Lung cancer is one of the most serious cancers worldwide and is the most common malignancy in cancer-related deaths, leading to approximately 1.4 million deaths per year [1
]. Lung cancer is considered to have the highest incidence and mortality with 1.8 million new cases and 1.6 million new deaths annually [2
]. Lung cancers are classified into two major forms, small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), based on microscopic appearance. NSCLC occurs more frequently and accounts for 85% of all cases of lung cancer [3
]. Treatment strategies for lung cancer include radiotherapy, chemotherapy, and surgery [4
]. The primary treatment approach for NSCLC is a surgical operation followed by chemotherapy to prevent recurrence [5
]. However, in terms of the initial outcome, treatment for NSCLC remains inefficient compared to that for SCLC. Additionally, surviving NSCLC tumors subsequently display acquired drug resistance caused by multidrug resistance (MDR), i.e., the ability of tumor cells to develop resistance to various drugs [6
]. Adriamycin (ADR) has been widely used as an anticancer drug for a large range of tumors, including lung cancer. Although SCLC is highly sensitive to ADR, NSCLC shows poor sensitivity to this chemotherapeutic agent [7
]. Thus, it is vital to find alternative approaches to reduce the side effects caused by ADR and enhance its efficacy in clinical use.
Cancer cells expressing a high protein level of ATP binding cassette (ABC) transporters can attenuate the efficacy of treatment by actively pumping drugs outs of the cells, leading to the MDR phenotype [9
]. Based on sequence homology and domain organization, these ABCs are subdivided into seven distinct subfamilies (ABCA–ABCG). Among these, multidrug resistance-associated protein 1 (MRP1), also known as ABCC1, was first identified from drug-resistant lung cancer cells that did not express ABCB1 (MDR1 or P-glycoprotein) [10
]. MRP1 plays a role in drug resistance in various cancer including NSCLC tumors [11
]. Regulation of the MRP1 gene at the 5′ untranslated promoter region is associated with various transcription factors, including neuroblastoma-derived MYC (MYCN) [12
]. Control of MRP1 expression can be viewed as a potential way to improve sensitivity to chemotherapy. Indeed, ADR resistance in human bladder cancer cells by resveratrol has been reported to be partially associated with an alteration of MRP1 [14
]. However, the regulation by phytochemicals of MRP1 and its underlying mechanism in drug-resistant cancer cells remains to be clarified.
Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone; NBT) is a major component of citrus fruits, particularly the peels of oranges (Citrus sinensis
]. NBT exhibits antiproliferative activities and suppresses invasion and migration of different cancer cell types, including human gastric adenocarcinoma, breast cancer, and lung cancer [16
]. It has also been reported that NBT can sensitize the growth inhibition activities of the chemotherapeutic drug fluorouracil (5-FU) without affecting normal cells [16
]. That study indicated that NBT may function by modulating and interacting with cellular targets that are associated with drug resistance. Consistent with these findings, more recently, NBT was shown to significantly sensitize ABCB1-overexpressing NSCLC cells to chemotherapeutic drugs by inhibiting the efflux function of ABCB1 [19
]. However, the mechanisms underlying the ability of NBT to increase sensitivity to chemotherapeutic drugs remain unclear, and additional research on the functions and mechanisms of NBT as a chemosensitizer is needed.
In the present study, we used transcriptome analysis to identify the differentially expressed genes in A549/ADR cells compared with parental A549 cells, and then performed in vitro and xenograft animal studies to evaluate the ability of NBT to exhibit chemosensitizing activity against ADR. We found that NBT suppressed the Akt/GSK3β/β-catenin/MYCN signaling pathway and inhibited the expression of MRP1, leading to increased accumulation of ADR. These results are the first to demonstrate the underlying molecular mechanism by which NBT sensitizes ADR-induced cytotoxicity.
2. Materials and Methods
2.1. Cell Culture
Human non-small-cell lung cancer (NSCLC) A549 cells were generously provided by Professor Min-young Kim at the Faculty of Biotechnology, Jeju National University, Republic of Korea. Cells were cultured in Ham’s F-12K (Kaighn’s) Medium (F12K) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere under 5% CO2 in an incubator. The ADR-resistant cell line was established from the parental cell line by step-dose selection in vitro. A549 cells were treated with ADR at concentrations ranging from 0.03 to 0.5 μM over a period of 3 months.
2.2. Cell Viability Assay
Antiproliferative activity was determined by a cell viability assay. The effect of the samples on the viability of various cancer cell lines was determined by an 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)-based assay. Exponential-phase cells were collected and transferred to a 96-well microtiter plate (5 × 104 cells per mL) to detect cytotoxicity. The cells were incubated for 2 days with various concentrations of ADR with/without NBT. After incubation, 0.1 mg MTT (Sigma, St. Louis, MO, USA) was added to each well, and the cells were incubated at 37 °C for 4 h, after which the medium was carefully removed. Dimethyl sulfoxide (DMSO) (150 μL) was added to each well to dissolve the formazan crystals. After the crystals had dissolved completely, the plates were read at 570 nm using a Sunrise microplate reader (Tecan Group, Ltd., Salzburg, Austria). The percentage cell viability was calculated by the formula: mean value of (control group—treated group/control group) × 100%. All results were examined in triplicate for each concentration.
2.3. Transcriptome Analysis
Total RNA was extracted from A549 cells and A549/ADR cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA purity and concentration were checked using a UV1800 Spectrophotometer (SHIMADZU, Kyoto, Japan). Then, 1 μg of the total RNA was used to construct a library using the Illumina TruSeq mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA). Poly-T oligo-attached magnetic beads were added to purify the poly-A-containing mRNA molecules. RNA-Seq was performed by Macrogen, Inc. (Seoul, Korea) according to the manufacturer’s instructions. Prior to the transcriptome assembly, duplicated sequences were removed from the raw reads using FastUniq [20
], and the human genome GRCh38 was indexed using Spliced Transcripts Alignment to a Reference (STAR) [21
]. Trinity was used to assemble the reads into transcriptomes [22
]. The abundance of each transcriptome was calculated using RNA-seq by Expectation Maximization (RSEM), which was used to determine significantly differentially expressed genes (DEGs) (p
< 0.001 and at least a twofold change) using EdgeR; these were annotated with Trinotate (https://trinotate.github.io/
2.4. Functional Annotation of Differentially Expressed Genes (DEGs)
We analyzed Gene Ontology (GO) using the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/
) to investigate the primary function of the differential expression of messenger RNA (mRNAs) in A549/ADR cells. Furthermore, we also applied the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to classify DEGs into different functional pathways [25
2.5. Analysis of the Effects of Drug Combinations
The Chou–Talalay method was utilized to calculate the combination index (CI) using CalcuSyn software (Biosoft, Ferguson, MO, USA). CI values of <1, 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively.
2.6. Intracellular Accumulation of ADR
A laser scanning confocal microscope Olympus FV1200 (Olympus Coporation, Tokyo, Japan) was used to measure the intracellular accumulation of ADR. A549 or A549/ADR cells were cultured on a cover glass (ISO LAB 20 × 20 mm). After 24 h of incubation, the cells were treated with ADR (0.5 μM) alone or in combination with NBT (50 μM) and incubated for 6, 12, and 24 h. Subsequently, the culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Cells were fixed in 4% formaldehyde for 20 min at room temperature and then washed twice with PBS. Nuclear DNA was stained with 10 μM Hoechst 33342. Imaging was carried out via fluorescence microscopy (Olympus Coporation, Tokyo, Japan) to compare the intracellular accumulation of ADR. For the flow cytometry analyses, ADR (0.5 μM) was added to A549 or A549/ADR cells and incubated with or without NBT (50 μM) for 6, 12, and 24 h. Cells were detached, re-suspended in 500 μL of PBS after washing in cold PBS, and analyzed by flow cytometry (BD FACS Aria, BD Biosciences, San Jose, CA, USA). MK571, a known MRP1 inhibitor, was used as a positive control.
2.7. Cell Cycle Analysis
Cells (5 × 104 cells/mL) were seeded 24 h before being treated with or without ADR for 48 h. After treatment, the cells were collected, fixed in 70% ethanol and kept at −20 °C. Before fluorescence-activated cell sorting (FACs) analysis, cells were washed in PBS (2 mM EDTA), resuspended in 0.5 mL PBS (2 mM EDTA) containing 1 mg/mL RNase and 50 mg/mL propidium iodide (PI), incubated in the dark for 30 min at 37 °C, and analyzed by FACScalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Data from 10,000 cells were collected for each sample.
2.8. Western Blot Analysis
Western blotting was performed as described previously [27
]. Briefly, cell lysates were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer. Most primary antibodies were used at 1:1000 dilution, except that β-actin (1:10,000) and anti-rabbit immunoglobulin G (IgG) secondary antibody (Vector Laboratories, Burlingame, CA, USA) were used at 1:5000 dilution. The membranes were analyzed using a BS ECL Plus kit (Biosesang Inc., Seongnam, Korea)
2.9. In Vivo Animal Studies
Mice were maintained and used for experiments according to a protocol approved by the Institutional Animal Care and Use Committee of Jeju National University (Jeju, Korea). Then, 1 × 106 A549/ADR cells resuspended in a mixture of 100 µL Matrigel (Sigma-Aldrich, St. Louis, MO, USA) in PBS were subcutaneously inoculated of into the flanks of 6-week-old athymic BALB/c female nude mice (n = 4/group). After the successful generation of tumor models, mice were treated with NBT (40 mg/kg), ADR (10 mg/kg) and their combination. The treatment was continued for up to 35 days. After that, the animals were sacrificed, and the tumors were removed from all animals and weighed.
2.10. Statistical Analysis
Results are expressed as mean ± standard deviation (SD). One-way analysis of variance using SPSS v 12.0 software was applied. All assays were performed in triplicate. Values of p < 0.05 were considered statistically significant.
Although chemotherapy agents have been used successfully in a variety of cancer treatments, chemotherapy resistance is a major obstacle to effective cancer treatment. ADR (or doxorubicin) is the most widely used anticancer drug for a wide range of tumors, including lung cancer, and resistance to ADR is a prime example of anticancer drug resistance. While ADR shows high activity against SCLC, it shows relatively limited efficacy with NSCLC, which accounts for 85% of all lung cancer patients [8
]. One cause of this low efficacy is acquired MDR. The overexpression of membrane transport proteins that effectively remove the chemotherapeutic drugs contributes to the mechanisms by which tumor cells acquire drug resistance. Among the most commonly known MDR-related membrane transporters are the ABC transporter superfamily, which includes P-gp (MDR-1) and multidrug resistant-associated protein (MRP-1). MRP-1, which was originally isolated from a doxorubicin-selected lung cancer cell line, mediates resistance to a broad range of anticancer drugs. As MRP-1 gene expression increases in various cancers including NSCLC, a combination of anti-cancer agents with MRP inhibitors to limit drug efflux is an obvious approach for the development of alternative chemotherapy treatments. Besides being inefficient, ADR also causes congestive heart failure when used at high doses, a major adverse effect [40
]. Therefore, there is a need for novel therapeutic strategies that can minimize the dose and reduce the cytotoxicity of doxorubicin, and enhance its therapeutic efficacy against NSCLC cells.
After the establishment of ADR-resistant A549/ADR cells by exposing A549 adenocarcinoma cells to increasing doses of ADR, we compared the ADR toxicity profiles of A549 cells and A549/ADR cells by an MTT assay and cell cycle and Western blot analyses (Figure 1
). A549/ADR cells showed higher cell viability, a reduced sub-G1 population, lower expression of pro-apoptotic (c-PARP, Bax) and higher expression of anti-apoptotic proteins (Bcl-xL). Among three commonly-upregulated ABC transporter genes in resistant cancer cell lines, we identified significant overexpression of MRP1 in A549/ADR cells (Figure 4
C). These results suggest that overexpression of MRP1 protein may underlie A549/ADR cells’ resistance to ADR and the concomitant anti-apoptosis. Thus, based on the assumption that modulation of MRP1 protein expression is crucial in overcoming drug resistance to ADR, we performed transcriptome analyses of RNA sequencing data to compare overall gene expression patterns between the two cell lines. The major differences between the two cell lines as analyzed by the GO database confirmed the drug-resistant characteristic of A549/ADR cells and further provided evidence of the unique characteristics of A549/ADR cells compared to A549 cells in terms of changes in gene expression (Figure 2
B). Previously, Fang et al. reported transcriptome analysis of cisplatin-resistant A549 in comparison with its parental cell line, demonstrating that the PI3K–Akt pathways, the mitogen-activated protein kinase (MAPK) pathway, and cell invasion pathways were enriched, and the highest number of DEGs were found in cisplatin-resistant A549 [41
]. In accordance with the cisplatin-resistant A549, cisplatin-resistant hepatocellular carcinoma HepG2 was also most enriched in PI3K–Akt and cancer pathways based on the KEGG transcriptome analysis [41
]. Interestingly, our KEGG pathway analysis also showed that the potentially oncogenic PI3K-Akt signaling pathway was significantly upregulated in the A549/ADR cell line compared to the A549 cell line (Figure 2
B). These results further supported the importance of the PI3K-Akt pathway in controlling resistance against various types of chemotherapeutic agents, including cisplatin and ADR, in various cancer types [42
]. However, in addition to the PI3K/Akt pathway and ABC transporters, we also witnessed changes in several proven multidrug resistance-associated pathways, including the extracellular matrix (ECM)-receptor interaction in A549/ADR cells and the AMP-activated protein kinase (AMPK) signaling pathway (Figure 3
As shown in Figure 4
, expression of MRP1 was greatly increased in association with decreased intracellular accumulation of ADR in A549/ADR cells. Based on these results and the results of the transcriptome analysis, we focused on the role of NBT in the regulation of the PI3K–Akt pathway and the ABC transporter (MRP1) to clarify the synergistic effects with ADR in A549/ADR cells. Interestingly, our results showed that the amount of MRP1 expression increased selectively among the ABC transporters, including MDR1 and ABCG2 (Figure 4
C). As modulation of the expression of the ABC transporter is quite complex, selective downregulation of this transporter could represent a promising approach to novel chemotherapies. In particular, MRP1 is well known to play a crucial role in multidrug resistance and is over-expressed in a variety of cancers. It has been reported that the expression of MRP1 is highly correlated with the expression of MYCN and that MRP1 can be regulated by MYCN at the transcription level [12
]. Interestingly, in our study, the level of MYCN was significantly higher in A549/ADR cells as compared to A549, and the expression of MYCN was down-regulated within 4 h following NBT treatment in A549/ADR cells (Supplementary Figure S2
). This allowed us to speculate that MYCN is an important regulator of MRP1 expression in A549/ADR cells.
Several researchers have reported on the role of MYCN in the progression of other tumors including neuroblastoma, and the development of new therapies targeting MYCN could be very attractive [45
]. However, MYCN has not attracted much attention in the treatment of lung cancer. Recently, Liu et al. found that up-regulation of MYCN expression was associated with a poor clinical outcome in NSCLC patients [47
]. Binding of the ubiquitin ligases to the N
-terminal conserved phosphodegron domain (CPD) is required for initiation of proteasomal degradation of MYC-family proteins. In oncogenesis, the stabilization of MYC-family proteins can be controlled by phosphorylation within this region at threonine 58 (T58) and serine 62 (S62) by GSK3β and MAPK, respectively [48
]. Phosphorylation at S62 serves as priming for GSK3β, which subsequently phosphorylates T58 to initiate Fbxw7-mediated degradation [51
]. GSK3β is in turn inhibited via phosphorylation by Akt. As a result, signaling via PI3-kinase and Akt stabilizes MYCN and protects it from proteasomal degradation [45
]. In addition to the translational regulation of MYCN by Akt–GSK3β signaling, the level of MYCN also can be regulated by GSK3β at the transcriptional level via a well-known modulator of β-catenin [53
]. Chromatin immunoprecipitation assays showed that β-catenin was associated with conserved DNA binding sites for T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins, which are located in the MYCN promoter, indicating direct regulation of the MYCN promoter by canonical Wnt signaling [54
]. Consistent with these previous studies, in our study, NBT induced down-regulation of MYCN either by Akt/GSK3β or by the Akt/GSK3β/β–catenin pathway axis (Figure 8
). However, further studies are required to confirm that the regulation of the MRP1 gene expression is directly associated with the MYCN transcription factor at its promoter region and to investigate how NBT plays an important role in this regulation.
Above all, in this study, in vivo experiments using a xenotransplant model showed that the combined treatment significantly decreased the tumor volume and weight, which is consistent with the in vitro results. Moreover, the weight of the mice showed that the combination treatment did not cause significant systemic toxicity to the mice. In conclusion, our results suggested that NBT can act as an effective chemosensitizer against ADR in the A549/ADR cell line.