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Comparative Study
. 2007 Jun 7:7:96.
doi: 10.1186/1471-2407-7-96.

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

Francesco Turturro  1 , Ellen Friday, Tomas Welbourne
Affiliations
Comparative Study

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

Francesco Turturro et al. BMC Cancer. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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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Figures

Figure 1
Figure 1
TXNIP and TRX expression in response to glucose assessed by gene expression profile (GEP) and semi-quantitative PCR. A) 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 harvested at 12 h based on previous growth curves obtained at specified glucose concentration and RNA was isolated, labeled and hybridized to Affymetrix Human U133A microarray chip. Average derived from duplictaes is shown as relative expression of TXNIP and TRX RNAs, respectively. B) TXNIP and TRX RNA message levels were detected by semi-quantitative PCR in MDA-MB-231 cells grown in the same conditions as in A. Average relative levels as compared to control β-actin RNA of TXNIP and TRX RNA messages from duplicates are represented. Representative gel electrophoresis of PCR products obtained from duplicate experiments is shown in the inset.
Figure 2
Figure 2
Time course of the TXNIP RNA expression and response to inhibition of glucose transporter. A) MDA-MB-231 cells were chronically grown at 5 mM and then switched to 20 mM glucose at t = 0. RNA was measured by semi-quantitative PCR at the indicated time points and shown as average ratio of control β-actin RNA levels derived from duplicates. Representative gel electrophoresis of PCR products obtained from 2 experiments is shown in the inset. B) Cells were grown chronically at 5 mM and then switched to 20 mM glucose at t = 0. RNA was measured by semi-quantitative PCR at 6 h and shown as average ratio of control β-actin RNA levels from duplicates. For inhibition of the glucose transporter study cells were pre-treated for 1 h with 300 μM phloretin. Representative gel electrophoresis of PCR products obtained from 2 experiments is shown in the inset.
Figure 3
Figure 3
TXNIP protein expression and time chase of the TXNIP protein expression in response to glucose. A) MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating as described. Cells were harvested and total proteins obtained from cell lysates were run on 10% SDS PAGE gel by electrophoresis and blotted with rabbit polyclonal antibody to TXNIP. Blots were stripped and reprobed for β-actin as loading control to estimate the average ratio of band intensity from duplicates as shown. Representative Western blot obtained from 2 experiments is shown in the inset. B) MDA-MB-231 cells were chronically grown at 5 mM and then switched to 20 mM glucose at t = 0 and treated as described. Cells were harvested at the time points indicated and total proteins obtained from cell lysates were run on 10% SDS PAGE gel by electrophoresis and blotted with rabbit polyclonal antibody to TXNIP. Blots were stripped and reprobed for β-actin as loading control to estimate the average ratio of band intensity from duplicates as shown. Representative Western blot obtained from 2 experiments at each time point is shown in the inset.
Figure 4
Figure 4
ROS and TRX activity in response to glucose and p38 MAPK inhibition. A) TXNIP RNA message levels were detected by semi-quantitative PCR in MDA-MB-231 cells grown either in 5 or 20 mM glucose chronically prior to plating. Average relative levels as compared to control β-actin RNA of TXNIP and TRX RNA messages from triplicates are represented. For inhibition of the p38 MAP kinase cells grown at 20 mM were pre-treated for 24 h with 20 μM SB203580. B) MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. Cells were assessed for ROS levels by DCFDA fluorescence staining and flow cytometry as shown in the inlet. For inhibition of the p38 MAP kinase cells grown at 20 mM glucose were treated for 24 h with 20 μM SB203580. Average mean fluorescence from triplicates is expressed per each group of cells. B) MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. Cells were assessed for TRX activity by the insulin disulfide reducing assay as described and the average OD 410 readings from triplicates are shown for each group of cells. For inhibition of the p38 MAP kinase cells grown at 20 mM were pre-treated for 24 h with 20 μM SB203580.
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