging systems tightly control cellular redox balance; however, their role in hyperglycemia-induced oxidative stress is unclear. A ubiquitous antioxidative mechanism for regulating cellular redox balance is thioredoxin, a highly conserved thiol reductase that interacts with an endogenous inhibitor, thioredoxin-interacting protein (Txnip). Here we show that hyperglycemia inhibits thioredoxin ROS-scavenging function through p38 MAPK-mediated induction of Txnip. Overexpression of Txnip increased oxidative stress, while Txnip gene silencing restored thioredoxin activity in hyperglycemia. Diabetic animals exhibited increased vascular expression of Txnip and reduced thioredoxin activity, which normalized with insulin treatment. These results provide evidence for the impairment of a major ROS-scavenging system in hyperglycemia. These studies implicate reduced thioredoxin activity through interaction with Txnip as an important mechanism for vascular oxidative stress in diabetes mellitus.
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              Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression.

              Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D(3) up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1alpha, 25-dihydroxyvitamin D(3) caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

                Author and article information

                BMC Cancer
                BMC Cancer
                BioMed Central (London )
                7 June 2007
                : 7
                : 96
                [1 ]Department of Medicine, Feist-Weiller Cancer Center, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, Louisiana, 70103, USA
                [2 ]Gene Therapy Program, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, Louisiana, 70103, USA
                [3 ]Department of Molecular and Cellular Physiology, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, Louisiana, 70103, USA
                Copyright © 2007 Turturro et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Research Article

                Oncology & Radiotherapy


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