Chrysin enhances sensitivity of BEL-7402/ADM cells to doxorubicin by suppressing PI3K/Akt/Nrf2 and ERK/Nrf2 pathway

https://doi.org/10.1016/j.cbi.2013.08.008Get rights and content

Highlights

  • Activation of Nrf2 is associated with drug resistance in BEL-7402/ADM cells.

  • Chrysin could reverse drug resistance by down-regulating PI3K/Akt/Nrf2 and ERK/Nrf2 pathway.

  • Chrysin can be used as an effective adjuvant sensitizer to combat chemoresistance.

Abstract

Nuclear factor-E2-related factor 2 (Nrf2) is an important cytoprotective transcription factor which plays a key role in antioxidant and detoxification processes. Recent studies have reported that development of chemoresistance is associated with the constitutive activation of the Nrf2-mediated signaling pathway in many types of cancer cells. Here, we investigated whether Nrf2 was associated with drug resistant in doxorubicin resistant BEL-7402 (BEL-7402/ADM) cells, and if chrysin could reverse drug resistance in BEL-7402/ADM cells. We found that remarkable higher level of Nrf2 and its target proteins in BEL-7402/ADM cells compared to BEL-7402 cells. Similarly, intracellular Nrf2 protein level was significantly decreased and ADM resistance was partially reversed by Nrf2 siRNA in BEL-7402/ADM cells. chrysin is a potent Nrf2 inhibitor which sensitizes BEL-7402/ADM cells to ADM and increases intracellular concentration of ADM. Mechanistically, chrysin significantly reduced Nrf2 expression at both the mRNA and protein levels through down-regulating PI3K-Akt and ERK pathway. Consequently, expression of Nrf2-downstream genes HO-1, AKR1B10, and MRP5 were reduced and the Nrf2-dependent chemoresistance was suppressed. In conclusion, these results clearly indicate that activation of Nrf2 is associated with drug resistance in BEL-7402/ADM cells and chrysin may be an effective adjuvant sensitizer to reduce anticancer drug resistance by down-regulating Nrf2 signaling pathway.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, causing more than 600,000 deaths annually around the world [1]. Chemotherapy is an important therapeutic strategy for HCC. Unfortunately, cancer cells often develop chemoresistance, which presents a major obstacle to the long-term efficacy of chemotherapeutic treatments. Therefore, it is urgently needed to develop new adjuvants that enhance chemotherapeutic efficacy and circumvent chemoresistance.

Nuclear factor-E2-related factor 2 (Nrf2) is a transcription factor which serves as a cellular sensor for oxidative and electrophilic insults [2], [3]. Under basal conditions, Nrf2 is retained in the cytosol by binding to Kelch-like ECH-associated protein 1 (Keap1). In response to oxidative/electrophilic stimuli, Nrf2 is released from Keap1, translocates into the nucleus, where it forms a heterodimer with its obligatory partner Maf and binds to the ARE sequence to activate transcription of a large number of cytoprotective enzymes including heme oxygenase-1 (HO-1), aldo–keto reductases and several ATP-dependent drug efflux pumps such as multidrug resistance-associated protein 5 (MRP5) [4], [5]. Nrf2 has been shown to be overexpressed in many types of cancer cells, indicating a potential role in tumor cell growth and survival [6], [7]. Somatic mutation or single-nucleotide polymorphism in Nrf2 and Keap1 genes resulted in constitutive activation of Nrf2 and promotion of tumor growth, as demonstrated by several groups [8], [9], [10]. This “dark” side of Nrf2 not only coped with the challenging tumor microenvironment, but also conferred chemo- and/or radio resistance during anticancer therapies [11]. Thus, potent and selective Nrf2 inhibitors would be useful for adjuvant medicine to subdue the development of cancer resistance to chemotherapy.

Aldo–keto reductase 1B10 (AKR1B10), also known as aldose reductase-like-1, is overexpressed in liver and lung cancers. Recent studies have showed that AKR1B10 can reduce the C13 ketonic group in daunorubicin and idarubicin [12], leading to chemoresistance of cancer cells to these cytostatic agents. In addition, AKR1B10 has been suggested to be involved in acquisition of the resistance of medulloblastoma to cyclophosphamide through a deactivation of a reactive cyclophosphamide metabolite aldophosphamide [13]. It also has been reported that AKR1B10 exert this antioxidant role in cancer cells resistant to other anticancer drugs, such as bleomycin [14], paclitaxel [15], and mitomycin c [16] that are known to produce ROS.

MRP5 is overexpressed in colon, lung, breast and pancreatic cancers. When transfected into drug-sensitive cells, MRP5 has been conferred resistance to antifolate drugs such as methotrexate and pemetrexed, and to nucleoside-based drugs such as 6-mercaptopurine, azidothymidine, 5-fluorouracil, and gemcitabine [17], [18].

Mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathways are important regulators of many fundamental biological processes, such as cell proliferation, apoptosis, migration, and differentiation. Furthermore, many researchers have been reported that these pathways can be involved in the development of chemoresistance and blockade of these pathways can sensitize a variety of cancer cells to chemotherapeutic drugs [19], [20], [21]. However, little is known regarding the molecular mechanism behind them.

Flavonoids, a diverse family of natural polyphenolic compounds commonly occurring in plants, showed strong anti-proliferative activity against many types of cancers and sensitized cancer cells to anticancer agents [22], [23]. Several flavonoid compounds have been reported to be Nrf2 inhibitor that can reverse drug resistance effectively, such as epigallocatechin 3-gallate (EGCG), luteolin, brusatol [24], [25], [26]. These prompted us to determine whether other flavonoids structurally related to them can sensitize cancer cells to anticancer drugs through antagonizing the Nrf2 signaling pathway.

Chrysin (5,7-dihydroxyflavone), a natural flavonoid that is found in many plant extracts, honey, and propolis, has been reported to possess antioxidant, anti-inflammatory, antiviral, and anti-cancer properties [27]. In this study, we reported that chrysin is a potent Nrf2 inhibitor. Chrysin could sensitize doxorubicin resistant BEL-7402 cells (BEL-7402/ADM) to ADM and its mechanism of sensitization could be associated with down-regulating PI3K-Akt/Nrf2 and ERK/Nrf2 signaling pathway, indicating the effectiveness of using chrysin as an adjuvant sensitizer to combat chemoresistance.

Section snippets

Materials

Chrysin was purchased from Shaanxi Huike Botanical Development Inc. (China), ADM from Zhejiang Haizheng Pharmaceutical Co., Ltd. (China). PI3K inhibitor (LY294002) and MEK1/2 inhibitor (U0126) was obtained from Beyotime Institute of Biotechnology. Antibody against Nrf2, MRP-5, Akt, p-Akt, ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, β-actin, and Lamin B were obtained from Santa Cruz Biotechnology. AKR1B10 and HO-1 antibody was from Epitomics. TRIzol Reagent were purchased from Gibco Invitrogen,

Nrf2 pathway is associated with chemoresistance in BEL-7402/ADM cells

To verify the drug resistance phenotype of BEL-7402/ADM cells, MTT assay was carried out. As shown in Fig. 1A, the IC50 value of ADM for BEL-7402/ADM cells was 5.13 μM and this value was 3.33 times higher than that for BEL-7402 cells (Table 1). Next, we tried to examine the expression of nuclear Nrf2 and its target genes in BEL-7402/ADM and the involvement of Nrf2 in the development of the acquired resistance. We found that the expression of nuclear Nrf2 protein and its downstream genes HO-1 was

Discussion

Drug resistance during chemotherapy is one of the major obstacles to successful treatment of many cancers. Several mechanisms are thought to account for the drug resistance phenotype, such as increased expression and activity of efflux transporter proteins, increased detoxification by metabolizing enzymes, enhanced DNA repair. Recently, Nrf2 has been proposed as a novel therapeutic target to overcome chemoresistance [28]. Therefore, identification of potent small molecule inhibitors of Nrf2 is

Conclusions

In conclusion, this study addressed that higher level of Nrf2 expressed in BEL-7402/ADM cells accounts for drug resistance, and low-cytotoxic concentrations of chrysin inhibites the expression of Nrf2 and its downstream genes HO-1, AKR1B10, and MRP5 by suppressing PI3K-Akt and ERK pathway and result in a reversal of drug-resistant phenotype eventually. Our finding on the inhibitory effect of chrysin on Nrf2 signaling might have significant implications for cancer therapy, and further studies in

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank Professor XB Wang (Laboratory of Chinese Herbal Pharmacology, Hubei University of Medicine) for the generous gift of BEL-7402, BEL-7402/ADM and MCF/ADM cells.

References (31)

  • K. Shen et al.

    Inhibition of IGF-IR increases chemosensitivity in human colorectal cancer cells through MRP-2 promoter suppression

    J. Cell. Biochem.

    (2012)
  • A. Singh et al.

    Expression of ABCG2 (BCRP), a marker of stem cells, is regulated by Nrf2 in cancer cells that confers side population and chemoresistance phenotype

    Mol. Cancer Ther.

    (2010)
  • S.L. Slocum et al.

    Nrf2: control of sensitivity to carcinogens

    Arch. Toxicol.

    (2011)
  • D.D. Zhang

    The Nrf2-Keap1-ARE signaling pathway: the regulation and dual function of Nrf2 in cancer

    Antioxid. Redox Signal.

    (2010)
  • N.J. Yoo et al.

    Somatic mutations of the KEAP1 gene in common solid cancers

    Histopathology

    (2012)
  • Cited by (161)

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text