Network pharmacology and molecular docking reveal the mechanism of action of Bergapten against non‑small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide, necessitating new treatment approaches with minimal side effects. In the present study, the potential of Bergapten (5-methoxypsoralen), a natural furanocoumarin compound, as a therapeutic agent against NSCLC was investigated by using network pharmacology, molecular docking and in vitro validation. Bergapten targets were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and SwissTarget databases, whilst lung cancer-related targets were sourced from GeneCards and DisGeNET. Protein-protein interaction analysis and molecular docking were performed to identify key targets. The inhibitory effects of Bergapten on lung cancer cells were assessed using Cell Counting Kit-8 assays, wound healing assays, cell migration experiments, flow cytometry and western blotting. SC79 was used to verify the regulation of Bergapten on the PI3K/AKT pathway. Network pharmacology identified 51 targets, one signaling pathway and four Gene Ontology projects associated with the action of Bergapten against NSCLC. Key targets identified included glycogen synthase kinase-3β, Janus kinase 2, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α and protein tyrosine kinase 2. In vitro experiments demonstrated that Bergapten significantly inhibited cell viability, promoted apoptosis, induced cellular senescence and inhibited the PI3K/AKT signaling pathway in NSCLC cells. In conclusion, Bergapten exerts its anti-NSCLC effects through the PI3K/AKT pathway, promoting cell senescence and inhibiting inflammation. These findings suggest that Bergapten has potential as a therapeutic agent for NSCLC.

Keywords: 5-methoxypsoralen; PI3K/AKT; cell senescence; non-small cell lung cancer.

Figure 1.

Drugs-potential targets network. (A) Network diagram of ‘5-methoxypsoralen-target’. (B) Venn diagram. (C) Network diagram of PPI of 5-methoxypsoralen against lung cancer. (D) Top 12 hub genes in PPI network. PPI, protein-protein interaction.

Figure 2.

Molecular model of 5-methoxypsoralen binding to its predicted protein target. Proteins (A) CASP3, (B) CCND1, (C) ESR1, (D) GSK3B, (E) JAK2, (F) NF-κB, (G) PIK3CA, (H) PTK2 and (I) TLR4 were demonstrated to be associated with 5-methoxypsoralen interactions, represented by the blue stick model. Lines represent residues in the binding site. The light dashed lines represent hydrogen bonds and the dark dashed lines demarcate π-π interactions. CASP3, Caspase 3; CCND1, Cyclin D1; ESR1, estrogen receptor 1; GSK3B, glycogen synthase kinase-3β; JAK2, Janus kinase 2; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α; PTK2, protein tyrosine kinase 2; TLR4, toll-like receptor 4.

Figure 3.

Effects of 5-methoxypsoralen on the viability and apoptosis levels of NCI-H1975, NCI-H1299 and NCI-H460 cells. (A) After 72 h of treatment with 5-methoxypsoralen, the viability of these cells was measured using the Cell Counting Kit-8 assay and the optimal drug concentration was determined. (B) Apoptosis levels was measured using flow cytometry after 72 h of treatment with 5-methoxypsoralen. n=3. ****P<0.0001.

Figure 4.

5-methoxypsoralen inhibits the migration of lung cancer cells. Representative images of wound healing assays using 40 or 50 µM 5-methoxypsoralen in (A) NCI-H1975, (B) NCI-H1299 and (C) NCI-H460 cells (magnification, ×200). (D) Quantification of migration efficiency in wound healing assay. n=3. ****P<0.0001.

Figure 5.

5-methoxypsoralen inhibits the migration of lung cancer cells. (A) Representative images of NCI-H1975, NCI-H129 and NCI-H460 cells treated with 40 or 50 µM 5-methoxypsoralen (magnification, ×200). (B) Fold change of migrated cell numbers. n=3. ***P<0.001; ****P<0.0001.

Figure 6.

PI3K/AKT signaling pathway and pro-aging serve important roles in the inhibitory effect of 5-methoxypsoralen on non-small cell lung cancer. (A) Western blot analysis used to determine the expression of P-PI3K, PI3K, P-AKT and AKT proteins in three types of cells after treatment 5-methoxypsoralen. (B) Quantification of western blot analysis in three types of cells after treatment 5-methoxypsoralen. (C) RT-qPCR analysis determined the P16, P21, MMP12, IL6 and IL8 mRNA levels after treatment with 5-methoxypsoralen. n=3. *P<0.05, **P<0.01; ***P<0.001; ****P<0.0001.

Figure 7.

SC79 (4 µg/ml) antagonizes the negative effects of 5-methoxypsoralen on non-small cell lung cancer. (A) ELISA was used to detect the AKT activation levels of three cell lines after treatment with 5-methoxypsoralen, 5-methoxypsoralen + DMSO and 5-methoxypsoralen + SC79. (B) RT-qPCR analysis determined the Caspase3, 7 and 9 mRNA levels after treatment with 5-methoxypsoralen, 5-methoxypsoralen + DMSO and 5-methoxypsoralen + C79. (C) RT-qPCR analysis determined the P16, P21, MMP12, IL6 and IL8 mRNA levels after treatment with 5-methoxypsoralen, 5-methoxypsoralen + DMSO and 5-methoxypsoralen + SC79. n=3. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; MMP12, matrix metallopeptidase 12; ns, no significant difference.