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