Tumor cells generate substantial amounts of reactive oxygen species (ROS), engendering the need to maintain high levels of antioxidants such as thioredoxin (Trx)- and glutathione (GSH)-dependent enzymes. Exacerbating oxidative stress by specifically inhibiting these types of ROS-scavenging enzymes has emerged as a promising chemotherapeutic strategy to kill tumor cells. However, potential redundancies among the various antioxidant systems may constrain this simple approach. Trx1 and thioredoxin reductase 1 (Txnrd1) are upregulated in numerous cancers, and Txnrd1 has been reported to be indispensable for tumorigenesis. However, we report here that genetic ablation of Txnrd1 has no apparent effect on tumor cell behavior based on similar proliferative, clonogenic, and tumorigenic potential. This finding reflects widespread redundancies between the Trx- and GSH-dependent systems based on evidence of a bypass to Txnrd1 deficiency by compensatory upregulation of GSH-metabolizing enzymes. Because the survival and growth of Txnrd1-deficient tumors were strictly dependent on a functional GSH system, Txnrd1−/− tumors were highly susceptible to experimental GSH depletion in vitro and in vivo. Thus, our findings establish for the first time that a concomitant inhibition of the two major antioxidant systems is highly effective in killing tumor, highlighting a promising strategy to combat cancer. Cancer Res; 70(22); 9505–14. ©2010 AACR.
Although the activated oncogene is indispensable for the maintenance of the malignant phenotype (1, 2), reliance on various pathways counteracting the oncogenesis-associated cellular stresses is commonly associated with tumorigenesis, collectively termed as “nononcogene addiction” (3, 4), which could be an Achilles' heel for cancer. Higher reactive oxygen species (ROS) production, constitutive oxidative stress, and overstrained antioxidant defense systems differentiate tumor cells from their untransformed counterpart (5), as the event of transformation is followed by increased production of ROS (6–9). This unique feature of tumor cells can be exploited for “selective toxicity” using the redox modifiers like l-buthionine sulfoximine (BSO), ascorbic acid, arsenic trioxide, imexon, phenethyl isothiocyanate, and motexafin gadolinium that selectively kill the tumor cells by perturbing the redox homeostasis (10).
The cellular redox homeostasis is mainly maintained by the thioredoxin (Trx)- and glutathione (GSH)-dependent systems. The Trx system, which consists of Trx, thioredoxin reductase (Txnrd), and Trx-dependent peroxidase (peroxiredoxins), is vital for antioxidant defense, DNA synthesis, and redox-regulated signal transduction, gene expression, and apoptosis (11). Whereas both Trxs and Txnrds are indispensable for embryonic development in mammals (12–16), cytosolic Trx (Trx1) and cytosolic Txnrd (Txnrd1) are essential for cell proliferation (13, 17), as they provide reducing equivalents to ribonucleotide reductase and are thus involved in the maintenance of the deoxynucleotide triphosphate pool (18, 19).
The critical involvement in sustaining high proliferation rates (13, 14, 16) marks the Trx1/Txnrd1 system as a promising drug target for cancer therapy (20). In fact, Trx1 expression level positively correlates with high proliferation capacity, low apoptosis, elevated metastasis (21), increased drug resistance (22), and decreased patient survival (23). By contrast, the role of Txnrd1 in cancer is less clear (24) and could be tumor origin dependent (20). Txnrd1 inactivation by chemical inhibition (25) or small interfering RNA (siRNA)–mediated knockdown (26–28) inhibits self-sufficiency of tumor cells, reverts the malignant phenotype, and sensitizes tumor cells toward the oxidative stress–inducing agents H2O2 and ionizing radiation (29). However, the lack of genetic models had thus far precluded to unequivocally assign a crucial role to Txnrd1 in tumorigenesis because of the intertwined functions of Trx1 and Txnrd1. Moreover, the degree of redundancies among various antioxidant systems in cancer has remained obscure. Using a genetic approach, we show that targeted disruption of Txnrd1 has hardly any effect on proliferation and clonogenic or tumorigenic potential of transformed cells. Furthermore, we identify distinct alterations in GSH metabolism, which compensate for the lack of Txnrd1, rendering knockout tumor cells highly susceptible to GSH deprivation.
Materials and Methods
Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich. pBJ3Ω c-myc and pUC EJ6.6 H-rasv12 were kindly provided by Dr. Hartmut Land. For gene transfer, the lentivirus vector system was used as described (30). Antibodies against human Trx and Txnrd1 were kindly provided by Dr. Vadim Gladyshev (Redox Biology Center). The Tat-Cre protein was a generous gift from Dr. W. Hammerschmidt (Helmholtz Zentrum München). Antibodies against glutathione S-transferases (GST), heme oxygenase-1 (HMOX1), and aldehyde oxidase 1 (AOX1) were kindly provided by Dr. Dolph Hatfield (NIH).
Isolation of mouse embryonic fibroblasts, establishment of transformed Txnrd1−/− cell lines, and reconstitution of Txnrd1 expression
Mouse embryonic fibroblasts (MEF), isolated from conditional Txnrd1 knockout mouse embryos (13), were transformed by cotransducing them with c-myc– and H-rasv12–expressing lentiviruses. In vitro deletion of Txnrd1 was achieved by treating the cells with Tat-Cre fusion protein (31). For reconstitution, the Txnrd1−/− cells were stably transfected with the tetracycline-inducible expression vector pRTS-1-SF-Txnrd1 (32). For induction, doxycycline (Dox), a semisynthetic tetracycline that is active at lower concentration and more stable than tetracycline, was used throughout this study.
Cell proliferation and cytotoxicity were measured by the MTT assay as described (ref. 33; Supplementary Data).
SDS-PAGE and Western blotting were performed as described previously (30).
Cell cycle analysis
The cell cycle analysis was done by the propidium iodide (PI) method (34), and data were analyzed by ModFitLT V3.0 software (Supplementary Data).
Soft agarose assay
Five hundred cells per well were plated in 0.3% soft agarose prepared in cell culture medium in a six-well cell culture plate and allowed to grow for 10 days. Colonies were fixed in methanol, stained with 0.5% crystal violet, and counted.
Quantitative reverse transcription-PCR
Quantitative reverse transcription-PCR (RT-PCR) was carried out by using the LightCycler FastStart DNA MasterPLUS SYBR Green I kit in combination with the LightCycler 1.5 System (Roche Diagnostic). Expression was normalized against 18S RNA and presented as fold change with respect to the parental cells.
Measurement of GSH concentration by high-performance liquid chromatography
GSH concentrations were measured by the isocratic high-performance liquid chromatography as described (35).
Determination of GSH reductase activity
GSH reductase (GR) activity was measured by monitoring the decrease in absorbance at 340 nm wavelength resulting from consumption of NADPH (36) in a 96-well plate (Supplementary Data).
Generation of Txnrd1-deficient B-cell lymphoma mice
To achieve B cell–specific deletion of Txnrd1, conditional Txnrd1 knockout mice (Txnrd1fl/fl; ref. 13) were bred with B cell–specific Cre-expressing mice (mb-1 Cre; ref. 37). To address the role of Txnrd1 in c-myc–driven B-cell lymphoma, Txnrd1+/fl;mb-1 Cre mice were crossed with λ-myc mice (38). The breeding scheme is shown in Fig. 3A.
Subcutaneous implantation of transformed cells into C57BL/6 mice
For s.c. tumor transplantation experiments, 8-week-old C57BL/6 mice were purchased from Janvier. Transformed cells (1 × 105) in a final volume of 100 μL were injected s.c. into the mice. After 12 days of tumor growth, mice were sacrificed, and the tumor mass was determined. Mice were kept under standard conditions with food and water ad libitum (Ssniff). All animal experiments were performed in compliance with the German animal welfare law and have been approved by the institutional committee on animal experimentation and the government of Oberbayern.
Treatment of Txnrd1−/− tumor-bearing mice with BSO
Transformed Txnrd1−/− cells (1 × 105) and the parental Txnrd1fl/fl cells were implanted s.c. into C57BL/6 mice. Tumors were allowed to settle for 3 days. Then, BSO (20 mmol/L) was provided in drinking water for 10 days. BSO-containing water was changed twice a week. At the end of the experiment, mice were sacrificed, and the tumor mass was determined.
Transformation and in vitro deletion of Txnrd1 in conditional Txnrd1 knockout MEFs
To address the supposedly vital role of Txnrd1 in oncogenesis by genetic means, we generated immortalized MEF cell lines from conditional Txnrd1 knockout mouse embryos (two Txnrd1fl/fl and one Txnrd1+/fl; fl, floxed). This became necessary as we had invariably failed to establish MEFs directly from Txnrd1−/− embryos (13). The gene targeting strategy previously used for targeted disruption of Txnrd1 is depicted in Fig. 1A (13). We used Txnrd1fl/fl and Txnrd1+/fl cells as parental cell lines to exclude potential clonal effects during isolation and subsequent culturing. Moreover, the floxed Txnrd1 allele was shown to behave like wild-type Txnrd1 allele (13). Therefore, we considered the Txnrd1fl/fl and Txnrd1+/fl cells as best available controls for the following experiments. The procedure of establishing transformed Txnrd1−/− cell lines is schematically depicted in Fig. 1B. Transformation of the immortalized fibroblasts was achieved by the well-established cooperative action of the two oncogenes c-myc and H-rasv12 (39). Outgrowing single-cell colonies in soft agar were individually picked and expanded to establish the clonally transformed cell lines. Expression of c-myc and H-rasV12 in these clones was confirmed by immunoblotting (Fig. 1C). Transformed Txnrd1−/− cell lines were established by treatment of the transformed cells with Tat-Cre protein followed by single-cell cloning (see Supplementary Data for detailed procedure). Knockout of Txnrd1 in the resulting clones was confirmed by PCR, RT-PCR, and immunoblotting (Fig. 1D). Transformed Txnrd1fl/fl parental cells and knockout cells derived from them are called Txnrd1fl/fl and Txnrd1−/− in the following, respectively.
Txnrd1 deficiency does not impair the proliferation of transformed MEFs
Several previous reports, including our unsuccessful attempts to establish Txrnd1−/− cell lines directly from knockout embryos (13), indicated an indispensable role for Txnrd1 in proliferation (13, 17) and tumorigenesis (25–28). Yet, by the procedure described above, we succeeded in generating Txnrd1−/− cell lines. A comparative analysis revealed that there was no difference in the proliferation rate of Txnrd1fl/fl and Txnrd1−/− cells (Fig. 2A). Because the cytosolic Trx system has been implicated in cell proliferation, for example, by providing reducing equivalents to ribonucleotide reductase (18, 19), we hypothesized that cell cycle phases might be altered in Txnrd1−/− cells. But there was no alteration in cell cycle distribution of Txnrd1−/− cells (G0-G1, 33.85 ± 4.7%; S, 52.33 ± 0.8%; G2-M, 13.81 ± 3.8%) compared with the Txnrd1fl/fl cells (G0-G1, 34.55 ± 1.1%; S, 48.76 ± 2.3%; G2-M, 16.68 ± 3.5%; Fig. 2B). This is in line with earlier reports that knockdown of Txnrd1 has no effect on cell cycle progression under normal culture conditions (27), whereas under serum-deprived conditions, transition through S phase is delayed (28).
siRNA-mediated knockdown (26–28) or chemical inhibition (25) of Txnrd1 has been reported to cause the reversal of malignant phenotype and loss of clonogenicity and tumorigenicity. At variance to this expectation, Txnrd1−/− cells were able to form colonies in soft agar to the same extent as Txnrd1fl/fl cells (153 ± 21 versus 215 ± 9; Fig. 2C). Moreover, Txnrd1−/− cells formed tumors of comparable mass (0.57 ± 0.18 g) on s.c. implantation into C57BL/6 mice like the Txnrd1fl/fl cells (0.59 ± 0.25 g; Fig. 2D). Similar results were obtained with another pair of cell lines (data not shown). This indicated that the loss of Txnrd1 had no effect on clonogenicity and tumorigenicity of transformed cells.
Txnrd1 is dispensable for c-myc–driven B-cell lymphomagenesis
Previously, we found that Txnrd1 is a target gene of the oncogene c-myc (40). To address the role of Txnrd1 in B-cell lymphomagenesis, we crossed the conditional Txnrd1 knockout mice (13) with λ-myc mice (38), which develop lymphoma of immature B-cell origin. B cell–specific deletion of Txnrd1 was achieved by crossing Txnrd1fl/fl mice with B cell–specific Cre-expressing mice (mb-1 Cre mouse; ref. 37). The mb-1–driven Cre is expressed at the very early pro-B-cell stage and is therefore the best available pan-B cell–specific Cre mouse strain. The breeding strategy and genotyping of the mice are depicted in Fig. 3A. To our surprise, there was no effect of Txnrd1 deletion on lymphoma development and survival in diseased mice as shown in Fig. 3B. Ten of 11 Txnrd1fl/fl;Cre+,myc+ mice developed tumors (median survival period of 117 ± 39.4 days) with comparable incidence with Txnrd1fl/fl;Cre,-myc+ mice (9 of 11 succumbed to the disease with a median survival of 108 ± 69.7 days; Fig. 3B). Txnrd1 knockout was confirmed by semiquantitative RT-PCR using primer pairs binding in exons 13 and 15 of murine Txnrd1 (Fig. 3C). Having shown that loss of Txnrd1 has no effect on tumor growth in two different tumor models, fibrosarcoma and myc-driven B-cell lymphoma, we hypothesized a compensatory upregulation of a yet unrecognized pathway as a plausible explanation.
Upregulation of GSH metabolizing enzymes is observed under Txnrd1 deficiency
To study the compensatory mechanisms rendering Txnrd1 dispensable for cell proliferation and tumor development, we used the Txnrd1-deficient transformed MEF cell lines for the subsequent analyses. Studies with mitochondrial Txnrd (Txnrd2) revealed that Txnrd2−/− cells rapidly die in response to GSH depletion by BSO, a highly specific and irreversible inhibitor of the rate limiting enzyme γ-glutamylcysteine synthetase (γ-GCS) in GSH biosynthesis (15). We therefore treated Txnrd1−/− and Txnrd1fl/fl cells with increasing concentrations of BSO. Like Txnrd2−/− cells, Txnrd1−/− cells readily died on treatment with 10 μmol/L BSO (3.06 ± 1.12% viability), whereas the viability of Txnrd1fl/fl cells was not affected even at 3-fold higher concentrations of BSO (100.48 ± 5.55% viability at 30 μmol/L BSO; Fig. 4A). We thus conclude that GSH is essential for proliferation and survival of Txnrd1−/− cells.
We hypothesized that upregulation or increased activity of GSH-metabolizing enzymes may compensate for the loss of Txnrd1 and thus determined the expression and activity of various key components of the GSH-dependent system in Txnrd1−/− cells. Txnrd1−/− cells exhibited a 3-fold (3.17 ± 1.5) increase in the transcript of the catalytic subunit (Gclc), an 8-fold (7.88 ± 1.76) increase of the modifier subunit (Gclm) of γ-GCS, and a 2.5-fold (2.46 ± 0.23) increase in GSH reductase (Gsr) transcripts compared with Txnrd1fl/fl cells (Fig. 4B).
These results were corroborated by measuring the intracellular GSH concentrations and GR activity in Txnrd1−/− cells. Total [GSH + oxidized GSH (GSSG)] and reduced GSH levels were 2.7-fold higher in Txnrd1−/− cells than in Txnrd1fl/fl cells (209.24 ± 68.26 versus 81.21 ± 19.87 μmol/L/mg protein for total and 186.81 ± 59.94 versus 74.16 ± 17.45 μmol/L/mg protein for reduced GSH). Likewise, GSSG was 3-fold increased (11.21 ± 4.47 versus 3.52 ± 2.64 μmol/L/mg protein) in Txnrd1−/− cells compared with Txnrd1fl/fl cells (Fig. 4C). However, this apparent increase in total (GSH + GSSG), reduced (GSH), and oxidized (GSSG) did not alter the GSH/GSSG redox couple considerably as the ratio of GSH/GSSG remained virtually constant in knockout and wild-type cells (16.6 versus 21.4). Additionally, Txnrd1−/− cells exhibited ∼2.5-fold higher GR activity than the Txnrd1fl/fl cells (2.05 ± 0.36 versus 0.87 ± 0.27 units/mg protein; Fig. 4D).
Reconstitution of Txnrd1 expression in knockout cells reverts the compensatory upregulation of GSH-metabolizing enzymes
To provide definitive proof that the increases in GSH levels and GR activity are indeed caused by the loss of Txrnd1 and are not due to clonal variations, Strep-FLAG–tagged wild-type mouse Txnrd1 (SF-Txnrd1) was expressed in Txnrd1−/− cells from the tetracycline-inducible expression vector pRTS-1 (32). Dox-inducible expression of SF-Txnrd1 was confirmed by immunoblotting (Fig. 5A). As illustrated in Fig. 5B, Txnrd1−/− cells survived treatment with 20 μmol/L BSO only when Dox (1 μg/mL) was added to the cell culture medium. This indicates that, in the absence of Txnrd1, survival and proliferation of cells relied on the GSH system.
Next, we asked whether reexpression of Txnrd1 in Txnrd1−/− cells may reduce the augmented GSH levels and GR activity (Fig. 5C and D). The total (444.30 ± 47.86 versus 164.14 ± 5.05 μmol/L/mg protein) and reduced (420.66 ± 41.04 versus 156.34 ± 1.02 μmol/L/mg protein) GSH concentrations in untreated Txnrd1−/− cells were 2.7-fold higher than in Dox-treated cells. Moreover, Dox treatment caused a 3-fold reduction in GSSG levels (3.90 ± 3.03 μmol/L/mg protein) compared with untreated cells (11.82 ± 3.41 μmol/L/mg protein; Fig. 5C). As observed before, when GSH and GSSG levels in knockout and control cells were compared, the GSH/GSSG ratio remained virtually unchanged on reexpression of Txnrd1 (35.5 in untreated cells versus 40.0 in Dox-treated cells). The drop in GSH levels was paralleled by an ∼50% reduction in GR activity after Dox addition (2.8 ± 0.3 versus 1.4 ± 0.3 units/mg protein; Fig. 5D). The reversal of augmented GSH levels and GR activity on reintroduction of Txnrd1 further lends support to our hypothesis that Txnrd1−/− cells upregulate the GSH-dependent pathway as a compensatory mechanism for the missing Txnrd1 function.
Upregulation of phase II enzymes in Txnrd1−/− cells
Sengupta and colleagues reported that targeted removal of the gene encoding selenocysteine-specific tRNA (Trsp) in liver leads to compensatory upregulation of phase II response genes including GSTs, HMOX1, and AOX1 (41). Given that Txnrd1 is a selenoprotein, we asked whether Txnrd1 disruption may also affect phase II gene expression in a manner similar to that observed in the selenoprotein-deficient liver. In fact, compensatory upregulation of phase II enzymes (GST and HMOX1) was observed in Txnrd1−/− cells, but not of AOX1 (see Fig. 4E).
Txnrd1−/− tumors are highly susceptible to pharmacologic GSH deprivation
If the growth of Txnrd1−/− tumor cells is dependent on the GSH-dependent system also in vivo, then pharmacologic intervention of GSH synthesis and/or metabolism might be of substantial therapeutic relevance. To test this, Txnrd1−/− tumor-bearing mice (see also Fig. 2D) were treated with 20 mmol/L BSO in the drinking water (Fig. 6A). As shown in Fig. 6B and C, Txnrd1−/− tumors were highly susceptible to BSO treatment in vivo and only gave rise to very small tumors (0.062 ± 0.05 g) compared with Txnrd1−/− tumors (0.48 ± 0.42 g) in untreated mice. The absence of Txnrd1 in these tumors was confirmed by Western blotting (Fig. 6D). Interestingly, no effect of BSO treatment was observed in Txnrd1fl/fl tumor-bearing mice (0.40 ± 0.20 g treated versus 0.38 ± 0.32 g untreated tumors). These studies suggest that the growth of Txnrd1-deficient tumors relies on a functional GSH-dependent system and that targeting Txnrd1 alone may not be sufficient to restrain tumor growth.
Compelling evidence established that Txnrd1 and in particular Trx1 are upregulated in numerous cancers (21). Given the procancerous function of the Trx system (21–23), both Txnrd1 and Trx1 are suitable candidates for cancer therapy. Here, we addressed two important questions: (a) can Txnrd1 deficiency be compensated by intrinsic mechanisms and (b) is the combined use of drugs simultaneously targeting Trx- and the GSH-dependent systems of potential pharmacologic value to combat cancer?
We show here for the first time that Txnrd1 is dispensable for aggressive tumors and provide evidence for a widespread redundancy between the two major antioxidant systems, the GSH- and Trx-dependent systems, in tumor growth. Although Txnrd1 has been widely regarded as an Achilles' heel for cancer, we failed to recapitulate the findings of previous reports using a well-defined genetic model system (13). Surprisingly, there was no difference in the proliferation, cell cycle distribution, clonogenicity, or tumorigenicity of c-myc– and H-rasv12–transformed Txnrd1−/− cells. Moreover, we could reproduce these results in a c-myc–driven B-cell lymphoma mouse model in which we could not find any effect of loss of Txnrd1 on lymphoma incidence and survival of the mice. Furthermore, the increased susceptibility of Txnrd1−/− cells toward GSH depletion indicated that the proliferation and survival of Txnrd1−/− cells was strictly dependent on the GSH system. The increased susceptibility of tumor cells toward the combined treatment with arsenic (III) oxide that has been reported to inhibit Txnrd and BSO (25) can be explained in the light of findings from our present study. A complex cross-talk between the Trx- and GSH-dependent systems has been reported in yeast (42), Drosophila melanogaster (43), and Arabidopsis thaliana (44); however, the compensatory upregulation of the GSH-dependent system was unprecedented in the context of mammalian tumor growth.
In Txnrd1−/− cells, a 2.5- to 3-fold upregulation in total, reduced, and oxidized GSH was observed, which was reversed on Txnrd1 reexpression. Remarkably, the GSH/GSSG redox couple and thus the redox potential of GSH/GSSG remained unaltered in knockout cells, which may be due to the corresponding increase in GR activity. By upregulating GR level and activity, Txnrd1−/− cells effectively maintain the steady-state redox potential and thereby avoid oxidative stress. Moreover, upregulation of GSH-synthesizing enzymes and phase II enzymes provides an additional mechanism for the protection of Txnrd1−/− cells against oxidative stress.
It should be stressed that cells with a genetic defect in Txnrd1, as reported here, had a better chance to adapt to Txnrd1-independent growth in vitro and in vivo than wild-type cells in which the enzyme was downregulated by siRNA or targeted by Txnrd inhibitors. This may in part explain the discrepancies between our findings and previous reports. Yet, only the genetic system provides the ultimate answer whether compensatory mechanisms exist that may bypass Txnrd1 inhibition and may favor tumor relapse after an initial response to chemotherapy.
Augmented expression of GSH-metabolizing enzymes and phase II enzymes is the probable adaptation mechanism by which Txnrd1−/− cells protect themselves from cell death. This induction is presumably mediated through the nuclear factor E2-related factor 2 (Nrf2) and the Kelch-like ECH associated protein 1 (Keap1) system (45). On oxidation of Keap1, Nrf2 is liberated from the cytosolic Keap1/Nrf2 complex and translocates into the nucleus. Nrf2 induces gene expression by binding to the “antioxidant response element” found in many phase II gene promoters including Gclc, Gclm, and GR (Gsr1) and GSTs (46). Indeed, knockout of all selenoproteins in liver triggers an Nrf2-dependent upregulation of antioxidant enzymes including HO-1, GCLC, and GSTP1 and combined liver-specific disruption of Nrf2 and Trsp causes hepatocyte dysfunction and impaired survival of compound mutant mice (47). Gene expression profiling of mice lacking liver selenoprotein synthesis revealed independently that many phase II enzymes are induced by selenoprotein loss (41). Hence, one may hypothesize that induction of a fraction of phase II enzymes on Trsp removal is mediated in part by Txnrd1 deficiency.
Although cancer cells are susceptible to pharmacologic ROS insults, an upregulation of antioxidant capacity as adaptive response to increased intrinsic oxidative stress might lead to emergence of drug resistance (48). A compensatory upregulation of the GSH-dependent system and phase II enzymes on Txnrd1 deletion and the high susceptibility of Txnrd1-deficient tumors toward pharmacologic inhibition of GSH suggest that simultaneous inhibition of more than one antioxidant systems is particularly efficient in killing tumor cells. Indeed, the redox modifiers like phenethyl isothiocyanate (8) and Motexifen gadolinium (49) have been reported to effectively kill tumor cells by causing the preferential accumulation of ROS, oxidative damage in mitochondria, inactivation of redox-sensitive molecules, and massive cell death.
Simultaneous inhibition of more than one antioxidant system for cancer therapy becomes even more important with the growing evidence that the two key redox systems display widespread redundant functions (35, 50). Recently, we showed that the cystine/cysteine cycle along with Txnrd1 cooperatively rescue GSH deficiency (35). Our data thus rule out a previously alleged indispensable role of Txnrd1 in cancer development. In light of our findings, the use of redox modifiers in cancer treatment should be exercised with great care due to the extensive cross-talk among the key redox systems.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Drs. V. Gladyshev, W. Hammerschmidt, and H. Land for providing the reagents as listed in Materials and Methods; Dr. Dolph Hatfield and Brad Carlson for the generous help for providing the antibodies against the phase II enzymes; Dr. Michael Reth for providing the mb-1 Cre mice; and Drs. Matilde Maiorino and Fulvio Ursini for fruitful discussions. P.K. Mandal thanks Dr. Derrick Rossi for his extended cooperation.
Grant Support: Deutsche Forschungsgemeinschaft (DFG) CO 291/2-1 (M. Conrad), Deutsche Krebshilfe e.V. 10-1979-Bo4 (G.W. Bornkamm), and DFG-Priority Programme SPP1190 (M. Conrad and H. Beck).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.