Nobiletin Inhibits Non-Small-Cell Lung Cancer by Inactivating WNT/β-Catenin Signaling through Downregulating miR-15-5p

Background Nobiletin is a natural compound with anticancer activity; however, the mechanism is not clear. Methods The inhibitory effect of nobiletin on non-small-cell lung cancer (NSCLC) cells was examined using soft agar, Transwell, and apoptosis analyses. Cancer stemness was measured by sphere assay. Genes and miRNAs regulated by nobiletin were identified by whole-genome sequencing. Protein levels were detected by western blot and immunofluorescence assays. Results Nobiletin significantly inhibited NSCLC cell colony formation and sphere formation and induced apoptosis. Nobiletin upregulated negative regulators of WNT/β-catenin signaling, including NKD1, AXIN2, and WIF1, while it inhibited the expression of β-catenin and its downstream genes, including c-Myc, c-Jun, and cyclin D1. Furthermore, we identified that GN inhibits miR-15-5p expression in NSCLC cells and that NKD1, AXIN2, and WIF1 are the target genes of miR-15-5p. Conclusions Nobiletin has a strong inhibitory effect on NSCLC, and nobiletin plays an anticancer role by inhibiting miR-15-5p/β-catenin signaling in NSCLC.


Introduction
Lung cancer is the most frequently diagnosed malignancy and the leading cause of cancer-related mortality worldwide [1][2][3]. Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers. Despite considerable advancements in diagnosis and treatment modalities, including surgery and chemoradiation therapy, the survival of patients with advanced stages of the disease remains unsatisfactory. Several oncogenes and tumor suppressor genes known to be associated with NSCLC have been investigated to analyze tumor behavior and determine their prognostic significance [4][5][6]. In this regard, there is a need to discover new agents for the treatment of this deadly disease. Many kinds of natural products have been proven to possess anticancer properties [7][8][9]. ese types of components can affect the overall process of carcinogenesis by several mechanisms. Among them, nobiletin has been known to have an anticancer effect, as it can inhibit the proliferation, invasion, or metastasis of tumor cells. Nobiletin is a flavonoid compound isolated from the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. Studies show that nobiletin exhibits an anticancer role in various cancers, including skin, prostate, colon, and breast cancer [10][11][12][13][14][15][16][17][18]. However, the anticancer mechanism of nobiletin is still unclear.
WNT/β-catenin signaling is a well-studied oncogenic pathway that plays an important role in the development and progression of various cancers. For example, there have been some reports that WNT/β-catenin might enhance the motility of malignant cells and tumor invasion, such as in breast cancer, melanoma, and gastric cancer [19][20][21][22][23]. As a result, many investigators have focused on the potential effect of some substances, such as flavonoids, on cancer prevention and therapy by inhibiting the WNT/β-catenin signaling pathway [1][2][3]. In fact, most reports show that inhibition of WNT/β-catenin signaling is a useful strategy for cancer treatment.
In this study, we demonstrated that nobiletin significantly inhibits NSCLC growth, metastasis, and stemness and stimulates NSCLC cell apoptosis. In addition, we demonstrated that nobiletin plays its role by inactivating Wnt/ β-catenin signaling by upregulating the expression levels of WIF1, AXIN2, and NKD1. We also identified that nobiletin inhibits the expression of WIF1, AXIN2, and NKD1 by inhibiting miR-15-5p in NSCLC cells.

Cell Lines and Cultures.
Cell lines H460 and A549 were obtained from ATCC (Manassas, VA, USA), and these cells were cultured in DMEM (HyClone, Shanghai, China) supplemented with 10% FBS (ScienCell Research Laboratories, California, USA) in a 5% CO 2 humidified atmosphere at 37°C. e cells were then treated with 2.5 µM nobiletin for 24 hours and then subjected to a soft agar assay. After 10 days, the cell cultures were treated with 10 µL of CCK-8 solution for 2 hours and incubated at 37°C for the determination of cell viability. A microplate reader was used to obtain the optical density (OD) at 40 nm. is research was approved by the Institutional Review Board of Chuncheon Sacred Heart Hospital.

Cell Viability and Soft Agar
Assay. For the cell viability assay, H460 and H549 cells were seeded in 96-well plates at a density of 5000/well. e next day, the cells were treated with nobiletin or PBS for 48 h, and cell viability was measured using a Cell Counting Kit-8 (MedChem Express, JN, USA). For the soft agar assay, cells were treated with the indicated concentration of nobiletin for 48 h, and then, cells were resuspended in 0.5 mL of 0.35% agar (Sigma) in the growth medium at a density of 5000 cells/well in 6-well plates. e agar-cell mixture was plated on the top of a solid layer of 0.8% agar in the growth medium. Colonies were counted 14 days later.

In Vitro Migration and Invasion
Assay. Cell migration and invasion assays were performed using a Transwell chamber. Cells were treated with the indicated concentration of nobiletin for 48 h. en, the cells were resuspended, and 2 × 10 4 cells in the serum-free medium were plated into the top chamber with or without gel coating. e cell growth medium with 10% FBS was added under the chamber. After 24 h, cells on the upper sides of the inserts were removed using cotton, and cells on the surface of the bottom sides of the inserts were fixed with 4% paraformaldehyde, followed by staining with 0.5% crystal violet. e cells in five random microscopic fields were photographed using an inverted phase-contrast microscope and counted.

Luciferase Reporter Assay.
e luciferase reporter assay was carried out as described by Fend [19].

Statistical
Analysis. All data are presented as the mean ± standard deviation (SD), and significant differences (P < 0.05) between groups were analyzed by Student's t-test using SAS statistical software version 6.12 (SAS Institute).

Nobiletin Inhibited the Growth of NSCLC In Vitro.
We first examined the inhibitory effects of nobiletin on NSCLC. Both H460 and A549 NSCLC cells were treated with nobiletin and then subjected to soft agar and apoptosis analyses. Our results showed that nobiletin significantly inhibited soft agar colony formation (Figures 1(a) and 1(b)) and significantly increased apoptosis in NSCLC cells (Figures 1(c) and 1(d)). Furthermore, western blot analysis confirmed the apoptosispromoting effects of nobiletin on NSCLC. Western blot analysis showed that nobiletin significantly increased proapoptotic protein levels, including cleaved PARP and cleaved caspase 3 (Figure 1(e)). Taken together, our findings suggested that nobiletin inhibits NSCLC by stimulating apoptosis.

Nobiletin Inhibited the Invasion and Migration of NSCLC.
Metastasis is a poor prognostic factor of NSCLC. erefore, we investigated the effect of nobiletin on the invasion and migration of A549 and H460 cells. Transwell chamber assays were conducted to evaluate the effects of nobiletin on invasion and migration. As shown in Figures 2(a) and 2(b), nobiletin significantly suppressed the invasive capacities of both A549 (P < 0.01) and H460 (P < 0.05) NSCLC cells compared to their control group. Consistently, migration assays also showed that nobiletin significantly inhibited the migration abilities of both A549 and H460 cells (Figures 2(c) and 2(d)), suggesting that nobiletin has powerful antimetastatic effects.

Nobiletin Inhibits WNT/β-Catenin Signaling in NSCLC.
To investigate the anticancer molecular mechanism of nobiletin in NSCLC, A549 and H460 cells were treated with nobiletin for 48 hours and then subjected to whole-genome sequencing. Nobiletin treatment altered the expression levels of many genes in both NSCLC cell lines (Figure 3(a)). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that nobiletin treatment significantly affected cancer stemness maintenance-related WNT/β-catenin signaling (Figure 3(b)). In fact, bioinformatics analysis of mRNA sequencing results showed that nobiletin upregulated negative regulators of WNT/β-catenin signaling, including NKD1, AXIN2, and WIF1 while inhibiting positive regulators of WNT/ β-catenin signaling, including WNT6 and Jun (Table 1). Consistently, gene set enrichment analysis (GSEA) showed that nobiletin treatment was negatively correlated with epithelial-mesenchymal transition (EMT) and cancer stemness in NSCLC (Figure 3(c)). Together, these findings suggested that nobiletin has anticancer effects by inhibiting WNT/β-catenin signaling.

Nobiletin Inhibits Cancer Stemness and WNT/β-Catenin
Signaling in NSCLC. To investigate whether nobiletin is directly involved in the inhibition of cancer stemness and WNT/β-catenin signaling, NSCLC cells were treated with nobiletin and then subjected to sphere formation assays, and the expression levels of marker proteins of cancer stemness and WNT/β-catenin signaling were detected. Our results showed that nobiletin significantly inhibited sphere formation of NSCLC cells (Figure 4(a)) and cancer stemness-related marker protein expression (Figure 4(b)). In addition, nobiletin treatment significantly inhibited β-catenin levels (Figures 4(c) and 4(d)) and downstream protein expression levels of WNT/ β-catenin signaling (Figure 4(d)). ese results suggested that nobiletin is directly involved in the inhibition of cancer stemness and the WNT/β-catenin signaling pathway in NSCLC.

Discussion
In recent decades, natural products have received widespread attention as anticancer drugs. ey have anticancer effects and fewer side effects [1][2][3][4]. However, natural products have not been widely used in clinical treatment because their mechanism of action is not clear. Nobiletin is a ubiquitous flavonoid derived from the peel of citrus fruits. Previous studies have demonstrated that nobiletin possesses therapeutic and biological activities, including antioxidant, anticancer, cardioprotective, and anti-inflammatory effects [10,11,[13][14][15][16][17][18]24], but the anticancer mechanism is still unclear.
Cancer stemness is a key factor for cancer progression. Studies show that increased cancer stemness stimulates cancer cell resistance to chemotherapy and enhances cancer cell invasiveness, thereby contributing to cancer progression [26], and increased cancer stemness also stimulates EMT [25]. EMT is a process in which epithelial tumor cells lose their cell polarity and cell-cell adhesion so that tumor cells gain migratory and invasive properties [27]. During EMT, epithelial cells lose epithelial markers, such as E-cadherin, which is the main molecule of stable epithelial adherens junctions, so that the invasion-metastasis cascade is promoted, resulting in the exit of tumor cells from the primary site, invasion to adjacent organs, intravasation, and distant metastasis via the blood or lymphatic system. Studies have shown that inhibition of cancer stemness and EMT can dramatically inhibit cancer progression [28,29]. Abnormally increased cancer stemness and EMT are caused by many factors in cancer, and hyperactivation of WNT/β-catenin signaling is one of them [30,31]. Here, our data showed that nobiletin inhibits NSCLC progression, cancer stemness, EMT, and WNT/β-catenin signaling, suggesting that nobiletin exerts its anticancer effects by inhibiting WNT/ β-catenin signaling-regulated cancer stemness and EMT.
Here, we also demonstrated that WNT/β-catenin signaling was inactivated by nobiletin. WNT/β-catenin  signaling is often hyperactivated in cancers by the downregulation of negative regulators, including NSCLC [32]. In addition, studies have shown that downregulated expression of the WNT/β-catenin signaling pathway is promoted by dysregulated miRNA expression in cancer [33]. Interestingly, studies have shown that natural products play an anticancer role by altering miRNA expression [34][35][36]. In this study, we reported for the first time that nobiletin suppresses miR-15-5p expression, thereby causing upregulation of miR-15-5p target genes, including negative regulators of WNT/β-catenin signaling, such as NKD1, AXIN2, and WIF1, which inactivate WNT/β-catenin signaling.

Conclusions
In conclusion, nobiletin inhibits NSCLC by inhibiting cancer stemness and EMT by inactivating the WNT/ β-catenin signaling pathway in NSCLC. Nobiletin inhibited miR-15-5p expression, thereby increasing negative regulators of the WNT/β-catenin signaling pathway and ultimately inactivating the WNT/β-catenin signaling pathway. Our results shed light on the mechanism of the anticancer effect of nobiletin and provide more inspiration for physicians in treating patients with NSCLC.

Data Availability
All data generated or analyzed during this study are included within this article.

Ethical Approval
is study was approved by the Institutional Review Board of Chuncheon Sacred Heart Hospital (2020-09-002), which waived the need for informed consent for the use of patient data due to the retrospective nature of the study. All methods were performed in accordance with the relevant guidelines and regulations.

Disclosure
is work was based on doctoral dissertation research completed at Kangwon National University of Korea.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.

Authors' Contributions
Hae Sung Kim contributed equally to this work. JWL proposed the topic and conceived and designed the study. SHH performed the experimental examinations, including PCR. JHH and WJC analyzed the data and helped with the interpretation. HSK and SSL collaborated with the corresponding author in the writing of the manuscript. All authors read and approved the final manuscript.