Regulation of ABCG2 by nuclear factor kappa B affects the sensitivity of human lung adenocarcinoma A549 cells to arsenic trioxide

https://doi.org/10.1016/j.etap.2017.12.011Get rights and content

Highlights

  • As2O3 enhanced the level of ABCG2 and activated NF-κB pathway in A549 cells.

  • Inhibition of ABCG2 increased the drug sensitivity of As2O3 to A549 cells.

  • As2O3 elevated the expression of ABCG2 through activation of NF-κB in A549 cells.

  • Regulation of ABCG2 by NF-κB affects sensitivity of A549 cells to As2O3.

Abstract

Arsenic trioxide (As2O3) is successfully used as an anticancer agent against acute promyelocytic leukemia and some solid tumors. However, the application of As2O3 is largely limited by its drug resistance in the treatment of non-small cell lung carcinoma (NSCLC). Therefore, it is an urgent task to enhance the sensitivity of lung cancer cells to As2O3. In this study, using human lung adenocarcinoma A549 cells as a cell culture model, we demonstrated that an adenosine triphosphate binding cassette (ABC) transporter, ABCG2, was significantly increased by As2O3 treatment, while other ABC transporters, ABCB1 and ABCC1 showed no remarkable change in the response to As2O3. After inhibition of ABCG2 by its specific inhibitor, the drug sensitivity of As2O3 to A549 cells was significantly enhanced, manifested by decreased cell viability and colony formation as well as the increased ROS production and cell apoptosis. To further understand the molecular mechanism underlying the elevation of ABCG2 expression in As2O3-treated cells, we detected the activation state of nuclear factor kappa B (NF-κB) pathway and its relationship with ABCG2 expression. Our results revealed that the increased expression of ABCG2 was regulated by NF-κB, and thus affecting the cell death of As2O3-treated A549 cells. These findings indicate that inhibition of NF-κB/ABCG2 pathway by specific inhibitors may be a new strategy for the improvement of As2O3 sensitivity in NSCLC treatment.

Introduction

Lung cancer is the leading cause of cancer-associated mortality throughout the world (Siegel et al., 2016). Non-small cell lung carcinoma (NSCLC) is the main histological subtype of lung cancer, and accounts for more than 80% of lung cancer (Chen et al., 2014). Surgery is commonly considered as an effective method for treatment of early NSCLC. However, for most advanced NSCLC, who missed the best surgery time, chemotherapy is the preferred approach to offer symptomatic relief and modest improvement in survival (Goldstraw et al., 2011; Reck et al., 2013). In addition, chemotherapy is also used pre-operatively and post-operatively for early NSCLC to inhibit proliferation of cancer cells (Felip et al., 2010). Until now, chemotherapy has been widely accepted as an irreplaceable method for NSCLC treatment (Davidoff et al., 2010). However, the utilization of the chemotherapy drugs is largely limited by the dose-dependent toxic side effects and drug resistance in NSCLC.

As a traditional Chinese medicine, arsenic trioxide (As2O3) has been developed as an anticancer agent against acute promyelocytic leukemia with low toxicity and high sensitivity (Burnett et al., 2015; Lo-Coco et al., 2013). A lot of studies have demonstrated that As2O3 can induce cell apoptosis and oxidative damage in some solid tumor cells at low concentrations (Chen et al., 2015; Jiang et al., 2013), indicating that As2O3 may be a potential treatment for solid tumors. Considering the limited number of effective drugs in lung cancer treatment, the therapeutic potential of As2O3 in NSCLC was studied in the last few decades (Lam et al., 2016; Zheng et al., 2015). Increasing evidence shows that As2O3 exerts promising treatment effects such as inhibition of proliferation, induction of cell apoptosis and oxidative stress on human NSCLC cell lines, but the sensitivity of NSCLC cells to As2O3 is still not high enough to achieve therapeutic purpose (Walker et al., 2016; Xie et al., 2015). Therefore, finding out the determinants that affect the sensitivity of human NSCLC cells to As2O3 is both necessary and desirable for the treatment of NSCLC.

Adenosine triphosphate (ATP) binding cassette (ABC) proteins refer to a large family of membrane transporters, utilizing ATP hydrolysis to power the export of a variety of substrates, such as peptides, phospholipids, drugs and toxicants (Chen et al., 2016c; Fletcher et al., 2010; Kathawala et al., 2015). It is noteworthy that ABC transporters are proved to be responsible for the efflux of arsenic-glutathione (GSH) conjugate in eukaryotes (Leslie, 2012). Moreover, trivalent inorganic arsenic (iAsIII) is thiol bound and can combine with intracellular glutamine to form AsIII(GS)3, which is subsequently identified and extruded out of the cells by ABC transporters (Leslie, 2012; Song et al., 2010). In this manner, the intracellular iAsIII concentration is reduced and the toxic effect of iAsIII on cells is attenuated (Leslie, 2012; Song et al., 2014). Therefore, ABC transporters are capable of conferring resistance to arsenic. Now, a series of ABC transporters have been found to participate in AsIII(GS)3 removal in human, such as ABCCs (Leslie, 2012) and ABCB6 (Chavan et al., 2011). However, the exact ABC transporter responsible for arsenic export in human lung cells is elusive and needs to be further studied.

As members of families of human ABC transporters, ABC sub-family B member 1 (ABCB1), ABC sub-family C member 1 (ABCC1) and ABC sub-family G member 2 (ABCG2), are reported frequently to mediate the drug resistance in cancer therapy (Chou et al., 2012; Liu et al., 2014; Singh et al., 2010), and therefore, their potential roles in As2O3 resistance of human lung adenocarcinoma A549 cells were undermined in this study. By using ELISA assay, our study demonstrated that the content of ABCG2 was increased more markedly than those of the other two transporters in response to As2O3 exposure, indicating that ABCG2 is the potential transporter responsible for arsenic export in the A549 cells. Our results further revealed that inhibition of ABCG2 by its inhibitor, novobiocin sodium, significantly enhanced the sensitivity of A549 cells to As2O3.

To better understand the molecular mechanism underlying the elevation of ABCG2 expression in As2O3-treated cells, the activation of nuclear factor kappa B (NF-κB) pathway was determined. We observed that NF-κB was activated by As2O3, and its activation led to the up-regulation of ABCG2 expression; Inhibition of NF-κB/ABCG2 pathway by specific inhibitors caused the increased level of cell death in A549 cells. Together, these results suggest that the regulation of ABCG2 by NF-κB affects the sensitivity of A549 cells to As2O3. Our findings provide a new potential strategy for ameliorating As2O3 resistance in the NSCLC treatment.

Section snippets

Chemicals and reagents

As2O3 was purchased from YiDa Pharmaceutical Co. Ltd. (Harbin Medical University, Heilongjiang, China) and novobiocin was from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); Pyrrolidinedithiocarbamic acid (PDTC), NF-κB p65 subunit nuclear translocation kit and Hoechst 33258 staining kit were all ordered from Beyotime Institute of Biotechnology (Jiangsu, China); Recombinant human tumor necrosis factor-α (TNF-α) was from Prime Gene Bio-Tech Co., Ltd. (Shanghai, China);

Effects of As2O3 on the contents of ABCB1, ABCC1 and ABCG2 in A549 cells

ABCB1, ABCC1 and ABCG2 are the most extensively studied ABC transporters involved in drug resistance (Li et al., 2016). Thus, to find out the critical ABC transporters contributing to As2O3 efflux in A549 cells, we firstly measured the protein contents of the three ABC transporters following As2O3 exposure (0–20 μM) for 6, 12 and 24 h using ELISA assay. As depicted in Fig. 1A, comparing with the controls, the contents of ABCB1 were significantly enhanced at all tested concentrations by 6 h

Discussion

With the fastest growing morbidity and mortality in many developed and developing countries, lung cancer has become a major burden of disease for human health (Chen et al., 2016b). Chemotherapy has been considered as the most effective treatment for advanced NSCLC, and more than 90% of patients need to receive chemotherapy (Howington et al., 2013). However, the major limitations of chemotherapy in NSCLC treatment are the side-effects and drug resistance (Gerber and Schiller, 2013). As a novel

Competing interests

The authors have declared that no competing interests exist.

Author contributions

Jiang, C. Chen and Z. Zhang wrote the manuscript. Z. Zhang designed the experiments. X. Jiang, C. Chen and S. Gu performed the experiments.

Acknowledgment

This study was supported by grants from National Science Foundation of China (No.81773380) to Zunzhen Zhang.

References (52)

  • K. Nakatomi et al.

    Transport of 7-ethyl-10-hydroxycamptothecin (SN-38) by breast cancer resistance protein ABCG2 in human lung cancer cells

    Biochem. Biophys. Res. Commun.

    (2001)
  • M. Reck et al.

    Management of non-small-cell lung cancer: recent developments

    Lancet

    (2013)
  • D.M. Smith et al.

    ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle

    Cell

    (2011)
  • Y. Su et al.

    Inhibition of efflux transporter ABCG2/BCRP does not restore mitoxantrone sensitivity in irinotecan-selected human leukemia CPT-K5 cells: evidence for multifactorial multidrug resistance

    Eur. J. Pharm. Sci.

    (2006)
  • C.-y. Zheng et al.

    Combination of arsenic trioxide and chemotherapy in small cell lung cancer

    Lung Cancer

    (2013)
  • H. Chavan et al.

    The ATP-binding cassette transporter ABCB6 is induced by arsenic and protects against arsenic cytotoxicity

    Toxicol. Sci.

    (2011)
  • Z. Chen et al.

    Non-small-cell lung cancers: a heterogeneous set of diseases. Nature reviews

    Cancer

    (2014)
  • C. Chen et al.

    Resveratrol protects against arsenic trioxide©induced oxidative damage through maintenance of glutathione homeostasis and inhibition of apoptotic progression

    Environ. Mol. Mutagen.

    (2015)
  • C. Chen et al.

    Nuclear translocation of nuclear factor kappa B is regulated by G protein signaling pathway in arsenite©induced apoptosis in HBE cell line

    Environ. Toxicol.

    (2016)
  • W. Chen et al.

    Cancer statistics in China: 2015

    CA. Cancer J. Clin.

    (2016)
  • C.-W. Chou et al.

    Tumor cycling hypoxia induces chemoresistance in glioblastoma multiforme by upregulating the expression and function of ABCB1

    Neuro-Oncol.

    (2012)
  • A.J. Davidoff et al.

    Chemotherapy and survival benefit in elderly patients with advanced non-small-cell lung cancer

    J. Clin. Oncol.

    (2010)
  • Y. Fang et al.

    PML-RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells

    Angiogenesis

    (2016)
  • E. Felip et al.

    Preoperative chemotherapy plus surgery versus surgery plus adjuvant chemotherapy versus surgery alone in early-stage non-small-cell lung cancer

    J. Clin. Oncol.

    (2010)
  • J.I. Fletcher et al.

    ABC transporters in cancer: more than just drug efflux pumps. Nature reviews

    Cancer

    (2010)
  • C. Garcia-Rodriguez Mdel et al.

    Antigenotoxic and apoptotic activity of green tea polyphenol extracts on hexavalent chromium-induced DNA damage in peripheral blood of CD-1 mice: analysis with differential acridine orange/ethidium bromide staining

    Oxid. Med. Cell. Longevity

    (2013)
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