Clinical applications and mechanisms of action of Danggui Beimu Kushen Wan on the targets and pathways implicated in prostate cancer: literature reviews and computational analyses
Creators: name: LI, Hong
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 420803
subject: Traditional Chinese medicine and treatments

description: Prostate cancer (PCa) is a cancer that occurs in the prostate with high morbidity and mortality. Routine treatments for PCa include active surveillance and observation, radiotherapy, surgery, androgen deprivation therapy, chemotherapy and immunotherapy. However, they are associated with many adverse events, such as fatigue, nausea and vomiting. Increasingly, PCa patients are seeking complementary and alternative medicine including Chinese herbal medicine for management.

Danggui Beimu Kushen Wan (DBKW), containing Angelicae Sinensis Radix (ASR), Fritillariae Thunbergii Bulbus (FTB) and Sophorae Flavescentis Radix (SFR), is a classic formula developed 1,800 years ago for patients with difficulty in urination. It has been widely used for urinary disorders including PCa in modern times. However, its applications and mechanisms of action have not been systematically investigated.

This project aimed to:
1. investigate the traditional evidence on the historical development of DBKW and its standard version and clinical applications;
2. assess the effects and safety of DBKW by systematically reviewing modern clinical studies;
3. explore the phytochemical and pharmacodynamics of DBKW's ingredients by reviewing modern experimental studies; and
4. examine the mechanisms of action of DBKW for PCa using computational analyses.

Review of classical literature (Phase 1)
The Encyclopaedia of Traditional Chinese Medicine was searched to identify the ingredients, dosages, aetiologies, pathogeneses, actions and indications related to DBKW documented in ancient texts. A descriptive summary is provided on their characteristics. A total of 41 texts in 36 classical books were included. Two aetiologies and 10 pathogeneses were investigated. All the identified formulas contained ASR, FTB and SFR in the ratio 1:1:1. The treatment dosage was 3 to 10 pills each time. The primary indication in Chinese medicine for DBKW was difficulty in urination with heat stagnation (Publications 1 and 2, Appendices A and B).

Review of DBKW for chronic prostatitis (Phase 2)
A search was conducted in 21 electronic databases to identify potential clinical and experimental studies, and 290 studies were identified. However, only one single case report related to PCa. Evidence showed that males who suffered from chronic prostatitis (CP) had a 30% higher probability of developing PCa, whereas there were no direct links between benign prostatic hyperplasia and PCa. Thus, I evaluated the clinical effects and safety of DBKW and its mechanisms of action for CP, instead of PCa for this analysis. Six clinical studies, including four randomised controlled trials (RCTs) and two case series, and eight experimental studies were included. Clinical studies indicated that DBKW used alone or as an adjunct therapy significantly reduced the symptom scores and decreased the pH of expressed prostatic secretions when compared to antibiotics or alpha-blockers. Experimental studies revealed that DBKW may have effects of anti-inflammation, anti-bacteria, antioxidation, sex hormone regulation and immunoregulation (Publication 3, Appendix C).

Review of anti-cancer activities of DBKW (Phase 2)
A search was performed in 21 electronic databases and 413 experimental studies were identified. Characteristics of the included studies and possible mechanisms of action were descriptively summarised. Five included pharmacological studies revealed that DBKW could inhibit the growth of tumours, regulate cell proliferation, induce tumour cell apoptosis, suppress the invasion and metastasis of cancer, enhance treatment effects and relieve the toxicity of chemotherapy.

Identification of compounds from DBKW (Phase 3)
Potentially active chemical constituents of DBKW were obtained from modern literature. Eleven databases were searched to identify potential studies. As a result, 42 studies were included for ASR, 65 for FTB, and 33 for SFR. A total of 818 chemical constituents were identified from three ingredients of DBKW, including 408 compounds from ASR, 133 from FTB, and 277 from SFR. There were 49 compounds found in two or three herbs from DBKW, including 7 essential oils, 5 organic acids, 6 nucleosides, 3 nucleobases, 12 amino acids, 1 carbohydrate and 15 elements (Publication 2, Appendix B).

Computational analysis of targets for PCa (Phase 3)
Potential targets of DBKW were identified: 14 from experimental studies through the search in 21 bibliographic databases, 21 from approved drugs in the DrugBank database and 6,247 from the Open Targets database. After cross-comparison, 28 common targets were identified, 26 of which had protein-protein interactions between each other. Thus, these 26 targets were selected for subsequent computational analyses including Go Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment to select targets and predict pathways for PCa using the ClueGo plugin and Enrichr database. These analyses predicted 67 biological processes and 4 molecular functions. The most significant biological process was regulation of lymphocyte apoptosis. A total of 93 biological pathways were clustered. Four of the top 10 pathways were cancer-related pathways. TP53 and CASP3 were the two most significant targets.

Molecular docking between DBKW compounds and PCa targets (Phase 4)
A total of 621 compounds from DBKW's ingredients were screened via molecular docking using the automated docking software PyRx and AutoDock Vina against 21 target proteins for PCa, predicting 13,041 docking results with an average binding score of -6.58 kcal/mol ranging from -2.4 to -13.3 kcal/mol. The visualisation of 3D docking positions and 2D ligand-receptor interaction diagrams were analysed using Discovery Studio 2019. Boxplots demonstrated that compared to the compounds from ASR and FTB, most compounds from SFR had lower binding scores. The top five compounds with high binding affinity were selected to generate a network using Cytoscape v3.7.2. The network indicated that compounds from all three herbs had high binding affinity against the 21 targets and may have potential biological activities with the targets, although most of the high-binding-affinity compounds were from SFR.

Conclusions
DBKW is a classic herbal formula which has been widely used for difficulty in urination in modern times involving PCa. Classical literature has provided fundamental evidence for symptomatic management. Modern clinical and experimental studies supported that DBKW may have anti-cancer, anti-inflammatory, anti-proliferation and anti-bacterial effects. Molecular docking indicated that chemical compounds from DBKW may interact with the 21 targets for PCa. DBKW contains multi-targeting agents that could act on more than one pathway of PCa at the same time. Further studies could focus on validating the in silico results via in vitro or in vivo studies.