Network pharmacology and experimental validation to explore the role and potential mechanism of Liuwei Dihuang Decoction in prostate cancer.

OBJECTIVE: To evaluate the anti-tumor effector of Liuwei Dihuang Decoction (LWDHD) in prostate cancer (PCa) and explore the potential mechanism using experimental validation, network pharmacology, bioinformatics analysis, and molecular docking. METHODS: CCK test, Clone formation assay and wound-healing assays were used to determine the effect of LWDHD on prostate cancer growth and metastasis. The active ingredients and targets of LWDHD were obtained from the TCMSP database, and the relevant targets were selected by GeneCards, OMIM and DisGeNET databases for PCa. The cross-targets of drugs and disease were imported into the STRING database to construct protein interactions. The network was also visualized using Cytoscape software and core targets are screened using the Network Analyzer plug-in. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed using R software. TCGA database was used to analyze the correlation of bioinformatics genes. AutoDock vina was used to predict the molecular docking and binding ability of active ingredients to key targets. Through WB and q-PCR experiments, the above gene targets were detected to verify the effect of LWDHD on PCa. RESULTS: CCK and scratch tests confirmed that LWDHD could inhibit the proliferation, invasion and migration of prostate cancer cells. Clone formation experiments showed that LWDHD inhibited the long-term proliferative capacity of PC3 cells. LWDHD and PCa had a total of 99 common targets, establishing a "drug-ingredient-common target" network. Through GO and KEGG enrichment analysis, PI3K/AKT, MAPK, TP53 pathway, MYC, TNF pathway and other signaling pathways were found. Bioinformatics analysis showed that MYC gene was highly expressed and CCND1 and MAPK1 were low expressed in prostate cancer tissues. In addition, TP53, AKT1, MYC, TNF and CCND1 were positively correlated with MAPK1, among which AKT1 and CCND1 were most closely correlated with MAPK1. Molecular docking results showed that quercetin, kaempferol, ß-sitosterol and other main active ingredients of LWDHD treatment for PCa were combined with core proteins MAPK1 and AKT1 well. WB and q-PCR results showed that LWDHD inhibited the expression of PI3K and AKT in PC3 cells. CONCLUSION: The mechanism of LWDHD therapy for PCa is a multi-target and multi-pathway complex process, which may be related to the biological processes mediated by MAPK1 and AKT1 pathways, such as cell proliferation and inhibition of metastasis, and the regulation of signaling pathways. The PI3K/AKT signaling pathway may be a central pathway of LWDHD to inhibit prostate cancer proliferation.

Two telomere-to-telomere gapless genomes reveal insights into Capsicum evolution and capsaicinoid biosynthesis.

Chili pepper (Capsicum) is known for its unique fruit pungency due to the presence of capsaicinoids. The evolutionary history of capsaicinoid biosynthesis and the mechanism of their tissue specificity remain obscure due to the lack of high-quality Capsicum genomes. Here, we report two telomere-to-telomere (T2T) gap-free genomes of C. annuum and its wild nonpungent relative C. rhomboideum to investigate the evolution of fruit pungency in chili peppers. We precisely delineate Capsicum centromeres, which lack high-copy tandem repeats but are extensively invaded by CRM retrotransposons. Through phylogenomic analyses, we estimate the evolutionary timing of capsaicinoid biosynthesis. We reveal disrupted coding and regulatory regions of key biosynthesis genes in nonpungent species. We also find conserved placenta-specific accessible chromatin regions, which likely allow for tissue-specific biosynthetic gene coregulation and capsaicinoid accumulation. These T2T genomic resources will accelerate chili pepper genetic improvement and help to understand Capsicum genome evolution.