Thioredoxin participates in a cell death pathway induced by interferon and retinoid combination

Apoptosis is a major mechanism of cancer cell destruction by chemotherapy and radiotherapy. The anthracycline class of antitumor drugs undergoes redox cycling in living cells producing increased amounts of reactive oxygen species and semiquinone radical, both of which can cause DNA damage, and consequently trigger apoptotic death of cancer cells. We show here that MCF-7 cells overexpressing thioredoxin (Trx) were more apoptotic in response to daunomycin. Thioredoxin (Trx) 2 is a low molecular mass protein (12 kDa) that is widely distributed; Trx is found within the cytoplasmic, membrane, extracellular, and mitochondrial cellular fractions (1, 2). The Trx system includes Trx, Trx reductase, and peroxiredoxins. Trx reductase is an efficient protein-disulfide reductase that uses NADPH as a source of reducing equivalents. Besides being an antioxidant itself (3, 4), Trx also plays an important role in regulating the expression of other antioxidant genes such as manganese superoxide dismutase (5). Trx overexpression also enhances the expression of peroxiredoxin that could reduce peroxides to molecular oxygen and H 2 O (6). Trx has been shown to regenerate oxidatively inactivated proteins (7,8). In addition to its role as an antioxidant protein, Trx has been shown to have growth promoting properties (9). In contrast, a recent study has demonstrated that overexpression of redox-active Trx could promote cell death via activation of caspase-8 (10). Additional studies have shown that Trx reductase is critical for cell death, and a Trx-dependent mechanism has been suggested (11). Recent studies also indicate that caspases, the executioner of cell death by apoptosis, could be activated by Trx due to its disulfide reducing properties (12). Caspases are rich in cysteine motifs that are required for their catalytic activity. Therefore, oxidation could inhibit caspase activity, which could be restored by the Trx system (12). Furthermore Trx also has been shown to promote p53-DNA binding due to its reducing actions on DNA-binding cysteine motifs on p53 (14). Taken together, accumulating evidence suggests that Trx is a multifunctional protein, which can participate in proliferation as well as cell death process. The antioxidative action of Trx could be due to its manganese superoxide dismutase inducing properties (5, 15) as well as direct scavenging of hydroxyl radicals or singlet oxygen.The anthracycline class of anticancer drugs such as doxorubicin or daunomycin has been shown to induce p53-dependent apoptosis in cancer cells (16,17). Additionally anthracyclines have also been shown to cause DNA damage, which increases p53 expression (18,19). p53 is a sequence-specific transcription factor, which can induce proapoptotic or suppress antiapoptotic genes in response to DNA damage or irreparable cell cycle arrest (20). Phosphorylation of p53 on the Ser 15 residue dissociates MDM2 and activates p53 as a transcription factor, which binds to various p53-dependent genes resulting in their activation or repression (20). While evaluating ...

Section: Thioredoxin (Trx)mentioning

confidence: 83%

Endogenous Thioredoxin Is Required for Redox Cycling of Anthracyclines and p53-dependent Apoptosis in Cancer Cells

Ravi

Muniyappa

Das

2005

“…A proposal with broader perspective for further investigation is that the redox-active protein thioredoxin, capable of reactivating oxidized caspases (60), might be a second crucial regulator of caspase activation and present in different amounts in different cell types. In fact, two recent studies showed that caspase-8-driven apoptosis in MCF-7 cells is promoted by overexpression of thioredoxin plus thioredoxin reductase, and, moreover, that thioredoxin antisense confers resistance against apoptosis induction (32,33). One can conclude from these and other studies that the delicate balance between physiological antioxidants (GSH and thioredoxin) and endogenously produced or exogenous oxidants (ROS and nitric oxide) finally determines whether caspase-initiated apoptosis can precede or is halted.…”

Section: Discussionmentioning

confidence: 93%

“…Vice versa, antioxidants were reported to enhance or facilitate apoptosis, e.g. the antioxidant ␣-liponic acid enhanced CD95-mediated apoptosis in Jurkat cells (31), and the antioxidant protein thioredoxin was necessary for apoptosis induction in MCF-7 cells (32,33). As a proposed mechanism, it has been demonstrated in many studies that under cell-free conditions, caspase activity or caspase activation requires a reducing environment: (i) oxidants such as NO or hydrogen peroxide blocked caspase-3 activity (23,34); (ii) the reductants GSH or DTT were necessary to ensure full caspase-3 activity (28); and (iii) the thiol oxidant disulfiram and oxidized glutathione (GSSG) directly inhibited apoptosome-mediated caspase-3 activation (22).…”

mentioning

confidence: 99%

Glutathione Dependence of Caspase-8 Activation at the Death-inducing Signaling Complex

Hentze¹,

Schmitz²,

Latta³

et al. 2002

Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cischloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathionedepleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.Elimination of functionally impaired or potentially dangerous cells by apoptosis is of paramount importance for the organism. Apoptosis mediated by the death receptor CD95 (also called APO-1 and Fas) has previously been investigated intensively, especially in the immune system and the liver. Triggering of CD95 by agonistic antibodies (␣CD95) or the naturally occurring CD95 ligand (CD95L) initiates trimerization of CD95

“…However, TNF-␣ also activates two transcription factors, AP-1 and NF-B, that induce the expression of genes that act to suppress TNF-␣-induced apoptosis, explaining why the apoptotic response to TNF-␣ usually depends on inhibition of pro-tein synthesis (36). Although several studies have addressed the issue, the role of Trx1 in apoptosis induced by oxidants or other agents remains unclear (37)(38)(39). We examined the effect of TRP14 or Trx1 depletion on TNF-␣-induced apoptosis in HeLa cells in which protein synthesis was blocked by cycloheximide.…”

Section: Roles Of Trp14 and Trx1 In Tnf-␣-induced Apoptosis Andmentioning

confidence: 99%

Roles of TRP14, a Thioredoxin-related Protein in Tumor Necrosis Factor-α Signaling Pathways

Jeong

Chang

Boja

et al. 2004

The possible roles of a 14-kDa human thioredoxin (Trx)-related protein (TRP14) in TNF-␣ signaling were studied in comparison with those of Trx1 by RNA interference in HeLa cells. Depletion of TRP14 augmented the TNF-␣-induced phosphorylation and degradation of I B␣ as well as the consequent activation of NF-B to a greater extent than did Trx1 depletion. Deficiency of TRP14 or Trx1 enhanced TNF-␣-induced activation of caspases and subsequent apoptosis by a similar extent. The TNF-␣-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), however, was promoted by depletion of TRP14 but not by that of Trx1. Unlike Trx1, TRP14 neither associated with nor inhibited the kinase activity of apoptosis signal-regulating kinase-1 (ASK1), an upstream activator of JNK and p38. In combination with the results in the accompanying paper that TRP14 did not reduce the known substrates of Trx1, these results suggest that TRP14 modulates TNF-␣ signaling pathways, provably by interacting with proteins distinct from the targets of Trx1. In an effort to identify target proteins of TRP14, a mutant of TRP14, in which the active site cysteine (Cys 46 ) was substituted with serine, was shown to form a disulfide-linked complex with LC8 cytoplasmic dynein light chain. The complex was detected in HeLa cells treated with H 2 O 2 or TNF-␣ but not in untreated cells, suggesting that LC8 cytoplasmic dynein light chain is a possible substrate of TRP14.The cellular redox state is affected not only by normal respiration and metabolism but also by the transient production of reactive oxygen species (ROS) 1 that results from the ligation of various cell surface receptors (1-3). Tumor necrosis factor-␣ (TNF-␣) was one of the first receptor ligands shown to generate ROS in nonphagocytic cells and is among the ligands whose signaling pathways have been studied most in relation to ROS production (4, 5). The binding of TNF-␣ to TNF receptor 1 (TNFR1) generates both proapoptotic and prosurvival signals by inducing the formation of a multiprotein signaling complex at the cell membrane. This complex in turn triggers the caspase cascade, the activation of specific kinases such as the I B kinase (IKK) complex and mitogen-activated protein kinases (MAPKs), and the transcription of various genes mediated by nuclear factor-B (NF-B) and activator protein-1 (AP-1) (6 -10). Many TNF-␣ signaling pathways are activated by ROS (7, 11-16) and attenuated by Trx overexpression (17-25).In the accompanying study (26), we identified and characterized a 14-kDa cytosolic protein, designated Trx-related protein 14 (TRP14). Here we studied the possible roles of TRP14 in TNF-␣ signaling in comparison with those of Trx1 by RNA interference (RNAi) in HeLa cells. Partial depletion of TRP14 or Trx1 by RNAi resulted in enhancement of TNF-␣-induced activation of caspases and NF-B. The TNF-␣-induced activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK, however, was augmented by depletion of TRP14 but not by that of Trx1. Furthermore, unl...