Predict the anti-cancer mechanism of astragalus membranaceus based on network ph

Jia Wang , Zhu Yang, Feng-Xi Long, Jin-Lin Wu, Ting Yu,Dong-Xin Tang

1Guizhou University of Traditional Chinese Medicine, Guiyang,550002, China.

2First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang, 550001, China.

3Tumor Inheritance and Scientific and Technological Innovation Talents Base of Traditional Chinese Medicine in Guizhou Province, Guiyang, 550001, China.

Abstract Objective: To explore the anti-cancer mechanism of active ingredients of Astragalus membranaceus (AM) through network pharmacology.Methods: TCMSP, PubChem, STICTH and GeneCards databases were used to predict and screen the main active ingredients and anti-cancer targets of AM. Active ingredient-target-disease network was constructed by Cytoscape 3.7.0 software, and protein interaction network was constructed by STRING platform. KEGG signaling pathway and GO biological process of targets were analyzed by Bioconductor database.Results: Twenty-four active ingredients were screened from AM, which acted on 106 cancer targets such as PTGS, NCOA2, ADRB2, PRSS1,NOS2, NOS3, GABRA1. Through these targets, the anti-cancer effect of AM mainly acts on small cell lung cancer, colorectal cancer, thyroid cancer, breast cancer, non-small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, gastric cancer, endometrial cancer, enriched in chemical carcinogenesis, Platinum drug resistance, Epstein-Barr virus infection, TNF signaling pathway, Toll-like receptor signaling pathway,p53 signaling pathway, VEGF signaling pathway, NF-kappa B signaling pathway, and PI3K - Akt signaling pathway.Conclusion: This study found that the main anti-cancer compounds of AM are kaempferol, quercetin, 7-O-methylisomucronulatol, formononetin, isorhamnetin, Calycosin, 3,9-di-O-methylnissolin. The main targets include PTGS, PTGS1, NCOA2, ADRB2, PRSS1, NOS2, NOS3, GABRA1, F2.The mechanisms involved in anticancer could be summarized as following: blocking the chemical carcinogenesis, reversing the platinum drug resistance, anti - Epstein - Barrvirus infection, and inhibiting cell proliferation related signaling pathways, such as TNF signaling pathway, Tolllike receptor signaling pathway, p53 signaling pathway, VEGF signaling pathway, NF-kappa B signaling pathway, PI3K - AKT signaling pathway.

Key words: Astragalus membranaceus, Main components, Anticancer,Network pharmacology

Introduction

H uangqi (Astragalus Membranaceus) was first recorded in the Shen Nong's Herbal Classic, which is the earliest extant work about traditional herbs and written in the Three Kingdoms period (AD220～280). Zhang Yuansu, the founder of Yi-Shui Clan believes that AM has the functions of tonifying the lung Qi and purging pathogenic fire of the lung.The Ben Cao Gang Mu (AD 1578) has also recorded that AM is one of the most effective tonics.Modern pharmacological studies have found that the main components of AM are polysaccharides, saponins, flavonoids, etc., which have many effects such as inhibiting tumor cell proliferation, inducing tumor cell apoptosis,regulating immunity, inhibiting tumor cell metastasis, and assisting chemotherapy drugs to reverse drug resistance [1-4], but the molecular mechanism of its active ingredients and multiple targets is unclear.

Abbreviations

Astragalus membranaceus(AM), oral bioavailability (OB), drug-like properties (DL), Serum albumin (ALB), RAC-alpha serine/threonine-protein kinase (AKT1), caspase-3 (CASP3), Estrogen receptor (ESR1), Mitogen-activated protein kinase (MAPK8), Myc proto-oncogene protein (MYC), Transcription factor AP-1 (JUN), Tumor necrosis factor(TNF), Prostaglan-din G/H synthase 2 (PTGS2), Nitric-oxide synthase,endothelial (NOS3), Aryl hydrocarbon receptor(AHR), Heme oxygenase 1 (HMOX1), Prostaglandin G/H synthase 2 (PTGS2), prostaglandin G/H synthase 1 (PTGS1),nuclear receptor coactivator2 (NCOA2), beta-2 adrenergic receptor (ADRB2), trypsin-1 (PRSS1), nitric oxide synthase inducible (NOS2), Thrombin (F2), gamma-aminobutyric Acid receptor subunit alpha-1 (GABRA1), Nitric oxide synthase, endothelial (NOS3), Actin, cytoplasmic 1(ACTB), Dipeptidyl peptidase IV(DPP4), Retinoic acid receptor RXR-alpha(RXRA), Acetylcholinesterase(ACHE), Apoptosis regulator Bcl-2(BCL2), Apoptosis regulator BAX(BAXb),Androgen receptor(AR), Estrogen receptor beta(ESR2), Retinoic acid receptor RXR-beta(RXRB), Cytochrome P450 1A1(CYP1A1), Cytochrome P450 1A2(CYP1A2), Cytochrome P450 3A4(CYP3A4), Cytochrome P450 1B1(CYP1B1),Caspase-8(CASP8), Caspase-9(CASP9),Nuclear receptor coactivator 1(NCOA1), Signal transducer and activator of transcription 1-alpha/beta(STAT1), Intercellular adhesion molecule 1(ICAM1), Transcription factor p65(RELA), Peroxisome proliferator activated receptor gamma(PPARG), Mitogen-activated protein kinase 14(MAPK14), Glycogen synthase kinase-3 beta(GSK3B), Progesterone receptor(PGR), Glutathione S-transferase Mu 2(GSTM2), Glutathione S-transferase Mu 1(GSTM1), Peroxisome proliferator activated receptor gamma( PPARG), Muscarinic acetylcholine receptor M1(CHRM1).

Network pharmacology is a new discipline based on the theory of systems biology and network analysis of biological systems to design the drugs. It comprehensively observes the intervention and influence of drugs on the disease network,and reveals the mystery of multi-molecular drugs synergistically acting on disease. The holistic and systematic characteristics are consistent with the mechanism of multi-component synergistic effects of traditional Chinese medicine, which provides new ideas and approaches for the modernization of traditional Chinese medicine [5-7]. Therefore, this study screened the active constituents of AM based on network pharmacology to analyzed the AM anticancer mechanism.

1 Materials and methods

1.1 Collection of chemical components of AM

Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP, http://ibts.hkbu.edu.hk/LSP/tcmsp.php) is a comprehensive Chinese medicine platform that contains specific information on the chemical composition and targets of traditional Chinese medicine, including more Chinese medicines. In this study, the chemical constituents of AM were collected by searching the TCMSP database, and the molecular structure was searched in Pubchem (http://pubchem.nlm.nih.gov/). The active components of AM in the databases of China Knowledge Network and Pubmed were also reviewed for any missing information.

1.2 Screening of active ingredients

As AM is mostly oral preparation, which needs to be absorbed, distributed, metabolized and excreted to reach the target organs, the pharmacokinetic parameters need to be satisfied. In order to screen potential active compounds of AM, oral bioavailability (OB) and drug-like properties(DL) was constructed to predict the ADME. OB refers to the relative amount and rate of drugs absorbed by the systemic blood circulation after oral administration [8]. The OB value is calculated by the computer prediction model OBioavail 1.1 [9]. OB ≥ 30% was used as a screening condition. DL refers to the similarity of a compound to a known drug, and DL ≥ 0.18(the average of the overall similarity) is considered to be similar to the drug in the Drugbank database. As some active ingredients of AM were not included in the conduction of DL ≥ 0.18. In order to screen out more active ingredients, DL ≥ 0.05 was used as the screening standard for candidate compounds.

1.3 Prediction of active ingredient targets

TCMSP database was searched to obtain the targets through compound names, STICTH database was also searched based on simplified molecular input line entry specification (SMILES) collected from Pubchem. Further,these targets were converted into Uniprot (http://www.uniprot.org/uploadlists/) code. Then the drug target was converted to standard genetic abbreviation via the UniProt database for further use.

1.4 Collection of tumor-related disease targets

Collect genes related to cancer in the Human Genome Annotation Database (GeneCards), using “Cancer” as a key word for data mining, then map the obtained oncogenes and drug targets to select common targets as the cancer treatment targets of AM.

1.5 Drug-ingredient-target-disease network construction and analysis

To elucidate the anticancer mechanism of astragalus, this study constructed a visual network map of drug-component-target-disease interactions. The AM-compound-target-tumor network map was conducted with Cytoscape 3.7.0. In the network,AM, compounds, targets, are represented by nodes, and the interaction between the two nodes is represented by an edge. The importance of each node in the network is assessed by topology parameter degree and betweenness centrality. The degree of a node indicates the number of edges connected to the node. The greater the degree, the more important the node is in the network, and the more biological functions involved. The betweenness centrality of a node refers to the proportion of the number of nodes passing through all the shortest paths in the network, reflecting the importance of the node transmitting information through the network. The larger of the betweenness centrality, the more important the node is in the network.

1.6 Target Protein Interaction (PPI) Network Construction

To analyze the interaction of the target protein of AM, the target protein interaction constructed using the STRING platform. The species is set to “Homo sapiens”and the lowest interaction threshold is set to medium confidence of 0.4, with the remaining parameters remaining at their default settings. The thickness of the PPI network connection indicates the strength of the interaction, and the degree of the target indicates the number of interacting targets.

1.7 KEGG Signal Pathway and GO Biological Process Analysis

The biological process of the drug target was studied by enriching the anti-cancer target of astragalus and enrichment analysis of the KEGG signaling pathway to study the major signaling pathways of drug targets through the Bioconductor database.

2 Results

2.1 Astragalus active ingredient screening

87 components were screened in the TCMSP database,but only 20 active constituents satisfied the screening conditions of OB ≥ 30% and DL ≥ 0.18. The main components of AM, such as flavonoids [10], saponins [11], polysaccharides [12], and amino acids [13] were not fully included in the study. Therefore, the screening conditions are adjusted to OB ≥ 30%, DL ≥ 0.05 to allow the main components included. 32 active ingredients of AM were got, and the molecular number, English name, OB value and DL value of each component were downloaded, as shown in Table 1.

Table 1 32 active ingredients of AM

MOL000392 formononetin 69.67 0.21 MOL000398 isoflavanone 109.99 0.30 MOL000401 astragalosideI 46.79 0.11 MOL000403 astragalosideII 46.06 0.13 MOL000405 astragaloside Ⅲ 31.83 0.10 MOL000414 Caffeate 54.97 0.05 MOL000417 Calycosin 47.75 0.24 MOL000422 kaempferol 41.88 0.24 MOL000432 linolenic acid 45.01 0.15 MOL000433 FA 68.96 0.71 MOL000434 acetylastragaloside I 43.54 0.09 MOL000435 acetylastragaloside I_qt 30.75 0.17 MOL000436(Z)-1-(2,4-d i h y d r o x y p h enyl)-3-(4-hydroxyphenyl)prop-2-en-1-one 87.51 0.15 MOL000438 (3R)-3-(2-hydroxy-3,4-dimethoxyphenyl)chroman-7-ol 67.67 0.26 MOL000439 isomucronulatol-7,2'-di-O-glucosiole 49.28 0.62 MOL000442 1,7-Dihydroxy-3,9-dimethoxy pterocarpene 39.05 0.48 MOL000098 quercetin 46.43 0.28

2.2 Prediction of active ingredient targets

A total of 556 targets were predicted with 496 from TCMSP database, and 60 from STICTH database. All these proteins were converted into the corresponding gene name and Uniprot code in the UniProt database.

2.3 Collection of tumor-related disease targets

22,560 cancer-related genes were obtained in the Gene-Cards database, and then the Uniprot coding of each gene was searched for, and the tumor genes without Uniprot coding were removed. Finally, a total of 17,714 tumor genes were obtained. The genes corresponding to the target of the active ingredient of AM were mapped to the tumor-related gene, and after removing the duplicated gene,a total of 106 genes were obtained, which were identified as anti-cancer target genes of AM.

2.4 Construction and analysis of AM-ingredient-target-tumor network

We found 8 of the 32 active constituents in AM without cancer-related targets. These compounds were 5'-hydroxyiso-muronulatol-2', 5'-di-O-glucoside, astragalosideI,astragalosideII, astragalosideIII acetylastragaloside I,acetylastragalosideI_qt, (3R)-3-(2-hydroxy-3,4-dimethoxyphenyl)chroman-7-ol, and isomucronulatol-7,2'-di-O-glucosiole. Their functions in cancer therapy need more researchs. The other 24 tumor-associated compounds were used as main anti-cancer components of AM. With the 106 cancer target proteins AM-components-cancer network was established. In these network, there are 132 nodes and 910 edges which representing the interaction of active ingredients with cancer targets, as shown in Figure 1. The top ranked compounds are kaempferol (degree =55), quercetin (degree = 55), 7-O-methylisomucronulatol(degree = 36), formononetin (degree = 36), isorhamnetin(degree = 34), Calycosin (degree = 25), 3,9-di-O-methylnissolin (Degree = 22) and so on (Table 2). The top targets include Prostaglandin G/H synthase 2 (PTGS2) (degree =21) and prostaglandin G/H synthase 1 (PTGS1) (degree= 19), nuclear receptor coactivator2 (NCOA2) (degree =14), beta-2 adrenergic receptor (ADRB2) (degree = 11),trypsin-1 (PRSS1) (degree = 10), nitric oxide synthase inducible (NOS2) (degree = 10), Nitric oxide synthase,endothelial (NOS3) (degree = 10), gamma-aminobutyric Acid receptor subunit alpha-1 (GABRA1) (degree = 10),Thrombin (F2) (degree = 9)etc., as shown in Table 3.

Figure 1 AM-component-target-cancer network mapNote: Purple nodes represent the active constituents of AM. Green nodes represent cancer targets.

Table 2 Main components of anti-cancer of Astragalus

Table 3 Main targets of anti-cancer in Astragalus

2.5 Target Protein-Protein Interaction (PPI) Network Construction

In order to further analyze the functions of predicted targets, protein-protein interaction were conducted with 106 predicted targets, after mapping, we found that two target proteins were not involved in protein-protein interaction(Figure 2) . In the PPI network, Serum albumin (ALB)(degree = 58), RAC-alpha serine/threonine-protein kinase(AKT1) (degree = 55), caspase-3 (CASP3) (degree = 40),Estrogen receptor (ESR1) (degree = 39), Mitogen-activated protein kinase (MAPK8) (degree = 39), Myc proto-oncogene protein (MYC) (degree = 39), Transcription factor AP-1 (JUN) (degree = 38), Tumor necrosis factor (TNF)(degree = 38), Prostaglan-din G/H synthase 2 (PTGS2)(degree = 37), Nitric-oxide synthase,endothelial (NOS3)(degree = 35), Aryl hydrocarbon receptor (AHR) (degree =34), Heme oxygenase 1 (HMOX1) (degree = 33), interacted strongly with other proteins and played key roles in this network (Figure 3).

Figure 2 Anti-cancer target protein PPI network

Figure 3 The core targets in PPI network

2.6 KEGG Signal Pathway and GO Biological Process Analysis

A total of 152 GO biological processes were obtained from the GO bioprocess enrichment analysis of 106 predicted targets. The top 20 biological processes are shown in Figure 4. According to the false discovery rate (FDR) ＜0.05, biological processes closely related to tumors were screened, which included the following functions, such as nuclear receptor activity, transcription factor activity,direct ligand regulated sequence-specific DNA binding,steroid hormone receptor activity, adrenergic receptor activity, oxidoreductase activity, acting on paired donors,with incorporation or reduction of molecular oxygen, RNA polymerase II transcription factor binding, transcription coactivator activity.

Figure 4 GO analysis of Anti-cancer potential target of AM

Figure 6 Mechanism of anti-cancer effect of AM

3 Discussion

It has been well reported that AM alone or in combination with chemotherapy drugs has therapeutic effects on malignant tumors such as liver cancer, gastric cancer and lung cancer [14]. In this study, we found that the main anti-cancer compounds in AM are kaempferol (degree =55), quercetin (degree = 55), 7-O-methylisomucronulatol(degree = 36), formononetin (degree = 36), isorhamnetin(degree = 34), Calycosin (degree = 25), 3,9-di-O-methylnissolin (Degree = 22). The anti-cancer effects of some ingredients in AM have been reported. Such as effects of quercetin, isorhamnetin, formononetin, calycosin and other flavonoids on regulating immune system, anti-oxidation and anti-apoptosis has been reported [15,16]. Quercetin can affect the proliferation, metastasis, apoptosis and metabolism of tumor cells through various signaling pathways in colorectal cancer, breast cancer and gastric cancer [17].Formononetin blocks cell cycle, inhibits tumor invasion,induces tumor cell apoptosis, and reverses tumor resistance [18]. Isorhamnetin exerts anti-breast cancer effects by inhibiting cell proliferation and promoting apoptosis[19]. Calycosin also inhibit the proliferation and migration of breast cancer cell [20]. These results suggested the predicted active ingredients in AM play great roles on cancer treatment.

In this study, 106 targets of 24 main ingredients were predicted. The main targets include PTGS, PTGS1,NCOA2, ADRB2, PRSS1, NOS2, NOS3, GABRA1, F2.

Through these targets, the anti-cancer effect of AM mainly acts on small cell lung cancer, colorectal cancer, thyroid cancer, breast cancer, non-small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, gastric cancer, endometrial cancer. AM exerts anti-small cell lung cancer and prevents recurrence and metastasis by acting on multiple targets such as AKT1, BCL2 and BAX, and regulating P53 and PI3K - Akt signaling pathways. By acting on targets such as BCL2, BAX as well as the PI3K - Akt signaling pathway, thereby AM may exert an anti-colorectal cancer effect. Studies have shown that Astragalus saponins regulates the development of colon cancer by regulating the expression of calmodulin-mediated glucose regulatory proteins [21], which can be complemented by modern medical research. AM also exerts anti-thyroid cancer effects by acting on RXRA, PPARG, RXRB, BAX, RXRG, and MYC targets. Studies have shown that Astragalus injection may have a certain inhibitory effect on the formation of lymphatic vessels in differentiated thyroid carcinoma and the metastasis of tumor cells through lymphatic vessels,but the specific mechanism is unknown [22]. This deserves further research. AM maybe exert anti-breast cancer effects by acting on targets such as AKT1, JUN, BAX, and MYC. Studies have shown that astragalus flavonoids can inhibit the growth and metastasis of breast cancer, and its mechanisms include promoting programmed cell death and regulating the expression of metastasis-associated genes[23]. It acts on non-small cell lung cancer by acting on targets such as RXRA, AKT1, RXRB, and BAX. Studies have shown that astragalus isoflavones inhibit proliferation of human non-small cell lung cancer by inducing cell cycle arrest and apoptosis [24]. It acts on anti-hepatocellular carcinoma by acting on targets such as AKT1, BAX, GSTP1,GSTM1, and MYC. Studies have shown that astragaloside IV inhibits the migration and survival of hepatocellular carcinoma cells by inhibiting RNA-ATB [25]. Studies have shown that Astragalus polysaccharide is the main active ingredient of Astragalus, which can improve the sensitivity of non-small cell lung cancer and hepatocellular carcinoma cells to chemotherapy drugs [26]. AM exerts anti-pancreatic cancer effects by acting on targets such as AKT1, ASP9, BAX, and STAT1. By acting on targets such as AKT1, BCL2, BAX, and MYC, it exerts anti-gastric effects. Zhu et al. [27] showed that isorhamnetin inhibited the proliferation of human gastric cancer cell line MKN28 and promoted its apoptosis through apoptosis-related proteins such as p53. Astragaloside IV has a relatively significant inhibitory effect on the proliferation of human gastric cancer cell line MKN-74 [28]. By acting on targets such as AKT1 and BAX, Astragalus exerts anti-endometrial cancer effects. Studies have shown that Astragalus polysaccharide can promote the expression of E-cadherin protein and gene, inhibit the expression of β-catenin protein and gene,and exert anti-tumor effect by regulating Wnt gene transduction pathway [29].

In this study, the anti-cancer target genes of Astragalus were enriched in chemical carcinogenesis, Platinum drug resistance, Epstein-Barr virus infection, TNF signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, VEGF signaling pathway, NF-kappa B signaling pathway, and PI3K-Akt signaling pathway. This suggests that the main mechanism of anti-cancer effect of AM is to interfere with signal transduction in tumor cells(TNF signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, VEGF signaling pathway,NF-KB signaling pathway, PI3K-AKT signaling pathway),blocking viral carcinogenesis and reverse transformation therapy resistance (Figure 6). Peripheral T-cell lymphoma is associated with Epstein-Barr virus infection, and the anti-cancer target gene of AM can regulate the Epstein-Barr virus infection signaling pathway, which provides ideas for the prevention of lymphoma. The Toll-like receptor signaling pathway is involved in the immune response and plays an important role in the development of colorectal cancer[30]. The active component of AM regulates the Toll-like receptor signaling pathway, which in turn exerts anticancer effects, providing an idea for the treatment of tumors.

In summary, this study used a network pharmacology approach to study the complex network relationship between Astragalus - ingredient - target - tumor. We found that the main anti-cancer compounds are kaempferol, quercetin,7-O-methylisomucronulatol, formononetin, isorhamnetin,Calycosin, 3,9-di-O-methylnissolin. The main targets include PTGS, PTGS1, NCOA2, ADRB2, PRSS1, NOS2,NOS3, GABRA1, F2. The anti-cancer effect of AM mainly acts on lung cancer, colorectal cancer, thyroid cancer,breast cancer, hepatocellular carcinoma, pancreatic cancer,gastric cancer, endometrial cancer, etc. The mechanisms involved in anticancer could be summarized as following:blocking the chemical carcinogenesis, reversing the platinum drug resistance, anti - Epstein - Barrvirus infection,and inhibiting cell proliferation related signaling pathways, such as TNF signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, VEGF signaling pathway, NF-kappa B signaling pathway, PI3K - AKT signaling pathway. However, these predicted results need further verification.