Shan-Qi Guo,Yao Zhang,Xiao-Jiang Li＊,Ying-Jie Jia＊

1Department of Oncology,First Teaching Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin,300381,China.

Objective: Prostate cancer (PCa) is the second most common male malignancy tumor in the world, and the occurrence rate and the mortality of PCa keeps increasing nowadays, exploration of accurate biomarker and predict clinical outcome of PCa is of great significance.Traditional Chinese medicine Huangqi(Astragalus membranaceus,AM) has been widely used for PCa treatment in China, while the pharmacological mechanisms are still unclear.The current paper intend to perform a network pharmacology analysis to reveal the mechanism of the effects of AM in PCa. Methods:Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) was used to obtain chemical constituents of AM.PCa-related target genes were collected via OMIM and GeneCards databases.PCa-AM common target protein interaction network was established by the STRING database.Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to further explore the PCa mechanism and therapeutic effect of AM.The network diagrams of the active component-action target and protein-protein interaction(PPI)networks were constructed using Cytoscape software.Results:A total of 20 active ingredients contained and 462 putative identified target genes were screened from AM,of which 46 overlapped with the targets of PCa and were considered potential therapeutic targets.The analysis of the network results showed that the AM activity components quercetin, kaempferol, isorhamnetin,3,9-di-O-methylnissolin and 7-O-methylisomucronulatol have a good binding activity with top ten screened targets,such as MYC, AKT1, CCND1, MAPK1,CASP3, EGFR, ESR1, VEGFA, ERBB2 and IL-6.GO and KEGG analyses revealed that these targets were associated with proteoglycans in cancer, virus infection, microRNAs in cancer, PI3K-Akt signaling pathway, and other biological process. Conclusion: This study demonstrated the multicomponent, multitarget, and multichannel characteristics of AM, which provided a novel approach for further research of the mechanism of AM in the treatment of PCa.

Key Words: Astragalus membranaceus, Prostate cancer, Bioinformatics, Traditional Chinese Medicine,Protein-protein interaction

Background

Prostate cancer (PCa) is a worldly common cancer in elderly men, the occurrence and general mortality of PCa keeps increasing.The mechanisms of PCa progression have been explored, and key progresses have been made in the treatment of PCa[1].Nowadays,complex Chinese medicines are increasingly concerned for the treatment of cancer including PCa.Considering low toxicity, non-apparent side effects, and obvious curative effects,Chinese medicines have been accepted by doctors, experts, and patients, to inhibit tumor growth and metastasis, prolong patients’ life span, as well as improve patients’life quality[2].

Huangqi (Astragalus membranaceus,AM) as a fundamentally representative herb of traditional Chinese medicine (TCM), contains numerous components such as polysaccharides, saponins,flavonoids,amino acids,trace elements et al.,and has a wide range of biological activities.AM is also well known as one of the important “Qi tonifying”adaptogenic herbs from the Chinese materia medica.“Qi tonifying” can promote blood flow to detoxicate and have an important effect in treating diseases.Meanwhile, the increase resistance to the immunosuppressive effects of antitumor therapies as well as ameliorate the toxic side effects of anticancer agents were recognized gradually [3].While it’s a pity that the key targets of AM for treating PCa were rarely known.Besides, few reports interpreted the active components of AM, the ingredient-targets and disease-targets of AM as a treatment of PCa were also rarely mentioned[4].

Our current study intended to seek critical targets and assessthe molecular mechanisms of AM for the treatment of PCa with network pharmacology.In addition, protein-protein interaction (PPI), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment of common targets were also analyzed.Our flowchart is shown in Figure 1.

Figure 1 Framework based on an integration strategy of network pharmacology.

Materials and methods

Active compound screening

Traditional Chinese Medicine System Pharmacology Database (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php )was used to collect the chemical constituents of AM,which could captures the relationships between herbal ingredients, targets, and diseases.Pharmacokinetic parameters such as oral bioavailability (OB), half-life(HL), drug-likeness (DL), Caco-2 permeability(Caco-2), and blood-brain barrier (BBB) can be provided.Ingredients meeting the demands of both OB≥ 30% and DL ≥ 0.18 were selected to find the effective components of AM.

Seeking out disease-related targets

Information on prostate cancer-associated target genes were gathered from databases of OMIM and GeneCards.OMIM (Online Mendelian Inheritance in Man)(https://omim.org/),an Online Catalog of Human Genes and Genetic Disorders.The GeneCards(https://www.genecards.org/) as a comprehensive database of functions including genomics, proteomics as well as transcriptomics.Therefore, we searched in these two databases with the keywords “Astragalus membranaceus” and “prostate cancer” to screen the targets related to PCa.Wayne diagram was used to show the important common targets for AM treatment of PCa.

Searching for common targets of AM and PCa

PPI network was derived based on STRING database(https://string-db.org/), which covered almost all functional interactions between the expressed proteins.Species are set as “Homosapiens” and the target interaction information was obtained according to the results of analysis.The interaction network drawing and analysis were achieved by Cytoscape (version 3.6.1;https://www.cytoscape.org/)software.

GO and KEGG pathway enrichment analysis

The biological process (BP), molecular function (MF),cell component (CC), and KEGG database(https://www.kegg.jp/) pathway enrichment analysis were using the DAVID system(https://david.ncifcrf.gov/ ).In the current research,GO functional annotation and KEGG pathway enrichment analyses were performed using R package,and thePvalue less than 0.05 was employed for further analysis.

Results

Identify active compounds of AM and PCa

The TCMSP databases was used to search the active compound targets of AM for each chemical component.Eighty-seven compounds were collected from the TCMSP, with the values of OB ≥30% and DL ≥0.18 properties, applied to screen the active compounds from AM.Finally, 16 candidate ingredients were selected out from AM compounds(Table1).In total,13 compounds and 91 targets were identified.To further understand the interaction relationship between the compounds and corresponding targets, we constructed a compound-target network, as shown in Figure 2.The compounds with more targets are quercetin,kaempferol, isorhamnetin and formononetin, which correspond to 43,13,6,and 6 targets,respectively.The result suggested that these four components probably served as significant therapeutic compounds in PCa.

Figure 2 Compound-target network of potential targets in Astragalus membranaceus.

Table 1 Basic information of Astragalus membranaceus compound ingredients

Common and key targets analysis by PPI network

With the keywords “Astragalus membranaceus” and“prostate cancer”, The targets related to PCa were screened via OMIM and GeneCards databases.The potential target gene in AM was mapped to PCa target gene and a Venn diagram was drawn(Figure 3).A total of forty-six potential genes associated with PCa were uploaded to the STRING database for analysis.The network of PPI (Figure 4) was established through the STRING database.The top ten targets MYC, AKT1,CCND1, MAPK1, CASP3, EGFR, ESR1, VEGFA,ERBB2 and IL6 have higher degree in this process,which explained their significance in the network.

Figure 4 (A) Common target protein-protein interaction network between prostate cancer and Astragalus membranaceus.(B) Information on potential targets and the topological attributes.

GO enrichment analysis

We imported the selected potential 46 target genes into the DAVID system for GO analysis.The results revealed that the functions of these potential targets are related to many BPs, MFs, and CCs, which may be important for the recurrence and development of PCa.GO terms were enrichment,and the top 20 entries were selected based onP＜0.05.The targets are closely related to ubiquitin-like protein ligase binding,DNA-binding transcription activator activity, RNA polymerase II-specific, phosphatase binding, cytokine receptor binding, ubiquitin protein ligase binding receptor ligand activity, and many targets were ranked highly as potential related genes(Figure 5).

KEGG pathway enrichment analysis

To further reveal the potential mechanism of the AM on the treatment effects of PCa, we conducted KEGG pathway enrichment analysis on 46 targets and finally screened out top 20 pathways based on the threshold ofP＜ 0.01.Numerous pathways for potential target genes were identified, such as Proteoglycans in cancer,Human cytomegalovirus infection, Human papillomavirus infection, Kaposi sarcoma-associated herpesvirus infection,MicroRNAs in cancer,PI3K-Akt signaling pathway,Prostate cancer and so on(Figure 6).

Figure 5 Enriched Gene Ontology terms for biological process (BP) of potential targets of the main active ingredients of Astragalus membranaceus.

Figure 6 Kyoto Encyclopedia of Genes and Genomes pathway analysis of putative target genes of Astragalus membranaceus.

Discussion

PCa is a major disease that affects men's health, and is the most common noncutaneous cancer in men worldwide.During the past decade, treatment strategies for patients with PCa mainly involving surgery, chemotherapy, radiotherapy, Endocrine therapy, immunotherapy and palliative treatment.Although knowing the indolent,non-invasive nature of most types of PCa, quite a number of patients died of PCa.Therefore, proper and reasonable therapeutic strategies were urgently needed[5–7].

TCM, which is the traditional medicine resources with unique advantages in China, treats diseases in a holistic model.TCM herbs and recipes were usually used to treat diseases according to syndrome differentiation.Nowadays, TCM for the treatment of cancers is getting more and more attention in the world.TCM herbs and recipes are multi-component,multi-target agents that making its pharmacodynamic component and molecular mechanism.Nevertheless,the mechanism of TCM still unclear [8].Thus, it is necessary to develop a new way to explain molecular mechanisms and pharmacological effects of TCM.Studies have indicated that TCM could inhibit tumor growth and metastasis, prolong patients’life span, and improve patients’ life quality.Astragalus is classified as an herb that tonify the qi.Its properties are sweet and slightly warm.Astragalusis typically prescribed as a dried root,powdered,or in a decoction.Classically,it is prescribed in combination with other Chinese medicinal herbs, depending on the desired therapeutic effect and the specific TCM diagnosis[9,10].

Several studies have verified that AM had the characteristics of suppress tumor proliferation.Cao et al.found that Astragalus injection apparently has attenuation and synergistic efficacy to platinum-based chemotherapy NSCLC patients [11].Through in vitro experiments, Zhang and colleagues discovered that APS treatment could reduce cell proliferation and morphological changes in bone marrow-derived MSCs that were co-cultured with A549 lung cancer cells[12].Zhou et al aimed to investigate the biological effects of extract from AM on breast cancer cell and its mechanism.They concluded that AM could inhibited cell proliferation and induced cell apoptosis effect of AM extract via PI3K/AKT/mTOR pathway [13],indicating the antitumor effect and mechanism of AM.Zhou and colleagues verified that APS may modulate immunity of host organism through activation of TLR4-mediated MyD88-dependent signaling pathway,enriching the mechanism of action of AM[14].Li et al suggested that APS can activate macrophages to release NO and TNF-α, which directly blocks cancer cell growth.The anti-breast cancer effect of APS and the in vivo mechanism will be further elucidated with a review to provide a therapeutic strategy for breast cancer [15].Wang et al showed that Astragalus extract enhances apoptosis in gastric cancer cells in vitro and in vivo, which is associated with decreased activation of IL-6/Stat3 signals, providing the molecular mechanism of AM [16].As for PCa, few studies focused on AM.While our previous findings suggested that Astragalus Polysaccharides which is one of the main composition of AM, could inhibits tumorigenesis and lipid metabolism through miR-138-5p/SIRT1/SREBP1 pathways in PCa [17].From the above studies,AM has anti-tumor properties,and there are few studies aimed at PCa, which is the significance meaning of the current study.

Our results showed that 20 active ingredients contained and 462 putative identified target genes were screened from AM, of which 46 overlapped with the targets of PCa and were considered potential therapeutic targets.The top ten targets MYC, AKT1,CCND1, MAPK1, CASP3, EGFR, ESR1, VEGFA,ERBB2 and IL6 have higher degree in this process.Adeline Berger and colleagues suggested that N-Myc and C-Myc share common functions but N-Myc may also regulate molecular programs that are not driven by C-Myc in PCa cells; this also help us predict the evolution of PCa and better guide the choice of future therapeutic strategies [18].Gao et al showed that metastatic human PC3 and DU145 PCa cells invade through Akt1-deficient human lung endothelial cell(HLEC)monolayer with higher efficiency compared to control HLEC [19], indicating that endothelial-specific loss of Akt1 promotes PCa metastasis in vivo via β-catenin pathway.Latest research revealed that AKT1/PIK3CA activating mutations are relatively common and depicted a distinct mCRPC molecular subtype with low-level AR copy gain.Clonal prevalence and evidence of mutant allele selection suggest PI3K pathway dependency in selected patients[20].Another study revealed miR-487a-3p could be suppressed PCa cell growth, migration, invasion by directly targeting cyclin D1 (CCND1) [21].Besides MYC, AKT1 and CCND1, other targets genes of AM and PCa also have been reported recent years,clarifying that AM might focus on the key targets of PCa and have effect on PCa in vivo and in vitro[22].

KEGG results showed primarily involving proteoglycans in cancer, Human cytomegalovirus infection, Human papillomavirus infection, Kaposi sarcoma-associated herpesvirus infection, microRNAs in cancer,PI3K-Akt signaling pathway,Prostate cancer etc.Moreover, GO enrichment analysis concluded that ubiquitin-like protein ligase binding, DNA-binding transcription activator activity, RNA polymerase II-specific, phosphatase binding, cytokine receptor binding, ubiquitin protein ligase binding receptor ligand activity,and many targets were ranked highly as potential related genes.These results further verified that the activation of proteoglycans, the effect of virus infection, and the effect of microRNAs and PI3K-Akt signaling pathway activation act as the critical mechanism of astragalus in the treatment of PCa.

In summary, the present study verifies the multicomponent, multitarget, and multichannel characteristics of AM, and provides a novel approach for further research of the mechanism of AM in the treatment of PCa.