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. 2012 Mar;11(3):604-15.
doi: 10.1158/1535-7163.MCT-11-0599. Epub 2012 Jan 16.

The relationship of thioredoxin-1 and cisplatin resistance: its impact on ROS and oxidative metabolism in lung cancer cells

Medhi Wangpaichitr  1 , Elizabeth J Sullivan, George Theodoropoulos, Chunjing Wu, Min You, Lynn G Feun, Theodore J Lampidis, Macus T Kuo, Niramol Savaraj
Affiliations

The relationship of thioredoxin-1 and cisplatin resistance: its impact on ROS and oxidative metabolism in lung cancer cells

Medhi Wangpaichitr et al. Mol Cancer Ther. 2012 Mar.

Abstract

Elimination of cisplatin-resistant lung cancer cells remains a major obstacle. We have shown that cisplatin-resistant tumors have higher reactive oxygen species (ROS) levels and can be exploited for targeted therapy. Here, we show that increased secretion of the antioxidant thioredoxin-1 (TRX1) resulted in lowered intracellular TRX1 and contributed to higher ROS in cisplatin-resistant tumors in vivo and in vitro. By reconstituting TRX1 protein in cisplatin-resistant cells, we increased sensitivity to cisplatin but decreased sensitivity to elesclomol (ROS inducer). Conversely, decreased TRX1 protein in parental cells reduced the sensitivity to cisplatin but increased sensitivity to elesclomol. Cisplatin-resistant cells had increased endogenous oxygen consumption and mitochondrial activity but decreased lactic acid production. They also exhibited higher levels of argininosuccinate synthetase (ASS) and fumarase mRNA, which contributed to oxidative metabolism (OXMET) when compared with parental cells. Restoring intracellular TRX1 protein in cisplatin-resistant cells resulted in lowering ASS and fumarase mRNAs, which in turn sensitized them to arginine deprivation. Interestingly, cisplatin-resistant cells also had significantly higher basal levels of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Overexpressing TRX1 lowered ACC and FAS proteins expressions in cisplatin-resistant cells. Chemical inhibition and short interfering RNA of ACC resulted in significant cell death in cisplatin-resistant compared with parental cells. Conversely, TRX1 overexpressed cisplatin-resistant cells resisted 5-(tetradecyloxy)-2-furoic acid (TOFA)-induced death. Collectively, lowering TRX1 expression through increased secretion leads cisplatin-resistant cells to higher ROS production and increased dependency on OXMET. These changes raise an intriguing therapeutic potential for future therapy in cisplatin-resistant lung cancer.

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Figures

Figure 1
Figure 1. CR lung cancer cells express lower levels of thioredoxin 1 protein
(A) Fluorometer analysis of H2O2 in various lung cancer cell lines detected by APFB probe indicates that CR lung cancer cell lines expressed higher basal levels of ROS (Mean SD of three experiments). (B) Immunoblot of TRX1 and TRX2 in lung cancer cell lines showed that resistant variants expressed lower levels of TRX1 while no significant changes in TRX2 occurred. Actin was used as a loading control. (C) RT-PCR of TRX1 in lung cancer cell lines indicated that all 4 cell lines possess similar levels of TRX1 mRNA expression. (D) Using thioredoxin reductase 1 activity assay, CR cells possess lower TRX1 activity when compared to their parental cells (*p<0.001, **p=0.003). (E) The concentration of extracellular TRX1 in culture medium. CR cells secreted greater levels of TRX1 when compared to their parental counterparts (*P=0.001, **P=0.002). (F) Immunohistochemistry of TRX1 in mouse xenograft tissue showed that SR2 possess lower levels of TRX1 protein. (G) The concentration of extracellular TRX1 in mouse serum. Mice bearing SR2 xenografts were found to have higher levels of TRX1 in the serum. The value (ng/ml) represents the average of 3 mice per group. (p=0.03; SCLC1 vs. SR2). (H) Immunocytochemistry of TRX1 in lung cancer cell cultures also showed decreased levels of TRX1 in CR cells (200X).
Figure 2
Figure 2. Down-regulation of TRX-1 results in increased ROS production, sensitivity to elesclomol, and resistance to cisplatin in a SCLC1 cell line
We selected SCLC1 which possess the highest amount of TRX1 while its cisplatin resistant variant (SR2) had the lowest amount of TRX1 to do the study. (A1&A2) Immunoblot of TRX1 and TRX2 in SCLC1scramble (control), SR2, SCLCsiTRXC1, and SCLCsiTRXC2 cells Two different siRNAs were able to down-regulate TRX1 by 80% at 48 hr. post transfection while no effect occurred in TRX2. Actin was used as a loading control. (B1&B2) Down-regulation of TRX1 resulted in significant ROS production. (*p=0.02, **p=0.035). (C1&C2) Growth inhibitory effect of cisplatin for 72hrs showed that down-regulation of TRX1 in SCLC1 cells resulted in resistance to cisplatin treatment (*p=0.02, **P=0.001, ***p<0.05, ****p=0.04). (D1&D2) Growth inhibitory effect of elesclomol for 72hrs showed that down-regulation of TRX1 in SCLC1 results in increased sensitivity to elesclomol treatment (*p=0.01, **P=0.003, ***p=0.12, ****p=0.04). (Mean SD of three experiments)
Figure 3
Figure 3. Over-expression of TRX-1 results in decreased ROS production, resistance to elesclomol, and sensitivity to cisplatin in the SR2 cell line
(A1&A2) Immunoblot of TRX1 in SCLC1, SR2, SR2TRX+C1, and SR2TRX+C2 cells. Both TRX1 over-expressing clones expressed about 3–4 fold increase in TRX protein when compared to SR2. Actin was used as a loading control. (B1&B2) Over-expressing of TRX1 resulted in decreased ROS production in SR2 cell lines (*p=0.002, **p=0.01). (C1&C2) Growth inhibitory effect of cisplatin for 72hrs showed that over-expressing of TRX1 in SR2 resulted in increased sensitivity to cisplatin treatment (*p=0.001, **p=0.03, ***p<0.05, ****p<0.05). (D1&D2) Growth inhibitory effect of elesclomol for 72hrs indicated that over-expressing of TRX1 in SR2 resulted in resistance to elesclomol treatment (*p=0.007, **P=0.006, ***p=0.008, ****p=0.004). (Mean SD of three experiments)
Figure 4
Figure 4. CR cells consume more oxygen and have higher mitochondrial activity than parental cells
(A) SCLC1 and SR2 were assayed for baseline oxygen consumption. Live cells were counted and placed in an oxygen chamber. The amount of oxygen consumed per cell in 10min was measured. The rate of oxygen consumption (O2 nM/mL/cell/min) was four times higher in SR2 cells than SCLC1 cells. (B) SCLC1 and SR2 were incubated with 100nM of MitoTracker. Bar graph represents the relative fluorescent units/cell via fluorometer plate reader. SR2 cells possess higher number of active mitochondria when compared to SCLC1 (p<0.001). (C) Flow analysis of MMP in SCLC1 vs. SR2, using 50nM of TMRE. SR2 possess significant higher levels of MMP (p<0.001). (D) Media from the SCLC1 vs. SR2 cells was collected and used in a chromatogenic assay to measure amounts of lactic acid (nM/well/cell). SR2 cells produced less lactic acid than CR cells (Mean SD of three experiments; p<0.05).
Figure 5
Figure 5. CR cells preferentially used OXMET which partly reversed upon over-expression of TRX1
(A&B) Relative mRNA levels of FH and ASS. Total RNAs extracted from these cells were reverse-transcribed and subsequently used as template for real-time quantitative PCR. GAPDH was used as internal control. The results shown in the graph were calculated with the ΔΔCt method by setting the FH or ASS mRNA level of SCLC1 as 1. Relative abundance of FH and ASS mRNA were higher in CR cells, but decreased with TRX1 over-expression. (C) Comparison of growth inhibition of SCLC1, SR2, SR2TRX+C1, and SR2TRX+C2 in arginine free media supplement with citrulline. At 72 hrs, only 30% of SCLC1 survive compared to 60–65% of TRX1 over-expressing clones, and 80% of SR2 were viable. (D) Immunoblot of key enzymes (ACL,ACC, FAS) in the FA synthesis pathway. CR cells had relative higher levels of expression in all of these proteins than parental cells. Over-expression TRX-1 protein can partly suppress these FA synthesis enzymes. Actin was used as a loading control. (E) Bar graph indicates the relative adjusted density of indicated protein expressions in each cell line. SR2TRX+C2 yielded similar results (data not shown)
Figure 6
Figure 6. Downregulation of ACC enhances cell death in cisplatin resistant cells
(A) Cells were treated by TOFA to inhibit ACC and cytotoxicity was assessed. Data are shown as % cell death as compared to untreated samples for each cell line. Over-expressing TRX1 protected CR cells against TOFA induced cell death. (B) Down regulation of ACC by siRNA (only 1nM is used to minimize the off-target effect) Immunoblot of ACC in SR2scramble, and SRsiACC cells showed more than 90% decrease in ACC 48hrs post-transfection. Actin was used as a loading control. (C) Comparison of cell death in SR2scramble and SRsiACC, ACC knocked down resulted in significant increase in cell death. (D) A proposed model: Acquired resistance to cisplatin disrupted the redox system through inhibition of TRX1 system (TrxR1/TRX1) causing TRX1 secretion. Decreased TRX1 resulted in accumulation of cellular ROS. Further increased ROS using elesclomol in these cisplatin resistant (CR) cells can push them beyond their tolerance limit which ultimately leads to cell death. Decreased TRX1 levels may involve metabolic reprogramming by switching CR cells from glycolysis toward OXMET. Inhibiting key metabolic enzymes in FA synthesis pathway led to significant cell death in CR cells.
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