Cucurbitacin B as a Chinese Medicine Monomer Inhibits Cell Proliferation, Invasion, and Migration in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor in southern China. Cucurbitacin B (CuB) is a tetracyclic triterpene compound isolated from Cucurbitaceae plants which has anti-in ﬂ ammation and antitumor properties and low toxic side e ﬀ ects. In this study, we use a series of wet experiments and network pharmacology analyses to explore the e ﬀ ects of CuB on cell proliferation, migration, invasion, and apoptosis of highly metastatic 5-8F NPC cells. The ﬁ ndings suggest that CuB inhibits NPC cells in a time- and dose-dependent manner and that cancer migration and invasion abilities decrease signi ﬁ cantly after CuB treatments. Mechanistically, CuB could increase the proportion of cells in the G2/M phase and reduce it in the G0/G1 phase, leading to apoptosis. The network pharmacological analyses and wet experiments uncovered that the MAPK pathway is a central target by pathway enrichment analysis, a ﬀ ecting the fate of cancer cells and in ﬂ uencing proliferation and apoptosis. Taken together, our study reveals that CuB could e ﬀ ectively inhibit 5-8F NPC cell proliferation, migration, and invasion via cell cycle blockage and cell apoptosis. Collectively, we have shown that CuB is a promising anti-NPC candidate compound for future preclinical study.


Introduction
Nasopharyngeal carcinoma (NPC) is the most common malignant tumor of the nasopharynx. Although global NPC incidence is less than 1/100,000, there are significant regional variations and a much higher incidence rate in North Africa and Southeast Asia, especially in the southern provinces of China. In 2018, there were 129,079 new NPC cases and 72,987 deaths around the world. Of these, more than 45% of the new cases were in China, several times higher than the global average incidence rate, and in some provinces, the rate exceeded 30/100,000 [1,2]. There are a number of pathological forms including the undifferentiated state [3], the hidden onset, mostly lymph node metastasis, and distant metastasis [4][5][6]. With the development of intensitymodulated radiotherapy (IMRT) treatment, patients with early stage NPC (stage I) who underwent radiation therapy alone experienced a 5-year survival rate (OS) of over 90% [7,8]. High survival rates are also seen in patients with stage II cancer who underwent radiotherapy alone, but 10-15% of patients develop recurrence and distant metastasis [9]. Combined chemotherapy can improve patient survival rate and reduce the rate of recurrence and metastasis, but the toxicity and side effects of chemotherapy drugs seem to do more harm than good [10,11]. Therefore, there is an urgent need to seek safe and effective radiotherapy adjuvant drugs with low toxicity and side effects which will help to reduce the recurrence and distant metastasis.
A number of studies have shown that many active components in Chinese herbal medicine have antitumor effects and that they are economical and multitarget, have low toxicity and side effects and, as such, are a current focus of antitumor drug research. Cucurbitacin is one of the main bioactive compounds found in Cucurbitaceae, such as cucumber [12]. It has been extracted from a variety of plants, has a wide range of pharmacological activities [13], such as cytotoxic, anti-inflammatory, and anticancer effects [14], and has been used as folk medicine for hundreds of years in countries such as India and China. Among the Cucurbitacin compounds, Cucurbitacin B (CuB) is one of the most widely used active substances for in vivo and in vitro tumor inhibition studies. With low toxicity and side effects, it is a promising potential antitumor herbal drug [15]; however, no studies have been conducted on its effect on NPC.
In this study, we examined the effect of CuB on the highly metastatic NPC 5-8F cells and reveal its possible mechanism of action using in vitro wet experiments and bioinformatics analyses. Our findings suggest that CuB could effectively inhibit 5-8F cell proliferation, migration, and invasion via cell cycle blockage and apoptosis. The network pharmacological analyses uncover that the MAPK pathway is a central target. Our findings show that CuB is a promising anti-NPC candidate compound and should be the focus of future research.

Material and Methods
2.1. Materials. 5-8F cells were purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences. CuB was purchased from MCE (United States). 1640 medium and FBS fetal bovine serum were purchased from Gibco (USA), Trypsin solution and DMSO were purchased from Amresco (USA), and BCA Protein Content Assay Kit and AnnexinV-FITC Apoptosis Assay Kit were purchased from Nanjing Kaiji Biotechnology Co., Ltd. CCK-8 reagent was purchased from Dojindo (Japan).

Cell
Culture. NPC 5-8F cells were cultured in RPM-1640 containing 10% fetal bovine serum at 37°C and 5% carbon dioxide. The medium was changed daily, and the cells were isolated with 0.25% trypsin.
2.3. CCK-8 Assay. Cells were seeded into 96-well plates and cultured for 24, 48, and 72 h. Cytotoxicity was detected by the CCK method. Absorbance at 450 nm was measured by an automatic enzyme plate reader (Bio-Tek, VT, USA). Cell mortality was calculated according to the following formula: inhibition rate ð%Þ = ðaverage A450 in the control group − average A450 in the experimental groupÞ/ðaverage A450 in the control group − average A450 in the blank groupÞ × 100%. Each experiment was performed in triplicate.

Transwell Migration and Invasion
Assay. 5-8F cells in each group were resuspended in serum-free medium and a 2 × 10 5 cells/mL cell suspension was prepared. A Transwell chamber was inoculated with 200 μL cell suspension in the upper chamber and 600 μL serum-free medium in the lower chamber. The cells were incubated at 37°C for 24 h, and after which, sterile cotton swabs were used to remove the upper 5-8F cells and were thoroughly washed in PBS. The cells were fixed in 4% paraformaldehyde for 30 min and stained in 0.1% crystal violet for 10 min. Three fields were randomly selected under the microscope for photography, and the number of stained cells in crystal violet was calculated as the number of migrated cells. For the invasion assay, RPMI 1640 medium was added at a ratio of 1 : 5 to Matrigel, and the upper chamber of Transwell was coated. After drying, the same protocol as the cell migration assay above was performed, and the number of invading cells was counted.

Flow
Cytometry Analysis of Apoptosis. The Annexin V-FITC double-staining method was used to detect the effect of CuB on 5-8F cell apoptosis. 1 × 10 6 cells were seeded into each well of a six-well plate, and different CuB concentrations (0, 200, 400, and 800 nM) were added after the cells adhered to the wall. Cells were incubated for 48 h, then harvested, resuspended in cold PBS, and stained with the Annexin-FITC apoptosis detection kit according to the manufacturers' instructions. Cell apoptosis was then analyzed by flow cytometry. The experiment was performed in triplicate.
2.6. Flow Cytometry Analysis of Cell Cycle. 5-8F cells in the logarithmic growth stage were seeded into 6-well plates, with a total volume of each well of 2 mL and cell density of 1 × 10 6 cells/L. After the cells were treated with CuB at final concentrations of 0, 200, 400, and 800 nM for 48 h, they were collected, rinsed twice with 0.01 mol/L PBS (pH = 7:2) for precooling, and fixed overnight with 75% cold ethanol at 4°C. Before detection, the fixation solution was removed and propidium iodide (PI) was added and the samples stained at 4°C for 30 min. 1 × 10 4 cells from each group were collected for flow cytometry detection. The MFLT SAAINC software was used to analyze the cell cycle phase ratio and early cell apoptosis rate. The experiment was performed in triplicate.

Bioinformatics
Analysis. The PubChem database (https:// pubchem.ncbi.nlm.nih.gov) was used to find the 3D compound structure of CuB, the PharmMapper database (http://www .lilab-ecust.cn/pharmmapper) was used to obtain CuB targets, and the GeneCards (http://www.genecards.org) database was used to retrieve target NPC-related diseases. The online tools from (http://bioinformatics.psb.ugent.be/webtools/Venn) were used to make maps of NPC and CuB targets. The PPI analysis of protein interactions was performed using String (https:// string-db.org/cgi). The Cytoscape 3.6.1 software was used to construct the CUB-nasopharyngeal carcinoma target network map, and cytoHubba's MCC method was used to search for core genes. The David web site (https://david.ncifcrf.gov/) was used for GO enrichment analysis on the common targets and the Kobas online database (http://kobas.cbi.pku.edu.cn/ kobas3) was used for KEGG pathway enrichment analysis.

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Journal of Nanomaterials 2.8. Western Blot. Total protein (40 μg) generated by cell lysis was separated on a 12% acrylamide gel (80 V, 30 min; 120 V, 60 min), then transferred to PVDF membrane (270 mA, 65 min), blocked with 5% skim milk for 1 h, washed three times with 0.1% TBST, and incubated in the primary antibody overnight at 4°C. The membrane was then washed three times with TBST, 5 min each, and then incubated with diluted fluorescent secondary goat anti-rabbit antibody for 1 h. The membrane was washed three times in TBST, 5 min each, and the Odyssey infrared fluorescence film scanner was used to sweep the film, adjust the required parameters, and save the picture. The antibodies used in this study were GADPH (Wuhan ProteinTech Company (China)), P-ERK and ERK (ABCAM (USA)), and BCL2 and BAX (Cell Signaling Technology (USA)). All the images were analyzed with ImageJ to calculate the gray value of the relevant target proteins.
2.9. Statistical Analysis. SPSS 25.0 was used for all statistical analysis. Measurement data was expressed as x ± s. A t-test was used for the comparison of two sample means, and one-way analysis of variance was used for comparison of means between groups. A P value < 0.05 indicates statistical significance. For all the data presented in this study, * represents P < 0:05, * * represents P < 0:01, and * * * represents P < 0:001. All experiments were repeated three times.

CuB Inhibits NPC Proliferation.
To explore the anticancer effects of CuB, we cultured 5-8F NPC cells (a highly metastatic cell line). The CCK8 assay results showed that CuB had a concentration-time-dependent effect on NPC 5-8F cell proliferation ( Figure 1). In detail, the inhibition rate of cell proliferation was 9:25 ± 2:58%, 19

CuB Suppresses Cell Migration and Invasion in NPC.
Since 5-8F is a highly metastatic cell line and metastasis is a clinical concern that needs to be addressed, we examined whether CuB has some effects on cell abilities such as migration and invasion. The results of the scratch experiment ( Figure 2) showed that the migration rates of 5-8F cells were 12:84 ± 2:37%, 10:12 ± 1:75%, To further validate these observations, Transwell migration experiments were performed. The results showed that the number of transmembrane cells in control and CuB treatment groups (50 nM, 100 nM, and 200 nM) was 386 ± 7, 187 ± 9, 142 ± 2, and 105 ± 10, respectively. Similarly, the Transwell-invasion experiment showed that the number of transmembrane cells in the control group and the CuB treatment groups (50 nM, 100 nM, and 200 nM) was 305 ± 26, 155 ± 4, 115 ± 9, and 29 ± 14, respectively. These results showed that as the CuB concentration increased, the migration and invasion ability of 5-8F cells decreased significantly (P < 0:05) (Figure 3). These results showed that CuB had some effect on metastatic abilities, such as migration and invasion, in NPC.

CuB Induces Cell Apoptosis in NPC.
After seeing that cell proliferation, migration, and invasion were all significantly inhibited, we next explored whether CuB could affect NPC cell fate. Flow cytometry showed that after 48 h treatment with CuB at 200, 400, and 800 nM, the apoptosis rates were 15:58 ± 3:09%, 24:6 ± 1:83%, and 31:76 ± 5:06%, respectively. Compared with the control group (6:25 ± 1:42%), the apoptosis rate was significantly increased and was correlated with concentration (P < 0:05) (Figure 4). These data suggest that CuB could induce cell apoptosis in NPC and that this is closely related to drug concentration.

Bioinformatics Analysis Uncovers MAPK as the Main
Target of CuB. To explore the overall anticancer effects of CuB against NPC, we performed bioinformatics analysis using a network pharmacology approach. In total, 357 CuB action targets were obtained from the PharmMapper database and 1786 target genes from the GeneCards database. Among them, 116 common targets were validated (Figure 6(a)). The PPI analysis showed that MAPK1 was the most correlated gene among all the target genes, with a total of 30 interrelated genes (Figures 6(b) and 6(c)). MCC analysis of the common target genes with the Cytoscape plug-in showed that MAPK1 had the highest score ( Figure 6(d)). Using P < 0:01 as the threshold, 119 terms were obtained by GO enrichment analysis, of which, MAPK1 played a role in 28 terms and participated in three functions: BP, CC, and MF ( Figure 6(e) shows the three functions of MAPK1 with the top 5 terms). The 28 functions in which MAPK1 is involved are shown in Figure 6(f). Using P < 0:01 as the threshold, MAPK1 (ranked by P value) was involved in 101 of the 159 pathways identified by the KEGG enrichment analysis. Figure 6(g) shows the MAPK1 position in the ranking by P value of the top 15 pathways. MAPK1 mainly participates in biological processes through the MAPK pathway, and Figure 6(h) shows all the common target genes enriched in the MAPK pathway. Western blotting showed that the phosphorylation level of ERK (downstream of the MAPK pathway) decreased in a dose-dependent manner after CuB treatment (Figures 7(a) and 7(b)). After CuB treatment, levels of the proapoptotic protein Bax increased and of the apoptosis regulator Bcl-2 decreased (Figures 7(a) and 7(b)). Overall, the bioinformatics analysis and wet experiments show that MAPK is the central target of CuB against NPC.

Discussion
Previous studies have shown that there is no obvious toxicity to normal cells at CuB concentrations lower than 5-10 μM [16]. Wakimoto et al. showed that when nude mice transplanted with MD-MB-231 tumor cells were injected with CuB (1 mg/kg) intraperitoneally for 6 weeks, the tumor volume reduced by 55% compared to the control group while body weight did not reduce [17]. At present, no national food and drug administration has included CuB in the list of drugs or harmful substances [18]. The Australian Therapeutic Goods Administration has approved unrestricted use of     Journal of Nanomaterials   9 Journal of Nanomaterials CuB and encourages its use in combination with other drugs [19], and China has developed calabash tablets, 60 percent of the active ingredient is CuB, which are used in the adjuvant treatment of hepatitis and primary liver cancer with the main side effect being mild gastrointestinal reactions. These studies confirm that CuB should be considered safe within therapeutic concentration ranges. In this study, when incubated with 5-8F cells for 24, 48, and 72 h, the IC50 of CuB was 1435:84 ± 272:36 nM, 622:94 ± 68:67 nM, and 314:79 ± 30:48 nM, respectively. Overall, this information suggests that CuB has obvious anti-NPC potential at safe dosage levels.
Studies have shown that CuB can inhibit the invasion and migration of a variety of tumor cells, but the specific mechanism remains unclear. Promkan et al. found that CuB could effectively inhibit the invasion and migration of BRCA1 defective breast cancer cells [20], and Liang et al. showed that it could destroy the cytoskeleton of breast cancer cells, altering their biomechanical properties, and inhibiting their migration and invasion [21]. In non-small cell lung cancer, Garg et al. found that CuB inhibited cell invasion and migration by decreasing mortalin, hnRNP-K, vascular endothelial growth factor, matrix metalloproteinase 2, and fibronectin [22], and Piao et al. found that CuB could inhibit tumor invasion and migration by inhibiting tumor cell angiogenesis [23]. In this study, we showed that CuB could also inhibit invasion and migration of NPC 5-8F cells, which is consistent with the results of previous studies.
Numerous studies have shown that CuB can inhibit the growth of many human cancer cells, including breast [24,25], prostate [26], lung [27], ovarian [28], liver [29], pancreatic [30], larynx [31], kidney [32], and skin cancer [33]. Although many studies have demonstrated the anticancer activity of CuB, the mechanism of its action is still unclear. It has been reported that CuB inhibits the growth of tumor cells and induces apoptosis by inhibiting the activation of signal transduction and transcriptional activator 3 (STAT3) and regulating its downstream genes such as cyclin D1, cyclin D2, and apoptosis-related genes like Bcl-2 and Bax [34,35]. In this study, we found that MAPK1 is one of the most important targets of CuB through network pharmacology analysis.
Mitogen-activated protein kinases (MAPK) are the largest subfamily of serine/threonine protein kinases and are involved in many signal transduction pathways and play important roles in protein renewal, cell growth, transcription factor activation, chromatin modification, and gene expression. The MAPK pathway is a well-known stress sensing signal transduction pathway, which can respond to extraand intracellular stress signals and transmit environmental and self-derived signals into cells. Relevant studies have indicated that CuB is an inhibitor of JAK-STAT3, Wnt, PI3K/Akt, and MAPK signaling pathways, which play an important role in apoptosis and survival of cancer cells [36].
In human chronic myelogenous leukemia studies, CuB inhibited MAPK/ERK pathway activation in various human chronic myelogenous leukemia cells and inhibited STAT3 activation [37]. The analysis in this study showed that there was inhibition of MAPK/ERK activation, which led to the arrest of the G2/M cell cycle and apoptosis. Silva et al. reported that CuB can inhibit PI3 kinase and MAPK pathways, delay cell migration, and reduce cellular invasive potential [38]. It could also induce dose-and time-dependent apoptosis, inhibit MMP release and FAK activation, and downregulate Akt, ERK, and NF-κBp65 phosphorylation. Zhang et al. found CuB had anticancer effects in the SH-SY5Y human neuroblastoma cell line [39]. CuB induced G2/M cell cycle arrest and apoptosis. This was accompanied by downregulation of CDK1 and cyclin B1, essential proteins in the cell cycle process. Another study has also reported that CuB inhibits proliferation and induces apoptosis of human osteosarcoma cells by regulating the JAK2/STAT3 and MAPK pathways [40]. In this study, MTT and Annexin V/propidium iodide staining showed that CuB (20-100 μM dose) significantly reduced cell viability and induced apoptosis and inhibited cell migration.
In conclusion, CuB's antitumor effect is mainly attributed to  Journal of Nanomaterials its induction of cell cycle arrest and apoptosis. The underlying mechanism of action may be attributed to the changes in key proteins involved in the regulation of cell cycle or apoptosis.
In conclusion, our study has shown that the Chinese medicine monomer CuB is a promising anticancer compound which affects cell proliferation, invasion, and migration in NPC cells. Mechanistically, its anticancer effects involve the cell cycle blockade and cell apoptosis, mediated through the MAPK signaling pathway. This shows that CuB is a promising agent for future NPC preclinical research and that this study also provides evidence that it might become part of a useful cancer therapy strategy.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on the reasonable request.