Original ContributionIncreased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells
Introduction
Breast cancer is the most common malignancy in Western women. The ability to reduce breast tumor growth through the administration of anti-estrogens has played a key role in the endocrine therapy of breast cancer. A non-steroidal antiestrogen, tamoxifen (TAM), is the most widely used antiestrogen in estrogen receptor-positive breast cancer patients [1]. Although most patients are initially responsive, resistance to TAM is a critical problem for anti-estrogen therapy [2].
To establish a TAM-resistant breast cancer cell line, Gottardis and Jordan [3] administered tamoxifen long-term to nude mice bearing MCF-7 cells, and the isolated MCF-7 cells became tamoxifen-resistant. In addition, continuous culture of human breast cancer cell lines with tamoxifen leads to the development of tamoxifen-resistant cell lines. Long-term culture of MCF-7 cells with tamoxifen or 4-hydroxytamoxifen also results in the formation of TAM-resistant cells [4], [5]. We have also established an MCF-7 derived TAM-resistant cell line (TAMR-MCF-7 cells) by long-term (> 9 months) culture of MCF-7 cells with 4-hydroxytamoxifen [6].
Cancer cells can resist oxidative stress by the rapid induction of ROS-protecting enzymes. Glutathione (GSH) is a non-protein sulfhydryl small molecule that scavenges free radicals and electrophiles [7]. γ-glutamylcysteine ligase (GCL), which is composed of two subunits, a catalytic heavy chain (γ-GCLh) and a regulatory light chain (γ-GCLl), is the rate-limiting enzyme for the biosynthesis of GSH [8]. Both γ-GCL subunits are induced by oxidative stress, including phenolic antioxidants, radicals, and GSH depletion via the activation of NF-E2-related factor 2 (Nrf2) or nuclear factor-κB [9], [10], [11]. In addition to GSH, thioredoxin is ubiquitously expressed thiol-reducing antioxidant system [12]. Cysteine-containing peroxiredoxins also represent a new type of peroxidase and these are coupled with thioredoxin and thioredoxin reductase [13]. Recent studies have also shown that the expression of thioredoxin or peroxiredoxin1 is dependent on Nrf2/antioxidant response element (ARE) pathway [14], [15].
The transition of chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is accompanied by the enhancement of cellular radical scavenging systems [16], and tamoxifen may cause apoptosis in estrogen receptor-negative human cancer cells by reactive oxygen species (ROS) formation [17], [18]. Given this role for oxidative stress in the anti-cancer effect of tamoxifen, the expression levels of anti-oxidant proteins in TAM-resistant breast cancer cells may change. Here, we show that the expression of γ-GCLh, heme oxygenase-1 (HO-1), thioredoxin and peroxiredoxin1 is higher in TAMR-MCF-7 cells than in MCF-7 cells, and show that γ-GCLh expression is controlled by sustained Nrf2/Antioxidant Response Element (ARE) activation, but not by NF-κB pathways activation. Moreover, we also reveal that ablation of Nrf2 partially restores TAM-responsiveness in TAMR-MCF-7 cells.
Section snippets
Materials
Anti-γ-GCLh was supplied by Neomarkers (Westinghouse, CA). Antibodies against peroxiredoxin1 and thioredoxin were obtained from Lab Frontier (Anyang, South Korea). Anti-HO-1 was from Assay Designs (Ann Arbor, MI). Horseradish peroxidase-conjugated, donkey anti-rabbit IgG, anti-goat IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). 5-Bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium was from Life
Anti-oxidant proteins are overexpressed in tamoxifen-resistant breast cancer cells
We have previously shown that TAMR-MCF-7 cells are resistant to 4-hydroxytamoxifen-mediated cell death [6]. In the present study, the acquisition of tamoxifen resistance in TAMR-MCF-7 cells was confirmed using an MTT-based proliferation assay. 4-hydroxytamoxifen (3 μM) treatment in control MCF-7 cells significantly inhibited cell proliferation but not in TAMR-MCF-7 cells (Fig. 1A).
In Western blots, basal γ-GCLh protein expression was higher in the TAMR-MCF-7 cells than control MCF-7 cells (Fig.
Discussion
Tamoxifen remains a commonly-prescribed drug for the treatment and prevention of ER-positive breast cancer, as the drug increases survival and helps maintain disease-free status [40]. Tamoxifen works principally through ER antagonism, but also involves ER-independent pathways. Tamoxifen induces apoptosis by increasing oxidative stress, which may mediate its anti-tumor effect [17], [41], [42]. We found that the expression of anti-oxidant proteins including thioredoxin, peroxiredoxin1 and γ-GCLh
Acknowledgments
This work was supported by a grant from the Korean Research Foundation (KRF; Basic Science Research Program E00082, 2005; Kang KW).
References (52)
- et al.
Beneficial effect of adjuvant tamoxifen therapy in primary breast cancer patients with high oestrogen receptor values
Lancet
(1985) - et al.
Tamoxifen (‘Nolvadex’): a review
Cancer Treat. Rev.
(2002) Glutathione: an overview of biosynthesis and modulation
Chem. Biol. Interact.
(1998)- et al.
Increase of intracellular glutathione by low-level NO mediated by transcription factor NF-kappaB in RAW 264.7 cells
Biochim. Biophys. Acta
(2005) - et al.
Reactive oxygen species, antioxidants, and the mammalian thioredoxin system
Free Radic. Biol. Med.
(2001) - et al.
2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications
Trends Mol. Med.
(2005) - et al.
Upregulation of thioredoxin system via Nrf2-antioxidant responsive element pathway in adaptive-retinal neuroprotection in vivo and in vitro
Free Radic. Biol. Med.
(2007) - et al.
Role of the Nrf2-antioxidant system in cytotoxicity mediated by anticancer cisplatin: implication to cancer cell resistance
Cancer Lett.
(2008) - et al.
Tumor necrosis factor increases hepatocellular glutathione by transcriptional regulation of the heavy subunit chain of gamma-glutamylcysteine synthetase
J. Biol. Chem.
(1997) - et al.
Constitutive and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase heavy subunit gene is regulated by a distal antioxidant response element/TRE sequence
J. Biol. Chem.
(1997)
Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2
J. Biol. Chem.
Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene
J. Biol. Chem.
Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein
Biochim. Biophys. Acta
Nuclear factor kappa B dependent induction of gamma glutamylcysteine synthetase by ionizing radiation in T98G human glioblastoma cells
Free Radic. Biol. Med.
Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein-1
J. Biol. Chem.
Inhibition of ERK and p38 MAP kinases inhibits binding of Nrf2 and induction of GCS genes
Biochem. Biophys. Res. Commun.
Estrogen induces death of tamoxifen-resistant MCF-7 cells: contrasting effect of the estrogen receptor downregulator fulvestrant
J. Steroid Biochem. Mol. Biol.
Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells
J. Biol. Chem.
Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages
J. Biol. Chem.
Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-beta
J. Biol. Chem.
Nrf2, not the estrogen receptor, mediates catechol estrogen-induced activation of the antioxidant responsive element
Biochim. Biophys. Acta
Expression of glutathione and gamma-glutamylcysteine synthetase mRNA is Jun dependent
Biochem. Biophys. Res. Commun.
Transcriptional regulation of the heavy subunit chain of gamma-glutamylcysteine synthetase by ionizing radiation
FEBS Lett.
Characterization of gamma-glutamylcysteine synthetase-heavy subunit promoter: a critical role for AP-1
FEBS Lett.
Development of tamoxifen-stimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen administration
Cancer Res.
Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling
Cancer Res.
Cited by (141)
Molecular mechanisms of ROS-modulated cancer chemoresistance and therapeutic strategies
2023, Biomedicine and PharmacotherapyThe beneficial effects of tamoxifen on arteries: A key player for cardiovascular health of breast cancer patient
2023, Biochemical PharmacologyIdentification of Nrf2/Keap1 pathway and its transcriptional regulation of antioxidant genes after exposure to microcystins in freshwater mussel Cristaria plicata
2023, Developmental and Comparative Immunology
- 1
These two authors contributed equally to this work.