Original Contribution
Luteolin inhibits Nrf2 leading to negative regulation of the Nrf2/ARE pathway and sensitization of human lung carcinoma A549 cells to therapeutic drugs

https://doi.org/10.1016/j.freeradbiomed.2011.03.008Get rights and content

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor regulating the expression of a battery of cytoprotective genes. Constitutive Nrf2 activation in many tumors enhances cell survival and resistance to anticancer drugs. Using a cell-based ARE-reporter assay we discovered that the flavonoid luteolin is a potent Nrf2 inhibitor. Luteolin inhibited ARE-driven gene expression redox-independently. In non-small-cell lung cancer A549 cells, which possess constitutively active Nrf2, luteolin elicited a dramatic reduction in Nrf2 at both the mRNA and the protein levels, leading to decreased Nrf2 binding to AREs, down-regulation of ARE-driven genes, and depletion of reduced glutathione. After transcription was blocked with actinomycin D, 1 μM luteolin decreased the Nrf2 mRNA level by 34% in 30 min, indicating its role in accelerating Nrf2 mRNA turnover. At physiological concentrations, luteolin significantly sensitized A549 cells to the anticancer drugs oxaliplatin, bleomycin, and doxorubicin. However, knockdown of Nrf2 using siRNA essentially abolished the induced sensitivity by the flavonoid, implying the importance of inhibiting Nrf2 for its activity. Our study demonstrates that an Nrf2 inhibitor can enhance the responsiveness of cancer cells to chemotherapeutic drugs and indicates the potential application of luteolin as a natural sensitizer in chemotherapy.

Section snippets

Chemicals and cell culture

Unless otherwise stated, all chemicals were from Sigma–Aldrich Co., Ltd. (Shanghai, China), and all antibodies were from Santa Cruz Biotechnology (Shanghai, China). Antibody against actin was from Sigma. Antiserum against aldoketo reductases 1C1 and C2 (AKR1C) or KEAP1 was provided by Professor John Hayes and Dr. Mike McMahon (University of Dundee, Dundee, UK) [19], [20].

MCF7 (human breast carcinoma), Caco2 (human colon cancer), and A549 (NSCLC) cell lines were obtained from the ATCC. AREc32,

Luteolin is an inhibitor of ARE-driven gene expression

Previously we reported a stable cell line, AREc32, which carries a luciferase reporter gene under the control of the ARE [21]. Both the basal and the inducible ARE-luciferase activities in these cells are primarily regulated by Nrf2 [21]. Therefore, this cell line provides an ideal cell-based assay to screen for antagonists of Nrf2. Twelve common flavonoids were screened (Table 1). The bioavailability of flavonoids is expected to be relatively low and they have been detected in human plasma in

Discussion

Drug resistance during chemotherapy is the major obstacle to successful treatment of many cancers. It has long been recognized that GSH homeostasis, which is critically regulated by Nrf2, plays an important role in cancer treatment. Elevated GSH levels, typically associated with higher levels of GSH-related enzymes and drug transporters, have been reported in many types of tumor [37], [38]. The involvement of Nrf2 in chemoresistance was further highlighted by recent evidence that frequent

Acknowledgments

We thank Professor C. Roland Wolf (University of Dundee, UK) for the generous gift of plasmid pRS-hNrf2. We also thank Professor John D. Hayes and Dr. Mike McMahon (both at the University of Dundee), for providing antiserum against AKR1C or KEAP1, and Dr. R. Rice (University of California, Davis, CA, USA) for the provision of plasmid pGL3-RARE. We are grateful to Professor I.C. Bruce (Zhejiang University School of Medicine) for critically reading the manuscript. This work was supported by the

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