To Reveal the Potential Mechanism of Quercetin against NSCLC Based on Network Pharmacology and Experimental Validation

Purpose: This study aimed to initially clarify the potential mechanism of quercetin in the treatment of non-small cell lung cancer (NSCLC) based on network pharmacology, molecular docking and in vitro experiments. Method: TCMSP, SwissTargetPrediction, TCMIP, STITCH, and ETCM databases were applied to obtain the targets of quercetin. NSCLC-related targets were retrieved from GeneCards, OMIM, PharmGKB, TTD, and NCBI databases. Their intersection targets were imported into the STRING database to construct a protein-protein interaction (PPI) network and core targets were identified through the Cytoscape 3.10.0 soft and the CytoHubba tool. Furthermore, Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the intersection targets. A compound-targetspathways network was subsequently constructed to screen for key targets and pathways. Molecular docking was performed with Discovery Studio software to verify the interactions between quercetin and core targets. In vitro validations were conducted employing CCK-8 assays, flow cytometry, and Western blotting (WB). Results: 193 potential targets of quercetin for treating NSCLC were obtained. The top ten core targets identified within the PPI network included TP53, HSP90AA1, AKT1, JUN, SRC, EGFR, ACTB, TNF, MAPK1, and VEGFA. GO analysis yielded 2319 items, and KEGG analysis resulted in 211 enriched pathways. Molecular docking results demonstrated a high affinity of quercetin towards the core targets. Based on the compound-targets-pathways network and molecular docking, the PI3K/AKT/P53 pathway and its key-related proteins (PIK3R1, AKT1, and TP53) were selected for further validation. Quercetin(20 and 40 μg/mL) significantly decreased the viability of A549 NSCLC cells but not BEAS-2B normal cells via CCK-8 assays. Flow cytometry and WB analyses further corroborated that quercetin could promote apoptosis of A549 cells by downregulating and upregulating the expression of Bcl-2 and Bax (P<0.05), respectively. Notably, quercetin did not significantly alter the total protein levels of PI3K, AKT, and P53 but downregulated the phosphorylation levels of PI3K and AKT (P<0.05) and upregulated the phosphorylation level of P53 (P<0.05). Conclusion: Quercetin exhibits therapeutic potential in NSCLC by regulating the PI3K/AKT/P53 pathway to promote cell apoptosis. result: A total of 193 potential targets of quercetin in the treatment of NSCLC were obtained. The top ten core targets of PPI network were TP53, HSP90AA1, AKT1, JUN, SRC, EGFR, ACTB, TNF, MAPK1 and VEGFA. 2319 items were obtained by GO analysis and 211 items were obtained by KEGG analysis. The molecular docking results showed that quercetin had high affinity with the core targets in PPI network. It was finally determined to verify the PI3K/AKT/P53 signaling pathway and the key-related-proteins (namely PIK3R1, AKT1 and TP53) via the compound-targets-pathways network and molecular docking analysis. Experiments in vitro showed that quercetin could promote the apoptosis of NSCLC cells by down-regulating the protein levels of Bcl-2, Phosphorylation of PI3K and Phosphorylation of AKT, and up-regulating the protein levels of Bax and Phosphorylation of P53.