Berbamine Inhibits Cell Proliferation and Migration and Induces Cell Death of Lung Cancer Cells via Regulating c-Maf, PI3K/Akt, and MDM2-P53 Pathways

Berbamine (BBM) is a natural product isolated from Berberis amurensis Rupr. We investigated the influence of BBM on the cell viability, proliferation, and migration of lung cancer cells and explored the possible mechanisms. The cell viability and proliferation of lung cancer cells were evaluated by MTT assay, EdU assay, and colony formation assay. Migration and invasion abilities of cancer cells were determined through wound scratch assay and Transwell assay. Cell death was evaluated by cell death staining assay and ELISA. The expressions of proteins were evaluated using western blot assay. A xenograft mouse model derived from non-small-cell lung cancer cells was used to detect the effect of BBM on tumor growth and metastasis in vivo. Both colony formation and EdU assays results revealed that BBM (10 μM) significantly inhibited the proliferation of A549 cells (P < 0.001). BBM (10 μM) also significantly inhibited the migration and invasion ability of cancer cells in wound scratch and Transwell assays. Trypan blue assay and ELISA revealed that BBM (20 μM) significantly induced cell death of A549 cells. In xenograft mouse models, the tumor volume was significantly smaller in mice treated with BBM (20 mg/kg). The western blotting assay showed that BBM inhibited the PI3K/Akt and MDM2-p53 signaling pathways, and BBM downregulated the expression of c-Maf. Our results show that BBM inhibits proliferation and metastasis and induces cell death of lung cancer cells in vitro and in vivo. These effects may be achieved by BBM reducing the expression of c-Maf and regulating the PI3K/Akt and MDM2-p53 pathways.


Introduction
Lung cancer is the leading cause of cancer-related death in both men and women. e incidence of lung cancer in China in 2014 was 73.3 per 100,000, ranking the first among malignant tumors, and lung cancer is the leading cause of cancer-related death in both males and females [1]. Nonsmall-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers and is usually diagnosed at an advanced stage with metastasis [2]. Treatments for patients with NSCLC at advanced stage have little therapeutic effect, and relapse often follows cancer therapy. erefore, new strategies to NSCLC treatment are urgently needed.
Chinese medicine has provided therapeutic options for patients with advanced tumors. Berbamine (BBM) is derived from a traditional Chinese medicine of Berberis amurensis Rupr. BBM has been widely used in Asian countries for patients with leukopenia and did not show any obvious side effects [3]. Recent studies have shown that BBM has antioxidation, immunoregulation, and antiarrhythmia activities [4][5][6][7]. BBM was also revealed to have potential antitumor function [8][9][10]. Studies demonstrated effects of BBM on cell activity and migration, with mechanisms involving Bcl-2/ Bax, and BBM was also revealed as an inhibitor of Stat3 [11] and NFκB [4,12,13]. However, few reports have examined the antitumor effects of BBM on lung cancer. us, more studies are needed to study the effects and potential mechanism of BBM in NSCLC.
Abnormality of the PI3K/Akt signaling pathway is related to tumor progression. Aberrant activation of the PI3K/ Akt signaling pathway occurs in a wide range of human tumors [2]. erefore, researchers have investigated therapeutic strategies to target the PI3K/Akt signaling pathway. For example, Shakti et al. found that bergapten inhibited liver carcinogenesis by downregulating the expression of PI3K [14]. Wu et al. found that high expression of PI3K is associated with metastasis and cisplatin resistance in NSCLC [15]. In addition, activation of both the Lyn/PI3K/Akt and MAPK/ERK pathways by green barley extract mitigated the cytotoxicity in human lymphocytes undergoing aggressive oxidative stress [16]. Amplification of PI3K and overaction of Akt or mutations in some of the regulatory components in the PI3K/Akt pathway are the main reasons of metastasis and drug resistance [17]. us, better understanding of the PI3K/Akt signaling pathway is helpful to identify potential targets for lung cancer treatment.
MDM2 is a novel downstream factor of PI3K [18]. Elevated MDM2 levels promote ubiquitination and degradation of E-cadherin [12], which in turn promotes cancer cell invasion [19]. e p53 tumor suppressor plays a critical role in regulating cell death, cell cycle arrest, and apoptosis. MDM2 interacts with p53 to promote p53 degradation [20]. c-Maf is a member of the basic leucine zipper transcription factor family [21], and c-Maf is highly expressed in MDA-MB-231 and MCF breast cancer cell lines [19]. Zhang et al. found that knockdown of c-Maf led to the downregulation of MDM2 in human gastric cancer cells [22]. e in vitro study will help to elucidate the BBM's impact on cell viability, proliferation, invasion, and migration in lung cancer cells and indirectly explore BBM's effect on the cell cycle profile. In this study, we examined the potential anticancer effects of BBM on NSCLC. We evaluated the impact of BBM on the cell viability, proliferation, invasion, and migration in lung cancer cells. We also investigated the effect of BBM on tumor growth and metastasis in nude mice and the potential involvement of the PI3K/Akt and MDM2-p53 signaling pathways and c-Maf in the effects of BBM.

Colony Formation Assay.
Colony formation assay was performed as previously described [26]. A549 cells and PC9 cells were inoculated into 6-well plates and treated with vehicle or different concentrations of BBM (10, 20, and 40 μM) for 24 h. e medium was then gently replaced with complete medium (with 10% FBS). Cells were cultured, and the medium was replaced every 3 days. After 8 days, the cells were stained with crystal violet.

EdU Cell Proliferation Assay.
e cell proliferation was evaluated by the EdU Apollo-567 In Vitro Kit (Ribobio, Guangzhou, China, cat. no. C10310-1) [26]. Briefly, A549 cells and PC9 cells (8 × 10 3 cells/well) were plated into 96well plates with vehicle or BBM (10, 20, and 40 μM) and cultured for 24 h. en EdU was added to each well and incubated at 37°C for 2 h. e cell nuclei were stained with Hoechst 33342 at 25°C for 30 min. e fluorescence of cells was observed using an inverted fluorescence microscope (Leica, Germany).

Trypan Blue Staining
Assay. Cell death was evaluated by the Trypan Blue staining assay as previously described [27]. A549 cells and PC9 cells (8 × 10 4 cells/well) were seeded into a 6-well plate and treated with vehicle or different concentrations of BBM (10, 20, and 40 μM). e cells were cultured at 37°C for 48 h. Trypan blue staining was performed according to the manufacturer's instructions. e cells were visualized under a light microscope (Leica DMi 4000).

Cell Death Assay.
Cell death was detected using the Cell Death Detection ELISA Kit [28]. is assay is based on the quantitative sandwich immunoassay using antibodies against histones and DNA. e presence of mono-and oligonucleosomes in the cell lysates indirectly represents the apoptosis of cells. A549 cells and PC9 were seeded into the 6-well plates and cultured with or without different concentrations of BBM for 24 h. en, the RIPA lysis buffer (with 1 mM PMSF) was added to the well, and the supernatant was collected for testing. e mono-and oligonucleosomal fragmented DNA was detected according to the manufacturer's instructions.

Wound Scratch Assay.
Cell migration was evaluated by the wound scratch assay [29]. A549 cells and PC9 cells were seeded into a 6-well plate and cultured for 24 h. A 200 μl plastic tip was used to generate a straight line in the cell monolayer. Fresh serum-free medium with vehicle or different concentrations (10, 20, and 40 μM) of BBM were added to each well. e cells were imaged at 0 h and 24 h in the same position of the wound. e migration distance was measured by NIH Image J software. Mitomycin (2 μg/ml) was always added to exclude the proliferation of the cells.

Transwell
Assays. Transwell assays [29] were performed to evaluate the metastasis of cancer cells. Briefly, A549 cells and PC9 cells (4 × 10 4 cells) were seeded into the upper chamber of a Transwell insert (8 μm pore, Corning, America, cat. no. 3412). e cells were allowed to invade or migrate through the chambers at 37°C for 24 h. Cells that invaded or migrated to the surface of the lower chambers were stained and counted. Mitomycin (2 μg/ml) was added in all experiments to exclude the proliferation of cells.

Experimental Animals.
Male BALB/c nude mice (6 weeks old, weighing 18-20 g) were obtained from Gem-Pharmatech Co., Ltd. (Jiangsu, China). e mice were housed in polystyrene, well aerated cages with a 12 h light/ dark cycle. e animals were maintained on a standard pelleted diet and were provided with free access to food and water ad libitum. All studies were performed with the approval of ARRIVE Guidelines (Animal Research: Reporting of In Vivo Experiments) and approved by the Animal Care and Use Committee of Soochow University.
2.11. In Vivo Assay. A549 cells were transplanted to the right axillary of the nude mice [30]. When the tumor volume (W 2 * L/2, where W � width and L � length) reached 150 mm 3 , the mice were randomly divided into three groups: the control group (Ctrl) and the experimental groups (20 mg/kg and 40 mg/kg). Each group had 6 mice. e mice were treated intraperitoneally with isotonic saline or different concentrations of BBM (diluted with isotonic saline) for 10 days, and they were treated daily. e body weight and tumor volume of the mice were recorded every 6 days. At the end of the experiment, the mice were harvested for western blotting assay. e weight and the metastatic nodules of lungs were recorded. e visceral tissues were removed for histopathological examination.

Histopathological Studies.
We used the Hematoxylin/ Eosin Staining Kit to study the histopathological change of various organs [30]. e tissues were fixed and sectioned at 3-4 μm, and the slices were stained with the Hematoxylin/ Eosin Staining Kit (Beyotime, China, cat. no. C0105 M) according to the manufacturer's instructions. Histopathological changes were examined by microscopy (Leica, Germany).
2.14. Statistical Analysis. Data are presented as mean-± standard deviation (SD). e statistics were analyzed using SPSS software. Statistical differences were evaluated by Student's t-test or one-way ANOVA method; the accepted level of significance was P < 0.05.

Effect of BBM on the Cytotoxicity of Lung Cancer Cells.
e in vitro cytotoxicity of BBM was evaluated by MTT assay, colony formation assay, and EdU assay. e results showed that BBM effectively inhibited the growth of A549 cells and PC9 cells in a time-and dose-dependent manner (Figure 1(a)). e IC 50 values for BBM against A549 cells and PC9 cells at 72 h were 8.3 ± 1.3 μM and 16.8 ± 0.9 μM, respectively ( Figure 1(b)). e results of the colony formation assay showed that colony numbers were significantly decreased among BBM-treated cells compared with the control group ( Figure 1(c)). In A549 cells and PC9 cells, BBM at 10, 20, and 40 μM decreased the number of the colonies in a dose-dependent manner (P < 0.001; Figure 1  cells (Figure 1(e)). BBM at 10, 20, and 40 μM led to a decrease of EdU-positive cells compared with the Ctrl group (P < 0.001, Figure 1(f )). Collectively, these results indicated that BBM inhibited the proliferation of lung cancer cells.

Effect of BBM on the Cell Death of Lung Cancer Cells.
We examined the effect of BBM on cell death of lung cancer cells using Trypan Blue Dye staining assay and the Cell Death Detection ELISA Kit. e results showed that BBM induced cell death in both A549 cells and PC9 cells (Figure 2(a)). BBM at 10, 20, and 40 μM increased the percentage of Trypan blue-positive in a dose-dependent manner in A549 cells and PC9 cells (P < 0.001, Figure 2(b)). ese results were further confirmed by an ELISA. BBM at 20 and 40 μM significantly increased the death of A549 cells (P < 0.05, Figure 2(c)). BBM at 10, 20, and 40 μM significantly increased the death of PC9 cells (P < 0.05, P < 0.05, and P < 0.001, Figure 2(c)).
ese results confirmed that BBM induces cell death of A549 cells.

Effect of BBM on Migration and Invasion of Lung Cancer
Cells In Vitro. e effects of BBM on the migration and invasion abilities of lung cancer cells were tested by the wound scratch assay and Transwell assay. e results showed that BBM inhibited wound closure in both A549 cells and PC9 cells (Figure 3(a)). BBM at 10, 20, and 40 μM significantly inhibited the migration of A549 cells in a dosedependent manner (P < 0.05, P < 0.001, and P < 0.001, respectively, Figure 3(b)). BBM at 10, 20, and 40 μM significantly inhibited the migration of PC9 cells in a dose-dependent manner (P < 0.001, respectively, Figure 3(b)). Transwell assays further showed that BBM significantly inhibited the migration and invasion of A549 cells and PC9 cells (Figure 3(c)). In migration assays, BBM at 10, 20, and 40 μM decreased the number of A549 cells on the surface of the lower chambers in a dose-dependent manner (P < 0.05, P < 0.001, and P < 0.001, respectively, Figure 3(d)), and BBM at 10, 20, and 40 μM decreased the number of PC9 cells on the surface of the lower chambers in a dose-dependent manner (P < 0.001, Figure 3(d)). In invasion assays, BBM at 10, 20, and 40 μM decreased the number of A549 cells on the surface of the lower chambers in a dose-dependent manner (P < 0.05, P < 0.01, and P < 0.001, respectively, Figure 3(e)), and BBM at 10, 20, and 40 μM decreased the number of PC9 cells on the surface of the lower chambers in a dose-dependent manner (P < 0.01, P < 0.001, and P < 0.001, respectively, Figure 3(e)). Together, these results demonstrated that BBM inhibits the migration and invasion of lung cancer cells.

Effect of PI3K Inhibitor LY294002 and MDM2-p53
Inhibitor AMG232 on the Cell Viability and Metastasis of Lung Cancer Cells. We used PI3K inhibitor LY294002 and MDM2-p53 inhibitor AMG232 to observe the anticancer actions of BBM. As the results showed, when the A549 cells and PC9 cells were treated with PI3K inhibitor LY294002, the cell viability was significantly inhibited compared to the Ctrl group (P < 0.001, Figure 5(a)), but there was no significant difference between the LY294002 group and the LY294002 + BBM group, and there was no significant difference on migration between the LY294002 group and the LY294002 + BBM group ( Figure 5(b)). When the A549 cells and PC9 cells were treated with MDM2-p53 inhibitor AMG232, the cell viability was significantly inhibited compared to the Ctrl group (P < 0.01 and P < 0.05, Figure 6(a)), and BBM + AMG232 could significantly inhibit the cell viability compared to the AMG232 group (P < 0.05, Figure 6(a)), and there was no significant difference on migration between the AMG232 group and the AMG232 + BBM group (Figure 6(b)).

Effect of BBM on the Growth and Metastasis of Tumors In Vivo.
To determine the effect of BBM on NSCLC in vivo, we implanted A549 cells into nude mice and treated the mice with BBM or vehicle. e body weight of the mice in the experimental groups showed no significant difference compared with the Ctrl group (Figures 7(a) and 7(b)). e tumor volume of the mice was significantly smaller in mice treated with BBM compared with Ctrl mice (P < 0.05, Figures 7(a) and 7(c)). BBM at 20 mg/kg and 40 mg/kg significantly decreased the lung weight (P < 0.01 and P < 0.05, respectively, Figures 7(d) and 7(e)). e tumor weight in the experimental group (40 mg/kg) was significantly decreased compared with the Ctrl group (P < 0.05, Figure 7(f )). In addition, the number of nodules per lung in the BBM group (20 mg/kg and 40 mg/kg) was significantly decreased compared with the Ctrl group (P < 0.001, Figure 7(g)). Histopathological evaluations revealed no changes in lungs, livers, hearts, and kidneys between experimental and Ctrl groups (Figure 8).

Effect of BBM on the Expressions of c-Maf, PI3K/Akt, and MDM2-p53
Signaling Pathways In Vivo. MDM2 is an oncoprotein associated with various malignancies; its overexpression is vital for aggressive metastasis [20]. To determine if BBM plays an anticancer role in vivo through the PI3K/Akt and MDM2-p53 signaling pathways, we examined the expressions of PI3K, p-Akt, Akt, MDM2, p53, caspase-3, c-Maf, Bcl-2, and Bax in tumors using western blot assay. BBM at 20 mg/kg and 40 mg/kg significantly reduced the expression of PI3K (P < 0.01 and P < 0.001, respectively, Figures 9(a) and 9(b)). BBM at 20 mg/kg and 40 mg/kg significantly reduced the Evidence-Based Complementary and Alternative Medicine

Discussion
Lung cancer is the leading cause of cancer-associated death among both men and women worldwide. In recent years, with the gradual elucidation of the molecular mechanism of NSCLC, more molecular-targeted drugs have been applied in clinical practice and achieved satisfactory results [31,32]. erefore, designing new drugs based on key target genes or proteins that play critical roles in NSCLC is important for the treatment of NSCLC in the future. Previous studies have demonstrated the antitumor effects of BBM in a variety of tumors, including breast cancer [33], myeloma [7,12], hepatoma [9,34], prostatic neoplasms [9], pancreatic carcinoma [35], and lung cancer [36]. All these findings indicate the potential of BBM for cancer treatment. Moreover, we demonstrated that BBM inhibited cell proliferation and metastasis of A549 cells and PC9 cells and inhibited migratory and invasive potential of these cells. Xenograft studies were also used to evaluate the effect of BBM on lung cancer. e results indicated that BBM significantly inhibited tumor growth and metastasis without manifesting changes in potential toxic signs such as diarrhea, movement disorders, or swelling, and the histopathological evaluations revealed no changes in lungs, livers, hearts, and kidneys between experimental and Ctrl groups. e PI3K/Akt pathway is associated with cell survival, invasion, and migration and plays key roles in various cancers [16,37,38]. In NSCLC, the PI3K/Akt/mTOR pathway has been heavily implicated in both tumorigenesis and the progression of disease [39]. e phosphorylation of Akt causes further phosphorylation of downstream effectors, such as mTOR and MDM2, which are closely associated with the apoptosis process [40]. Most of the studies showed that natural products exhibited anticancer actions via PI3K/Akt pathway [41]. Furthermore, numerous preclinical studies have shown that some herbs and natural phytochemicals can inhibit AKT activity directly [42,43]. e effects of BBM on lung cancer cells were deciphered on the alteration in the level of expression of PI3K/Akt-related markers. We also noted that BBM inhibited the expressions of PI3K and Akt. When we used the inhibitor of PI3K, there was no difference in the proliferation between the PI3K inhibitor group and the BBM + PI3K inhibitor group.
us, PI3K may be the target of BBM on the lung cancer cells, and BBM may inhibit the cell proliferation and metastasis through PI3K/Akt pathway.

Evidence-Based Complementary and Alternative Medicine
MDM2 is an oncoprotein that exhibits dynamic negative regulation of the tumor suppressor p53 [19]. MDM2 is often highly expressed in a variety of human cancers, and its overexpression promotes cancer cell's proliferation [25,44]. e MDM2-p53 pathway is important in regulating cell events, such as cancer cell death, cell cycle arrest, apoptosis, senescence, and DNA repair [25]. Moreover, MDM2 suppresses the migration of cancer cells and induces apoptosis of cancer cells [29,44,45]. Our results showed that BBM inhibited the migration and invasion of A549 cells and PC9 cells in vitro, and the number of nodules on lungs in a xenograft mouse model treated with BBM was significantly reduced in vivo; in addition, the expression of MDM2 was downregulated both in vitro and in vivo.
Besides, when we pretreated the cells with MDM2-p53 inhibitor, we found that the migration of the lung cancer cells showed no difference between the MDM2-p53 inhibitor group and the BBM + MDM2-p53 inhibitor group.
ese results indicate that BBM inhibits cancer cell metastasis and this effect may be achieved by modulating the expression of MDM2.
Recent studies revealed that c-Maf is overexpressed in a variety of cancers including NSCLC [46]. Studies showed that c-Maf is a downstream molecule of PI3K [47] and is indirectly associated with cell migration [48]. Our results showed that the expressions of PI3K and c-Maf were reduced by BBM. us, the mechanism of BBM inhibits lung cancer cells maybe through regulating the expression of   [22]. Divya et al. found that c-Maf was a downstream protein of PI3K/Akt pathway [49]. Our results revealed that migration and invasion were inhibited by BBM both in vitro and in vivo, and cell death was induced by BBM in vitro. e expressions of c-Maf were downregulated by BBM, and the expressions of p53 and cleaved-caspase-3/ caspase-3 were upregulated. ese results demonstrated that BBM inhibits the migration and induces the cell death of A549 cells. us, the expression of MDM2 may be regulated by c-Maf, and p53 may be regulated by MDM2.

Conclusion
Our study demonstrated that BBM suppresses the proliferation and migration of human lung cancer A549 cells. is effect may be through BBM regulating the expression of c-Maf by blocking the PI3K/Akt signaling pathway. e MDM2-p53 signaling pathway may be regulated by c-Maf ( Figure 10). With low toxicity characteristics, exploiting BBM alone or in combination with established chemotherapy regimens may provide a new treatment strategy for lung cancer. However, a noncancerous cell line for the in vitro studies should be included in the future, in particular,

Data Availability
e data used to support the findings of this study are included within the article.