Integrating network pharmacology, bioinformatics, and experimental validation to unveil the molecular targets and mechanisms of galangin for treating hepatocellular carcinoma.

Galangin, a flavonoid compound, is derived from Alpinia officinarum Hance. Previous studies have shown that galangin can inhibit the proliferation of hepatocellular carcinoma (HCC), but its mechanism is still unclear. This study aims to investigate the potential targets and molecular mechanisms of galangin on HCC through network pharmacology, bioinformatics, molecular docking, and experimental in vitro validation. In this study, network pharmacology was used to investigate the targets and mechanisms of galangin in the treatment of HCC. AutoDockTools software was used to simulate and calculate the binding of galangin to its core targets. GO and KEGG enrichment analyses were conducted in the DAVID database to explore the main biological functions and signaling pathways impacted by galangin intervention. In addition, bioinformatics was applied to examine the correlation between the differential expressions of the anti-HCC core targets of galangin and the survival of patients with HCC. Finally, the findings obtained from network pharmacology and bioinformatics were verified in cell experiments. A total of 67 overlapping target genes of galangin and HCC were identified. Through the analysis of the protein-protein interaction (PPI) network, 10 hub genes with the highest degree of freedom were identified, including SRC, ESR1, MMP9, CDK4, CCNB1, MMP2, CDK2, CDK1, CHK1, and PLK1. These genes were found to be closely related to the degradation of the extracellular matrix, signal transduction, and the cell cycle. GO and KEGG enrichment analyses revealed that galangin exerts an anti-HCC role by affecting various signaling pathways, including the cell cycle, pathways in cancer, and the PI3K-Akt signaling pathway. The results of molecular docking indicated a significant interaction between galangin and CCNB1, CDK4, CDK1, and PLK1. Bioinformatics analysis revealed that CCNB1, CDK4, CDK1, and PLK1 were upregulated in the liver of patients with HCC at both the mRNA and protein levels. Flow cytometry analysis showed that galangin induced G0/G1 phase arrest and cell apoptosis in HepG2 and Huh7 cells. Additionally, galangin suppressed the expression of key proteins and mRNAs involved in the cell cycle pathway. These results suggest that galangin inhibits the growth of HCC cells by arresting the cell cycle at the G0/G1 phase.

Li X ，Zhou M ，Chen W ，Sun J ，Zhao Y ，Wang G ，Wang B ，Pan Y ，Zhang J ，Xu J ... -  《BMC Complementary Medicine and Therapies》

Mechanism of emodin in treating hepatitis B virus-associated hepatocellular carcinoma: network pharmacology and cell experiments.

Hepatocellular carcinoma (HCC) is a pressing global issue, with Hepatitis B virus (HBV) infection remaining the primary. Emodin, an anthraquinone compound extracted from the natural plant's. This study investigates the molecular targets and possible mechanisms of emodin in treating HBV-related HCC based on network pharmacology and molecular docking and validates the screened molecular targets through in vitro experiments. Potential targets related to emodin were obtained through PubChem, CTD, PharmMapper, SuperPred, and TargetNet databases. Potential disease targets for HBV and HCC were identified using the DisGeNET, GeneCards, OMIM, and TTD databases. A Venn diagram was used to determine overlapping genes between the drug and the diseases. Enrichment analysis of these genes was performed using GO and KEGG via bioinformatics websites. The overlapping genes were imported into STRING to construct a protein-protein interaction network. Cytoscape 3.9.1 software was used for visualizing and analyzing the core targets. Molecular docking analysis of the drug and core targets was performed using Schrodinger. The regulatory effects of emodin on these core targets were validate through in vitro experiments. A total of 43 overlapping genes were identified. GO analysis recognized 926 entries, and KEGG analysis identified 135 entries. The main pathways involved in the KEGG analysis included cancer, human cytomegalovirus infection and prostate cancer. The binding energies of emodin with HSP90AA1, PTGS2, GSTP1, SOD2, MAPK3, and PCNA were all less than -5 kcal/mol. Compared to normal liver tissue, the mRNA levels of XRCC1, MAPK3, and PCNA were significantly elevated in liver cancer tissue. The expression levels of XRCC1, HIF1A, MAPK3, and PCNA genes were closely related to HCC progression. High expressions of HSP90AA1, TGFB1, HIF1A, MAPK3, and PCNA were all closely associated with poor prognosis in HCC. In vitro experiments demonstrated that emodin significantly downregulated the expression of HSP90AA1, MAPK3, XRCC1, PCNA, and SOD2, while significantly upregulating the expression of PTGS2 and GSTP1. This study, based on network pharmacology and molecular docking validation, suggests that emodin may exert therapeutic effects on HBV-related HCC by downregulating the expression of XRCC1, MAPK3, PCNA, HSP90AA1, and SOD2, and upregulating the expression of PTGS2 and GSTP1.

Wang Y ，Li S ，Ren T ，Zhang Y ，Li B ，Geng X ... -  《Frontiers in Cellular and Infection Microbiology》

Kaempferol from Alpinia officinarum hance induces G2/M cell cycle arrest in hepatocellular carcinoma cells by regulating the ATM/CHEK2/KNL1 pathway.

Alpinia officinarum Hance (A. officinarum), a perennial herb known for its medicinal properties, has been used to treat various ailments, such as stomach pain, abdominal pain, emesis, and digestive system cancers. A. officinarum is extensively cultivated in the Qiongzhong and Baisha regions of Hainan, and it holds substantial therapeutic value for the local Li people of Hainan. Kaempferol, a flavonoid derived from A. officinarum, has demonstrated anticancer properties in various experimental and biological studies. Nevertheless, the precise mechanisms through which it exerts its anti-hepatocellular carcinoma (HCC) effects remain to be comprehensively delineated. This investigation aims to elucidate the anti-HCC effects of kaempferol derived from A. officinarum and to delve into its underlying mechanistic pathways. Using ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) to identify active compounds in A. officinarum. HCCLM3 and Huh7 cells were used to study the anti-HCC effect of kaempferol from A. officinarum. The cytotoxicity and proliferation of kaempferol and A. officinarum were measured using CCK-8 and EDU staining. Wound-healing assays and three-dimensional tumor spheroid models were further used to evaluate migration and the anti-HCC activity of kaempferol. The cell cycle and apoptosis were evaluated by flow cytometry. Western blot and qRT-PCR were used to detect the expression of proteins and genes associated with the cell cycle checkpoints. Finally, bioinformatics was used to analyze the relationship between the differential expression of core targets in the ATM/CHEK2/KNL1 pathway and a poor prognosis in clinical HCC samples. UPLC-MS/MS was employed to detect five active compounds in A. officinarum, such as kaempferol. The CCK-8 and EDU assays showed that kaempferol and A. officinarum significantly inhibited the proliferation of HCC cells. A wound-healing assay revealed that kaempferol remarkably inhibited the migration of HCC cells. Kaempferol significantly suppressed the growth of tumor spheroids. In addition, kaempferol markedly induced G2/M arrest and promoted apoptosis of HCC cells. Mechanically, kaempferol significantly reduced the protein and mRNA expression levels of ATM, CHEK2, CDC25C, CDK1, CCNB1, MPS1, KNL1, and Bub1. Additionally, the combination of kaempferol and the ATM inhibitor KU55933 had a more significant anti-HCC effect. The results of bioinformatics showed that ATM, CHEK2, CDC25C, CDK1, and KNL1 were highly expressed in patients with HCC and cancer tissues, indicating that these genes have certain value in the clinical diagnosis of HCC. Collectively, our results revealed that kaempferol from A. officinarum inhibits the cell cycle by regulating the ATM/CHEK2/KNL1 pathway in HCC cells. In summary, our research presents an innovative supplementary strategy for HCC treatment.

Li X ，Zhou M ，Zhu Z ，Wang Z ，Zhang X ，Lu L ，Xie Z ，Wang B ，Pan Y ，Zhang J ，Xu J ... -  《-》

Identification of the molecular targets and mechanisms of compound mylabris capsules for hepatocellular carcinoma treatment through network pharmacology and bioinformatics analysis.

Traditional Chinese herbal formulas have been proven to exert an inhibitory effect on tumor. Compound mylabris capsules (CMC) has been used for treating cancer, especially hepatocellular carcinoma (HCC), for years in China. However, its therapeutic mechanisms on HCC remain unclear. This research aimed to elucidate the molecular targets and mechanisms of CMC for treating HCC. First, the bioactive ingredients and potential targets of CMC, as well as HCC-related targets were retrieved from publicly available databases. Next, the overlapped genes between potential targets of CMC and HCC-related targets were determined using bioinformatics analysis. Then, networks of ingredient-target and gene-pathway were constructed. Finally, cell experiments were carried out to examine the effects of CMC-medicated serum on HCC and validate the core molecular targets. In total, 151 bioactive ingredients and 255 potential targets of CMC were selected, 982 differentially expressed genes of HCC were identified. Among them, 34 overlapped genes were finally selected. In addition, 20 pathways and 429 GO terms were significantly enriched. Protein-protein interaction and gene-pathway networks indicated that Cyclin B1(CCNB1) and Cyclin Dependent Kinase 1(CDK1) were the core gene targets for the treatment of CMC on HCC. Moreover, in vitro studies showed that CMC-medicated serum significantly inhibited the viability of HepG2 cells. Furthermore, CMC downregulated CCNB1 and CDK1 expressions and induced G2/M phase cell cycle arrest. CMC plays a therapeutic role in HCC via multi-component, -target and -pathway mechanisms, in which CCNB1 and CDK1 may be the core molecular targets. This study indicates that the integration of network pharmacology and bioinformatics analysis, followed by experimental validation, can serves as an effective tool for studying the therapeutic mechanisms of traditional Chinese medicine.

Wei J ，Ma L ，Liu W ，Wang Y ，Shen C ，Zhao X ，Zhao C ... -  《-》

Network-based Pharmacology and In vitro Validation Reveal that Galangin Induces Apoptosis in Bladder Cancer Cells by Promoting the P53 Signaling Pathway.

Galangin is one of the flavonoids in Alpinia officinarum. It has various anti-tumor activities, but its anti-bladder cancer effect is unclear. To investigate the mechanism of action of galangin against bladder cancer using a network pharmacology approach. The TCM Systematic Pharmacology Database and Analysis Platform (TCMSP), SwissTargetPrediction database, and the Targetnet database were used to predict the targets of action of galangin. Bladder cancer-related targets were obtained through the GeneCards database. The intersection of the two was taken as the target of galangin's action against bladder cancer. The intersecting targets were screened for core targets using the STRING database and Cytoscape 3.9.0 software to build a protein-protein interaction (PPI) network of targets. The core targets were subjected to gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the online annotation and visual integration analysis tool DAVIDBioinformaticsResources (2021Update). A drug-disease-target-pathway network was constructed using Cytoscape 3.9.0 software. The antibladder cancer effect of galangin was observed by cell proliferation, and plate cloning assay; apoptosis of bladder cancer cells induced by galangin was detected by Hoechst33342 staining and flow cytometry; protein immunoblotting (Western-blot) was used to detect the effect of galangin on apoptosis-related proteins Bax, Bcl-2, Cleaved-PARP, p53 signaling pathway p53 and cytc. A total of 115 genes were obtained from galangin against bladder cancer, and 16 core targets were screened. The kEGG pathway enrichment analysis included Pathways in cancer, PI3K-AKT signaling pathway, p53 signaling pathway, etc. In vitro experiments showed that galangin could inhibit bladder cancer cell proliferation, induce apoptosis, upregulate the expression of apoptosis-related proteins Bax and Cleaved-PARP and downregulate the expression of Bcl-2; meanwhile, galangin could promote the upregulation of the expression of p53 and cytc proteins by activating the p53 signaling pathway. Galangin induced apoptosis in bladder cancer cells by activating the p53 signaling pathway.

Long X ，Chen L ，Yang J ，Dong T ，Cheng Q ，Wang W ，Zou Y ，Su Y ，Dai W ，Chen B ，Zhou X ... -  《-》