Network Pharmacology Combined with Proteomics Reveals That 3‑Acetyl-11-keto-β-boswellic Acid Inhibits the Progression of Prostate Cancer by Regulating the IL-17 Signaling Pathway

Abstract

Background: 3-acetyl-11-keto-beta-boswellic acid (AKBA) is a monomer extracted from the traditional Chinese herbs of Boswellia that has antitumor effects. However, the therapeutic effects and mechanisms of AKBA in prostate cancer (PCa) are unclear.

Objective: To predict the target of AKBA treatment of PCa using network pharmacology methods and validate the predicted targets through CCK-8, flow cytometry, cell scratch test, Transwell chamber assay, and proteomics.

Methods: The protein-protein interaction (PPI) network was established. Further analysis was performed by gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes for biological functionality and pathway enrichment. CCK-8 assay, flow cytometry, wound healing assay, and Transwell chamber assay were used to detect cell proliferation, apoptosis, invasion, and metastasis, respectively. Transcriptomics was used to detect the effects of AKBA on protein levels in PCa cells.

Results: 120 potential targets for the AKBA treatment of PCa were obtained. GO enrichment analysis revealed that the biological processes of AKBA treatment of PCa include the steroid metabolic process, drug response, and fatty acid metabolic process. The results of KEGG enrichment revealed that the IL-17 signaling pathway is the key pathway for the AKBA treatment of PCa. In addition, experimental results demonstrate that AKBA inhibits the proliferation of PCa cells, induces cell apoptosis, and suppresses cell invasion and metastasis. Proteomics identified 119 differentially expressed proteins, which were primarily enriched in pathways closely related to the phogosome, pathways in cancer, IL-17 signaling pathway, spliceosome, PPAR signaling pathway, and HIF-1 signaling pathway.

Conclusion: Through the methodologies of network pharmacology and transcriptomics, AKBA may exert its therapeutic effects on PCa by modulating the expression of the IL-17 signaling pathway.

1

Mechanistic study of the network pharmacological method to analyze AKBA for PCa.

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(A) Chemical structure of AKBA. (B) Venn diagram of AKBA and PCa intersection target sites.

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(A) PPI network of AKBA acting on PCa. (B) AKBA acts on 14 core targets of PCa.

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(A) GO enrichment function analysis histogram and bubble diagram. (B) Diagram of the KEGG IL-17 signaling pathway.

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AKBA inhibited proliferation and induced apoptosis in PCa Cells. (A) Cell viability of 22RV1 cells after treatment with different concentrations of AKBA for 48 h; (B) cell viability of PC3 cells after treatment with different concentrations of AKBA for 48 h; (C) morphological changes of 22RV1 cells under light microscopy after treatment with AKBA for 48 h; (D) morphological changes of PC3 cells under light microscopy after treatment with AKBA for 48 h; (E) apoptosis rate and quantitative analysis of 22RV1 cells after treatment with AKBA for 48 h, as detected by flow cytometry.

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AKBA inhibits the invasive and metastatic abilities of 22RV1 and PC3 cells. (A) Scratch test to detect the effect of 30 μM AKBA on cell migration ability of 22RV1 and PC3 cells at 48 h. (B) Wound healing rate of 22RV1 and PC3 cells treated with AKBA for 48 h. (C) Transwell assay to detect the effect of 30 μM AKBA on the invasion ability of 22RV1 and PC3 cells. (D) Cell counts on the Transwell chamber membrane for each group. Data were expressed using mean ± variance (n = 3, ***P < 0.05, **P < 0.01).

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Identification of DEPs. (A) Flowchart of proteomics detection; (B) bar chart and volcano plot of quantitative differences in proteins after AKBA intervention in PCa cells; and (C) heatmap of DEPs.

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Functional enrichment of DEPs. (A,B) Go enrichment analysis of DEPs; (C,D) KEGG enrichment analyses of DEPs; (E) distribution of subcellular location of DEPs; (F,G) enrichment analysis of domains of DEPs.