Anti-cancer Effects and Mechanisms of Diosmin in HepG2 Cells in Vitro Revealed by Network Pharmacology and Molecular Docking

Diosmin is able to exert anti-cancer effects on various cancer cells, including Caco-2 and HT-29. Herein, we set out to investigate the anti-cancer effects and action mechanisms of diosmin in the HepG2 cell line. We utilized the PharMapper server and Genecards database to identify the target proteins. The Autodock software was used to dock key targets with diosmin to identify action sites. MTT and Western blot assays were utilized to determine cancer cell proliferation, as well as expression of related target proteins in order to validate the antitumor effect of diosmin. Results demonstrated that 13 key targets and 14 tumor signaling pathways were involved in the anti-cancer effect of diosmin. In vitro experiments demonstrate that the number of HepG2 cells were signicantly reduced with increasing Bax/Bcl-2 expression ratio after 200 µg/mL of diosmin treatment. Concomitantly, the degrees of RAS p-ERK and p-MEK were signicantly decreased, whereas those of Caspase-3 was increased. In conclusion, antitumor effect of diosmin is a comprehensive effect of multi-cell signaling pathways, multi-biological processes and multiple targets. The MAPK signaling pathway is one important pathways by which diosmin exerts its anti-tumor effects. The study provides a rationale for the application of diosmin in the treatment of HepG2.

that diosmin has the opposite effect, such as antiproliferative and pro-apoptotic activity, on tumor cells. This includes oral cancer cells, colon cancer cells, breast cancer cells and liver cancer cells [15,16,17].
Nevertheless, the antitumor mechanism of diosmin remains unknown. In addition, research on proliferation inhibition of diosmin in HepG2 cells has rarely been reported. Network pharmacology and molecular docking technology are emerging and promising techniques in pharmacological research over recent years [18]. Molecular docking has been applied to evaluate the relationship between small molecule compound ligands and macromolecular bioreceptors via molecular mechanics energy analysis.
It has been widely used in virtual screening of target proteins, and elucidating the mechanisms of action.
The credibility of the results has been validated among various molecular pharmacological experiments [19].
Herein, we analyzed and identi ed potential target proteins of diosmin from the PharmMapper and Genecards database, the string database and Gene Ontology Resource. Moreover, we identi ed the main tumor suppressor pathway of diosmin using KEGG analysis. Furthermore, we performed molecular docking analysis to determine the site of action of the main target proteins, using the small molecule diosmin as substrate. Finally, western blot technology was utilized to determine the function of diosmin in inhibiting proliferation of HepG2 cells. These results provide a scienti c theoretical basis for the development of anti-cancer drugs through the use of diosmin as the lead or analogue. Fig. 1 depicts the owchart of our study.

Chemicals
The DNA content quantitation measurement kit, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The BCA protein assay kit was purchased from by Beyotime Biotechnology (Shanghai, China). The chemicals that were required for cell culture, including RPMI 1640 medium, heat-inactivated fetal bovine serum (FBS) and trypsin were bought from Hyclone Co., USA. Antibodies that targeted ERK, p-ERK, MEK, p-MEK, HRAS, Raf-1, Bax, Bcl-2, Caspase-3 and β-actin were purchased from Wanleibio Co. (Shenyang, China). Other chemicals were of analytical grade and purchased from local suppliers. The chemical structure of diosmin was obtained via a Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP). The molecular structure of diosmin was saved in a mol2 format. The molecular structure of diosmin was imported into the PharmMapper database, setting human protein targets only. Results were merged from database and duplicated targets were deleted to predict the potential targets of diosmin. To identify the potential targets for the anti-cancer effect of diosmin, we selected the Genecards  database, entered cancer, anti-cancer, tumor and anti-tumor, searched for existing cancer-related genes, integrated the data, and deleted the duplicate genes.

Screening of anti-cancer targets
2.2.3. Diosmin-anti-cancer target network structure Next, we entered the intersection target into the String database, limited the species to only human, and obtained the interaction between the target proteins that have potential anti-cancer effect of diosmin. The information obtained was then imported into the Cytoscape software (version 3.6.1) to construct a protein-coded interaction network. Within this network, a node represents a protein, and the degree value indicates the number of other nodes that are directly connected to the node. The larger the degree, the more important the node is within the network. Closeness centrality is reciprocal of the average geodesic distance between the node to additional nodes. Furthermore, it re ects the degree in the central position of this node within the network structure. As the betweenness centrality depicts the position of the node between two other nodes that do not have direct connectivity between them, it can affect these other two nodes. Therefore, betweenness centrality is an indicator that re ects the importance of the node, and namely the importance of protein. According to the degree value and betweenness centrality, the important proteins were selected. Then, we utilized the Disgenet database [20] in order to obtain information on the potential anti-cancer effect of diosmin.

KEGG pathway analysis
The biological information annotation Database for Annotation, Visualization and Integrated Discovery (DAVID) functions to integrate biological data and analysis tools in order to provide a systematic and comprehensive biological function annotation information for both large-scale gene and protein lists. The anti-cancer targets of diosmin were then chosen to identify biological processes and pathways that were involved using the DAVID database for Gene Ontology (GO) analysis. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for the differential target genes and drew a diagram of the anti-cancer pathway of diosmin.

Determination of the binding site between diosmin and target protein
We performed the docking study using AutoDock in order to nd a possible binding mode and the a nity power between diosmin and the target protein. The binding energy between diosmin and the protein was evaluated to select the main target protein and diosmin molecules in order to evaluate binding activity. The human hepatoma HepG2 cell line was acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (Hyclone) supplemented with 10% (v/v) heat-inactivated FBS. Cells were incubated at 37°C in a humidi ed atmosphere of 95% air and 5% CO 2 until they reached con uence [21]. Typically, cells were passaged using trypsin and maintained in RPMI 1640 medium.

Cell proliferation assay
The MTT assay is a colorimetric method to evaluate survival and cell proliferation. Succinate dehydrogenase in living cells was able to deoxygenate exogenous MTT into a water-insoluble, blue-violet formazan and deposit it into cells. Next, we utilized this assay to screen for the pharmacodynamic effects of diosmin on multiple human cancer cell lines [21,22].
HepG2 cells were incubated into 96-well culture plates at a density of 1×10 4 cells in each well. The cells were then treated with 0, 50, 100, 150, 200, 250, 300, 350, and 400 µg/mL diosmin while the control groups were treated with phosphate buffer saline (PBS). After 24 hours, the supernatant was removed and 100 uL of culture medium was added. Next, 20 uL of MTT (5 mg/mL) was added to each well and under the same conditions, incubated for 4 h. The supernatant was then discarded, and a 150 uL dimethyl sulfoxide (DMSO) was added for 10 min in order to dissolve the converted violet dye into a 96well plate. The absorbance was quanti ed through the use of an automatic microplate-reader with 490 nm wavelength. Cell viability was then expressed as a ratio of optical densities of the treatment group to normal group, and calculated according to the following formula:

Cell morphology analysis
The morphological changes of HepG2 cells were directly observed using an inverted microscope and further analyzed using HE staining. Use of HE staining causes the cytoplasm to be stained red and the nucleus to be stained blue. Changes of cell structure in uence the light-shielding rate, and the stained cell images appear under a light microscope. HepG2 cells treated with diosmin were collected after trypsinization, and then incubated at a density of 1×10 5 /mL on a coverslip, which was placed into a 6well plate. After corresponding time, the coverslip was taken out. Then, the cell samples were xed with 95% ethanol for 20 min, followed by HE staining and observation under a light microscope.

Colony formation assay
In brief, we seeded HepG2 cells in a 6 well-plate (400 cells per well), which was cultured in a humidi ed incubator at 37°C and 5% CO 2 . After 24 hours, the medium was replaced with RPMI 1640 supplemented with the nal concentration of 200 µg/mL diosmin. The culture medium was refreshed every 48 h for the next 14 days. The medium was then removed and 1 mL of methanol per well was added to x the cells. wash away the excess dye solution, and the colonies were observed under a light microscope (Photoelectric Instrument Co. Ltd, Chongqing, China).

Western blot for protein expression detection
Cells were plated into 6-well plates at a density of 1.0 × 10 6 cells per well. After adhering, cells were treated with diosmin for 24 hours. Proteins were then extracted from the treated and untreated cells, and the concentration was determined using the BCA protein assay kit. Protein samples were separated via a 12% sodium dodecyl sulfate-polyacrylamide electrophoresis, and then transferred to 0.22 µm NC membranes. The membranes were then blocked with 5% skimmed milk for one hour at 4℃. Primary antibodies against ERK, p-ERK, MEK, p-MEK, HRAS, Raf-1, Bax, Bcl-2 and Caspase-3 were diluted at a concentration of 1:500-1:1000 in blocking buffer. Membranes were then incubated with diluted primary antibodies overnight at 4°C, which was followed by incubation with secondary antibody for one hour at 37°C. After each membrane was washed three times with TBS containing 0.1% Tween-20 (TBST), it was treated with the DAB Substrate kit (Beyotime Biotechnology, Shanghai, China) and visualized using the Gel Image System. In order to compare the protein expression between untreated and treated cells with diosmin, data were normalized to loading control (β-actin) during densitometric analysis.

Statistical analysis
The results represent mean ± SD from at least three independent experiments. Data was analyzed using the SPSS 19.0 software. Only differences that were P<0.01 were considered statistically signi cant.

Diosmin's targets to exert anti-cancer effect
The molecular structure of diosmin was obtained from the TCMSP platform (Fig. S1), and downloaded in the mol2 format. The diosmin structure consists of two phenolic hydroxyl groups and two monosaccharide molecules, which refers to a avonoid glycoside compound. The molecular weight of diosmin is 608.60, and thus, it a small molecule substance. The autodock molecular docking software can be utilized to predict the interaction between the ligand diosmin and the receptor biological macromolecule.
The 300 potential activity targets of diosmin can be collected via the Pharmmapper database. In total, we obtained 27,228 targets related to anti-cancer effects. Using an intersection with the target of diosmin action from a previous analysis, 119 targets with t value > 3 were chosen to analyze the intersection between the target proteins. After importing the obtained target proteins into the String database, and hiding the targets without interactions, the nal 59 target proteins with interactions were obtained. The information is listed in Table 1.

Construction and analysis of protein interaction network
The cytoscape 3.6.1 software was used to construct the resulting interaction network diagram between target proteins (Fig. 2). The nodes in the gure represent related proteins, while the lines represent correlation between proteins. The larger the degree value, the larger the node, and the more importance it occupies within the entire network.
According to the data, the nodes with degree value>5.02, betweenness centrality>0.0354 and closeness centrality>0.3424 were chosen as the main nodes for subsequent analysis. Using the Disgenet database to search properties of the main node proteins, and the results are listed in Table 2. The data demonstrates that the anti-cancer process of diosmin largely involves a variety of enzymes (mitogenactivated protein kinase 1, superoxide dismutase 2, cyclin dependent kinase 2, dihydrofolate reductase), enzyme modulators, and interleukins 2.

Gene function and pathway analysis
After inputting the 13 important target proteins that were obtained from previous analysis into the shared data platform (https://david.ncifcrf.gov/) (DAVID Bioinformatics Resources 6.8) and setting the species as homo sapiens, GeneOntology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Path analysis was conducted.
GO analysis includes three parts, comprising of cellular component (CC), molecular function (MF) and biological process (BP). Overall, we obtained 35 items from the GO analysis, and 23 items had a corrected P value of less than 0.05. Among them, there are four CC items, which mainly involved the cytosol, nucleoplasm, extracellular exosome, and caveola. In addition, there was 1 MF item (ATP binding). There are also 18 BP items, which were found to be mainly related to positive regulation of cell proliferation, positive regulation of cell migration, MAPK cascade, and cellular response to insulin stimulus ( Fig. 4A; Table 3). After ltering for corrected P value less than 0.05, the KEGG pathway analysis obtained a total of 14 items ( Fig. 4B; Table 4). These included metabolic pathways with seven targets involved, PI3K-Akt signaling pathways with ve targets involved, FoxO signaling pathway with ve targets involved, insulin signaling pathway with four targets involved, purine metabolism pathway with four targets involved and other signaling pathways closely related to the anti-tumor effects of diosmin. By drawing the interaction network diagram of the rst three main pathways (Fig. S2), we can see that the MAPK signaling pathway is closely related to these three pathways, so it has become the most focused pathway.  Fig. 4).

Effect of diosmin on cell inhibition
Starting from a concentration of 150 µg/mL, diosmin exhibited inhibition on HepG2 cells in a concentration-dependent manner, with a signi cant effect at 200~400 µg/mL (P<0.01; Fig. 5A). Accordingly, we further examined the mechanical behavior of 200 µg/mL diosmin using morphological observation. H&E staining showed that the internal structure of the treatment group was disordered with the condensed and aggregated chromatin, ruptured cell membrane and blue-violet particles, which appeared inside the treatment group (Fig. 5B). Then, the cell membrane stretched into an irregular shape, the cell swelling ruptured, and produced vesicles. In addition, diosmin attenuated proliferative activity by reducing the size and number of colonies compared to the untreated controls in HepG2 by colony formation assay (Fig. 5C).
We measured expression of the main proteins of MAPK signaling pathway, RAS/Raf1/MEK/ERK obtained from the previous network pharmacological analysis. Compared to the untreated cells, we observed 0.64fold, 0.42-fold, 0.45-fold and 0.31-fold decrease in protein expression level in cells that were treated with 200 µg/mL of diosmin for p-ERK, p-MEK, HRAS and Bcl-2, respectively. There were 16.09-fold and 5.24fold increase for Bax/Bcl-2 ratio and Caspase-3 expression. These differences were statistically signi cant (P<0.01) (Fig. 5D). Furthermore, protein levels of Raf-1 were found to be increased by 1.05-fold in treated cells, though difference was not statistically signi cant (Fig. 5D). HepG2 cells is rarely reported, and there is no hints about the signaling pathway of action. The liver cancer has become the sixth most common cancer and the second leading cause of cancer deaths worldwide, so this study was conducted to study the effect of Diosmin on the proliferation of HepG2 cells. The results showed that diosmin over 150 µg/mL could inhibit proliferation. Moreover, compared with the blank group, the Diosmin-treated group not only destroyed cell morphology, but also signi cantly reduced the clone numbers of cancer cells.

Discussion
In order to explore the possible pathway of action, we performed network pharmacological analysis. The related pathways of diosmin's tumor suppressor effect were mainly PI3k-Akt signaling pathway, FoxO signaling pathway, and insulin signaling pathway, according to P value from the KEGG pathway analysis,.
Interaction network diagram showed MAPK signaling pathway was closely related to these three pathways. The MAPK signaling pathway has been reported in some studies as an effector on other types of cancer cells. Jiang et al. discovered that C-phycocyanin was able to induce MDA-MB-231 cell apoptosis through the MAPK signaling pathways [28]. Yang et al. indicated that Morusin inhibited cell growth and migration by down-regulating the ERK phosphorylation level proteins [29]. Yang et al. found that Fangchinoline derivatives induced cell cycle arrest and apoptosis in human leukemia cell lines via suppression of PI3K/AKT and MAPK signaling pathway [30].
The MAPK pathway consists of different signaling cascades, of which the Ras--Raf--MAPK/ERK kinase is one of the most dysregulated in human cancer. RAS is a small GTPase whose active form is combined with downstream effector molecules, including Raf and PI3K, in order to promote cell growth and proliferation. The acceleration of RAS signaling can cause tumorigenesis, invasion and metastasis of different cancers. In the T24 bladder cancer (BC) cell line in 1982, HRAS was the rst human oncogene to be reported, as it was found to be mutated or up-regulated across a variety of cancers [31]. Therefore, in order to verify the important role of MAPK pathway in diosmin inhibiting the proliferation of HepG2 cells, we detected the expression of main proteins involved in the MAPK signaling pathway, HRAS, Raf1, MEK and ERK. Expression of p-ERK, p-MEK and HRAS down-regulated signi cantly. Meanwhile, activated MAPK pathway up-regulated downstream multi-effect molecules Bax and Caspase-3 and down-regulated Bcl-2, thereby cause the apoptosis of HepG2 cells. Consequently, the RAS-MAPK signaling pathway was indeed an innegligible pathway for diosmin to exert its anti-tumor effect in HepG2 cells.

Conclusion
Herein, network pharmacology analysis demonstrated that the antitumor effect of diosmin comprised a comprehensive effect of multi-cell signaling pathway, multi-biological process and multi-target. In vitro experiments identi ed that diosmin exerts anti-cancer effects on HepG2 cells. Comprehensive analysis reveals that diosmin has an anti-cancer role most probably by MAPK signaling pathway. Cell experiments has veri ed diosmin inhibits transcription by decreasing phosphorylating level of ERK and MEK and promote apoptosis by increasing expression ratio of Bax/Bcl-2 and Caspase-3 (Fig. 6).

Declarations Con ict of interest
The authors declare no con ict of interest exists.  Figure 1 The owchart of network pharmacology-based strategy for the anti-cancer effect of diosmin. Firstly, the potential target proteins of diosmin were analyzed and identi ed using Network Pharmacology. Next, molecular docking analysis was carried out to determine the site of action of the main target proteins using the small molecule diosmin as a substrate. Finally, cellular inhibition assays were designed and performed using HepG2 cells in order to explore anti-cancer effects and mechanisms of diosmin.

Figure 2
The protein-protein interaction network. The nodes within this gure represent related proteins, and the lines represent correlation between the proteins. The larger the degree value, the larger the node, and the more important it occupies within the entire network.
Page 19/22   Culture media was changed every three days. Cells were incubated for two weeks to grow colonies at 37℃ under conditions of 5% CO2, xed with methanol, stained with crystal violet solution and dried overnight. The wells were detected using a camera. (D) Western blot analysis of key proteins in HepG2 cells after 24 h treatment or untreated with 200 μg/mL diosmin. Data were normalized to β-actin.