Investigation of Anti-Liver Cancer Activity of the Herbal Drug FDY003 Using Network Pharmacology

Globally, liver cancer (LC) is the sixth-most frequently occurring and the second-most fatal malignancy, responsible for 0.83 million deaths annually. Although the application of herbal drugs in cancer therapies has increased, their anti-LC activity and relevant mechanisms have not been fully studied from a systems perspective. To address these issues, we conducted a system-perspective network pharmacological investigation into the activity and mechanisms underlying the action of the herbal drug. FDY003 reduced the viability of human LC treatment. FDY003 reduced the viability of human LC cells and elevated their chemosensitivity. There were a total of 16 potential bioactive chemical components in FDY003 and they had 91 corresponding targets responsible for the pathological processes in LC. These FDY003 targets were functionally involved in regulating the survival, proliferation, apoptosis, and cell cycle of LC cells. Additionally, we found that FDY003 may target key signaling cascades connected to diverse LC pathological mechanisms, namely, PI3K-Akt, focal adhesion, IL-17, FoxO, MAPK, and TNF pathways. Overall, this study contributed to integrative mechanistic insights into the anti-LC potential of FDY003.


Introduction
Liver cancer (LC) is the sixth-most frequently occurring and the second-most fatal malignancy, responsible for 0.83 million deaths annually [1]. Molecular-targeted and immune checkpoint therapies serve as the main treatment strategies for LC in clinical settings [2]. However, such treatment options and their efficacy are largely restricted to a few patients, which emphasizes the need to design and develop effective therapeutics for LC treatment [2]. ere is growing recognition of herbal drugs as potent therapeutics for LC treatment to augment treatment efficacy while minimizing the adverse effects of anticancer strategies [3][4][5][6][7]. Herbal drugs possess beneficial properties, increasing survival and tumor response rates, enhancing the treatment efficacies and immune functions, increasing the quality of life, and inhibiting the development of side effects caused by anticancer treatments in patients with LC [3][4][5][6][7].

Herbal Drug Preparation.
For the preparation of FDY003, dried raw AcT (150.0 g), LjT (150.0 g), and Cm (100.0 g) were obtained from Hanpure Pharmaceuticals (Pocheon, Korea), mixed, and ground. e herbal mixture was dispersed in 70% ethanol (500 mL) and subjected to a 3 h reflux extraction process at −80°C. After purifying the herbal extracts with 80% and 90% ethanol solutions successively, they were lyophilized at −80°C and the resulting 50.4 g of freeze-dried extracts were stored at −20°C. We used distilled water to dissolve the prepared FDY003 samples before further experiments.

Analysis of Cell Viability upon Drug Treatment.
e viability of the drug-treated cells was analyzed by watersoluble tetrazolium salt (WST-1) experiments. Cells (1.0 × 10 4 ) were seeded and cultured in 96-well plates and treated with FDY003, sorafenib (Sigma-Aldrich, St. Louis, MO, USA), or both for 72 h. Next, cells were incubated for 2 h with WST-1 solution (Daeil Lab Service Co. Ltd., Seoul, Korea) at 37°C and 5% CO 2 . e cell viability was assessed by measuring the absorbance (450 nm) of the samples on an xMark microplate absorbance spectrophotometer (Bio-Rad, Hercules, CA, USA).

Screening for Bioactive Chemical
Components. First, we investigated comprehensive herbal medicine-related databases (e.g., a bioinformatics analysis tool for the molecular mechanism of traditional Chinese medicine [20], an anticancer herbs database of systems pharmacology [21], and the traditional Chinese medicine systems pharmacology [22]) and acquired large-scale data for the chemical composition of FDY003. For these components, we used pharmacokinetic parameters, namely drug-likeness, Caco-2 permeability, and oral bioavailability, for the screening of the bioactive chemical components, as previously described [14,22,23]. Drug-likeness determines whether a given chemical component can adequately serve as a drug based on its molecular structure and physicochemical properties [22,24]. A chemical component with a drug-likeness equal to or greater than 0.18 (the mean drug-likeness based on the currently available drugs) is considered to have adequate drug-like functions [22,24].
e Caco-2 permeability determines whether a given chemical component has adequate intestinal absorption and permeability [22,[25][26][27]; a chemical component with Caco-2 permeability equal to or greater than −0.4 is considered to have adequate intestinal permeability and absorption activity [28,29]. Oral bioavailability determines whether an orally administered chemical component has an adequate delivery rate to reach the target sites of drug action [22,30]. A chemical component with an oral bioavailability equal to or more than 30% is considered to have adequate absorption in the human body [22,30].

Target Screening.
To screen the targets of the identified bioactive chemical components of FDY003, we investigated their simplified molecular input line entry system notation using PubChem [28] and imported the information into the diverse tools and databases used to evaluate the molecular interactions between the chemical components and genes and proteins as follows: PharmMapper [29], Similarity Ensemble Approach [31], SwissTargetPrediction [32], and Search Tool for Interactions of Chemicals 5 [33]. Among the FDY003 targets, the genes and proteins significantly associated with the LC pathological mechanisms were examined using "liver cancer" and "hepatocellular carcinoma" as query keywords in the following tools and databases: Pharmacogenomics Knowledgebase [34], Online Mendelian Inheritance in Man [35], Human Genome Epidemiology Navigator [36], DisGeNET [37], Comparative Toxicogenomics Database [38], erapeutic Target Database [39], GeneCards [40], and DrugBank [41].

Analysis of Correlation between the Survival of Patients with LC and the Expression Profiles of FDY003 Targets.
e correlations between the survival of patients with LC and the expression profiles of LC-associated targets of FDY003 were analyzed using the Kaplan-Meier Plotter [45], a commonly used database for the survival analysis with large-scale gene expression profiles and survival information of patients with various cancer types obtained from e Cancer Genome Atlas (TCGA) [46], the Gene Expression Omnibus (GEO) [47], and the European Genome-phenome Archive (EGA) [48]. e survival analysis was performed using an auto-selected best cut-off and the results with p < 0.05 (log-rank test) were regarded to be statistically significant. e clinical information of the included patients for the survival analysis is provided in Supplementary Table S1.

Exploration of Functional Enrichment of FDY003 Targets.
e Gene Ontology (GO) and pathway enrichment of FDY003 targets were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) [49] and the Kyoto Encyclopedia of Genes and Genomes (KEGG) [50], respectively, and the results having p < 0.05 were regarded to be statistically significant.

Investigation of Binding Affinities between the Chemical Components and Targets of FDY003.
To assess the binding affinities between the chemical components and corresponding targets of FDY003, we calculated the binding energies. First, we imported the molecular structures of the bioactive components of FDY003 (obtained from the RCSB Protein Data Bank [51]) and their targets (obtained from PubChem [28]) into AutoDock Vina [52] and calculated the resulting binding energies. As suggested earlier [53,54], the binding affinities between the chemical components and targets were considered to be highly significant if their corresponding binding energies were ≤ −5.0.

Inhibitory Role of FDY003 in LC.
To assess the action of FDY003 against LC, HepG2 human LC cells were treated with FDY003 and/or sorafenib (a first-line therapeutic agent for LC treatment [2]), and the effects of the treatment on the cells were monitored. FDY003 significantly reduced the viability of HepG2 cells; the viability of the cells was further inhibited upon treatment with a combination of the herbal drug and sorafenib (Supplementary Figures S1A and S1B). ese observations suggest that FDY003 exhibits inhibitory and chemosensitizing effects on the LC cells.

Identification of Bioactive Components and Targets of FDY003.
As previously described, we selected FDY003 constituents with Caco-2 permeability ≥ −0.4, druglikeness ≥ 0.18, and oral bioavailability ≥ 30%, and considered them as bioactive components (Supplementary  Tables S2 and S3) [8,14,22,23]. Several components having potent anticancer functions against LC were also included in the list of bioactive components, despite their failure to meet the inclusion criteria (Supplementary  Tables S2 and S3). Using the databases for examining the molecular binding interactions between the chemical components and genes and proteins, a total of 379 targets were investigated. Among them, 91 were determined to be associated significantly with the LC pathological mechanisms based on previous cancer biology and oncological studies ( Figure 1, and Supplementary Tables S4 and S5). Analytical investigation of complex interactions among the therapeutic targets of a given drug is pivotal for understanding the underlying treatment mechanisms [55][56][57][58][59].

Network Analysis of Poly-Pharmacological Mechanisms
us, we constructed a PPI network consisting of large-scale interactions among the FDY003 targets to further dissect their pharmacological features ( Figure 3). For analyzing the network topology of the PPI network, we identified the network hubs having a relatively large number of interacting partners as compared to the nonhubs; they had a high probability of being effective drug targets [60,61]. As described previously, nodes with number of edges ≥ 2 × the average node degree were considered hubs [62,63]. As a result, the identified hubs were AKT1, AR, EGFR, ESR1, JUN, PIK3R1, SRC, TP53, and VEGFA ( Figure 3), which suggests that they were the primary nodes mediating the therapeutic roles of FDY003 in LC. Additionally, the expression status of these hubs could estimate the survival rate of patients with LC (Figure 4), which suggests their prognostic roles.
To understand the mechanisms of action of FDY003 in LC treatment, we evaluated the GO terms and pathways that were functionally enriched for the targets. e results showed that the FDY003 targets were functionally involved in the control of survival and proliferative behaviors, apoptosis, and cell cycle of LC cells (Supplementary Figure S2), which suggests the potential FDY003 mechanisms in the LC treatment. Additionally, FDY003 could target key signaling cascades connected to diverse LC pathological mechanisms ( Figure 5 and Supplementary Figure S2) as analyzed by the KEGG pathway enrichment investigation, which demonstrated that these pathways were important mediators of the FDY003 drug activity against LC.

Assessing the Binding Affinities of Chemical Components of FDY003 and eir Targets.
We performed a molecular docking study to assess the binding affinities and active binding sites between the chemical components of FDY003 and their targets. e resulting binding energies of the chemical component-target pairs were less than −5.0 kcal/ mol ( Figure 6 and Supplementary Figure S3 and Supplementary Table S6), which indicated their high pharmacological binding potentials.

Discussion
Globally, LC is the sixth-most frequently occurring and the second-most fatal malignancy, responsible for 0.83 million deaths annually [1]. Although the application of herbal drugs in cancer therapies has increased, their anti-LC activity and relevant mechanisms have not been fully studied from a systems view [3][4][5][6][7]. To address these issues, we conducted a system-perspective network pharmacological investigation into the activity and mechanisms of action of the herbal drug, FDY003, for LC treatment. Treatment with FDY003 significantly reduced the viability of human LC cells and elevated their chemosensitivity. A total of 16 potential bioactive chemical components of FDY003 and corresponding 91 targets were identified for the pathological process of LC. e FDY003 targets were functionally involved in the regulating survival, proliferation, apoptosis, and cell cycle of LC cells. Additionally, we found that FDY003 may target key signaling cascades connected to the diverse LC pathological mechanisms, namely, phosphoinositide 3-kinase (PI3K)-Akt, focal adhesion, interleukin (IL)-17, forkhead box O (FoxO), mitogen-activated protein kinase (MAPK), and tumor necrosis factor (TNF) pathways. Overall, this study added to the integrative mechanistic insights into the anti-LC potential of FDY003. e hub targets identified from the analysis of the FDY003-associated PPI network modulate crucial LC pathological mechanisms, and this targeting may result in effective therapeutic effects against LC. AKT1 is an important regulator of the tumorigenic processes, migration,     Evidence-Based Complementary and Alternative Medicine spreading, therapeutic sensitivity, resistance, survival, and proliferation of LC cells, and is a promising theragnostic target [64][65][66][67]. AR expression and activity influence angiogenesis, stemness, invasion, angiogenesis, epithelialmesenchymal transition (EMT), migration, and oncogenesis of LC cells, and it is a prognostic determinant and responsive marker for anticancer therapies [68][69][70][71][72][73]. EGFR is responsible for the modulation of proliferation, metastasis, migration, self-renewal potential, EMT, angiogenesis, anchorage-independent growth, invasion, and drug sensitivity of LC cells and tumors, and its expression and activity are implicated in the initiation, recurrence, progression, metastasis, and aggressiveness of LC in patients [74][75][76][77][78][79][80][81][82][83]. ESR1 is involved in the coordination of migration, angiogenesis, cancer stem-like properties, mobility, proliferation, and invasion of LC cells, and its genetic and expression status correlate with cancer susceptibility, tumor growth, metastasis, and prognosis of LC [84][85][86][87][88][89]. JUN is an oncogenic transcription factor that promotes tumor formation, chemoresistance, invasion, proliferation, migration, and metastasis, and serves as a marker for evaluating chemotherapeutic responses [90][91][92]. PIK3R1 is highly expressed and activated in LC cells and tumors, and it stimulates their migration, proliferation, invasion, and survival [93,94]. Its elevated expression level is associated with poor prognostic outcomes [93,94]. Src is highly expressed in LC cells and tumors, and its targeting can repress anoikis resistance, metastasis, treatment resistance, growth, stemness, invasion, tumorigenesis, migration, and mobility [95][96][97][98][99][100][101]. e expression and mutations of TP53 serve as biomarkers of prognosis, therapeutic response, aggressiveness, progression, and survival in LC [102][103][104][105][106][107]. VEGFA contributes to the malignant processes in LC by inducing angiogenesis, growth, invasion, prosurvival, metastasis, lymphangiogenesis, and migration of LC cells and tumors, and its expression and polymorphisms are related to the therapeutic responsiveness, prognosis, and cancer susceptibility of patients with LC [108][109][110][111][112][113].

Conclusions
In conclusion, we performed a system-perspective network pharmacological investigation into the activity and mechanisms underlying the effects of the herbal drug, FDY003, for LC treatment. FDY003 could reduce the viability of human LC cells and elevate their chemosensitivity. A total of 16 potential bioactive chemical components of FDY003 that pharmacologically regulate diverse LC-related drug targets and signaling cascades were identified. Further studies should focus on enhancing the therapeutic potential of herbal drugs as efficacious clinical agents for anticancer treatment, including investigations into their mechanisms of action in the modulation of a variety of cancerous and protumorigenic phenotypes and therapeutic sensitivities.

Data Availability
All data generated or analyzed during this study are included in this published article and its supplementary materials file.