Original Contribution
Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells

https://doi.org/10.1016/j.freeradbiomed.2008.05.011Get rights and content

Abstract

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (γ-glutamylcysteine ligase heavy chain (γ-GCLh), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and γ-GCLh protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-β-estradiol or ICI-182,780 did not significantly change γ-GCLh expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.

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).

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    These two authors contributed equally to this work.

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