Nrf-2/Gst-α mediated imatinib resistance through rapid 4-HNE clearance

https://doi.org/10.1016/j.yexcr.2017.03.004Get rights and content

Highlights

  • Nrf-2 was more abundant in K562R cells and bone morrows of CML patients.

  • Nrf-2 targeted Gst-α serves as a detoxifying agent via facilitating 4-HNE export.

  • Inhibition of 4-HNE clearance restored sensitiveness of K562R cells to imatinib.

Abstract

The advent of imatinib mesylate (IM) has dramatically improved the outcome of patients with chronic myeloid leukemia, but drug resistance, particularly in advanced stage of disease, portents eventual relapse and progression. To identify the candidate molecule responsible for resistance during IM treatment, an IM-resistant K562 cell line was generated by culturing in gradually increasing dose of IM. The expression of Nrf-2 and its downstream target, Gst-α, were significantly induced in these cells. GST-α, in turn, mediated cell survival by maintaining intracellular low level of 4-HNE. Inhibition of Nrf-2 effectively reduced the expression of Gst-α, resulting in accumulation of 4-HNE and elevated sensitiveness to IM. Moreover, in IM-sensitive K562 cells enforced Gst-α expression strikingly protected cells from the insult of IM. Finally, we also examined the levels of Nrf-2 in clinical bone morrow samples. Nrf-2 and Gst-α were more abundant in bone morrow of CML patients compared with that of healthy donors. In addition, Nrf-2 and Gst-α were further up-regulated in samples of patients with weak response to IM. In conclusion, our study shows that rapid clearance of 4-HNE by Nrf-2/GST may represents a novel molecular basis of IM resistance in CML.

Introduction

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder associated with an acquired chromosomal translocation, t (9,22) which leads to the formation of bcr/abl fusion gene, coding for Bcr/Abl fusion protein [1]. This tyrosine kinase with constitutive activity is capable of activating multiple downstream signaling pathways, resulting in deregulation of cell cycle control and resistance to apoptosis. The advent of imatinib mesylate (IM), a specific tyrosine kinase inhibitor (TKI) that targets Bcr/Abl fusion protein, has revolutionized the treatment of CML [2]. Despite the great achievement, CML is currently considered incurable in most patients largely due to the development of drug resistance [3]. IM resistance resulted from mutations in the bcr/abl gene could be overcame by application of the more potent second-generation TKIs, such as nilotinib [4] and dasatinib [5]. The outcome of TKIs treatment is limited in patients with blast crisis CML (BC-CML) in which the leukemic cells may not depend solely on Bcr/Abl tyrosine kinase for survival, as a result of additional molecular alternations [6]. For instance, Hsp-70 over-expression, which is independent of Bcr/Abl in some patients without mutations in bcr/abl gene, contributes to cell survival of the TKIs treatment, even the tyrosine kinase was fully blocked [7]. Therefore, TKIs alone may not be sufficient to eliminate cancer cells since Bcr/Abl is not necessary for their survival. Targeting multiple pathways simultaneously by partnering existing TKIs with novel agents will bring new hope for overcoming drug resistance and finally eradicate the entire leukemic cell population.

Recently, contribution of cellular anti-oxidation in chemo-resistance has been intensively studied. Generation of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide are considered inevitable in aerobic life. Overproduction of ROS promotes peroxidation of lipid in cellular membranes. 4-HNE (4-Hydroxynonenal) is one of most bioactive products and has been widely accepted as a cytotoxic molecule by forming covalent adducts with cellular macromolecules, such as proteins, nucleic acids to affect their functions [8]. Several signaling pathways have been shown to be modulated by 4-HNE, for example, 4-HNE induces caspase-3 and −9 activation and of release of cytochrome c from mitochondria [9]. Moreover, 4-HNE suppresses AKT activity and induces P53 expression [10], [11]. In order to counteract the insults of ROS, mammals have evolved a battery of defensive enzymes, GST (glutathione S-transferase), one of the best documented enzymes, exerts detoxifying role by catalyzing the formation of 4-HNE-GSH conjugate, promoting its extracellular export [12]. The CNC (cap' n′ collar) family member Nrf-2 (nuclear factor erythroid-2 related factor 2) transcription factor is the master regulator of GST genes. Under basal circumstance, Nrf-2 maintains inactive and is interacts with Keap-1 (Kelch-like ECH-associated protein 1) in cytoplasm [13]. In response to oxidative stress, the interaction equilibrium is disrupted and Nrf-2 trans-locates into nucleus to trigger targeted genes transcription by binding to ARE regulatory DNA sequence [14]. Various studies have demonstrated that constitutively activated Nrf-2 confers resistance to chemotherapies [15], [16], [17], [18]. Therefore, the role of Nrf-2 in IM resistant CML cells is of great interest.

In the present study, we focused on the abundance of Nrf-2 in IM resistant K562 cells and clinical bone morrow samples from patients with CML at different stages, our data suggested that Nrf-2 may serve as a novel target for treatment of CML with drug resistance.

Section snippets

Main reagents

Imatinib mesylate was purchased from Selleck, antibodies to 4-HNE was purchased from Abcam (Cambridge, MA, USA), Keap-1, Nrf-2, c-Abl,H2AX, γ-H2AX were purchased from cell signaling technology (Danvers, MA, USA). Gst-α,Gst-μ,Gst-π and HPR-conjugated secondary antibodies were products of Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell counting kit CCK-8 was purchased from Selleck Chemicals (Houston, TX, USA). Co-Immunoprecipitation Kit was purchased from Pierce™ (Rockford, IL, USA).

Cell culture

K562

Generation and characterization of K562R cells

To mimic the development of resistance to TKIs during the process of treatment, we constructed an IM-resistant K562 (K562R) cell line by culturing in gradually increasing dose of IM. The initiating concentration of IM was 0.1 μM. To calculate to resistance index (RI), CCK-8 assay was then performed. IC50 of K562 and K562R cells were 0.93 and 6.14, respectively (Fig. 1a); the RI of K562R was 6.6. FCM results showed significant reduction of apoptotic rate in K562R cells treated with 1 μM IM for 24 h

Discussion

In this study, we have identified the abnormal activity of Nrf-2 and its implication in IM resistant cell line and bone morrow of CML patients. As a pivotal sensor of chemical-induced oxidative stress, Nrf2 functions to restore homeostasis by up-regulating antioxidant and other cytoprotective enzymes. Dysfunction of Nrf-2 and its implication in chemoresistance have been intensively studied during the past decade. Indeed, several lines of investigation suggest that Nrf-2 pathway activation is

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Science Foundation of China Grant No. 81600134 to Fang Wang.

References (29)

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