Uncovering the Pharmacological Mechanism of 2-Dodecyl-6-Methoxycyclohexa-2,5 -Diene-1,4-Dione Against Lung Cancer Based on Network Pharmacology and Experimental Evaluation

Abstract

Background: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1,4-Dione (DMDD) was purified from the roots of Averrhoa carambola L. Previous research demonstrated that DMDD is a small molecular compound with significant therapeutic potential for tumors. However, the potential targets and pharmacological mechanism of DMDD to treat lung cancer has not been reported. Methods: We employed network pharmacology and experimental evaluation to reveal the pharmacological mechanism of DMDD against lung cancer. Potential therapeutic targets of DMDD were screened by PharmMapper. Differentially expressed genes (DEGs) in The Cancer Genome Atlas (TCGA) lung cancer data sets were extracted and analyzed by GEPIA2. The mechanism of DMDD against lung cancer was determined by PPI, gene ontology (GO) and KEGG pathway enrichment analysis. Survival analysis and molecular docking were employed to obtain the key targets of DMDD. Human lung cancer cell lines H1975 and PC9 were used to detect effects of DMDD treatment in vitro. The expression of key targets after DMDD treated was validated by Western Blot. Results: A total of 60 Homo sapiens potential therapeutic targets of DMDD and 3,545 DEGs in TCGA lung cancer datasets were identified. Gene ontology and pathway analysis revealed characteristic of the potential targets of DMDD and DEGs in lung cancer respectively. Cell cycle and pathways in cancer were overlapping with DMDD potential targets and lung cancer DEGs. Eight overlapping genes were found between DMDD potential therapeutic targets and lung cancer related DEGs. Survival analysis showed that high expression of DMDD potential targets CCNE1 and E2F1 was significantly related to poor patient survival in lung cancer. Molecular docking found that DMDD exhibited significant binding affinities within the active site of CCNE1 and E2F1. Further tests showed that DMDD inhibited the proliferation, migration and clone formation in lung cancer cell lines (H1975 and PC9) in a dose and time dependent manner. Mechanistically, DMDD treatment decreased the expression of CDK2, CCNE1, E2F1 proteins and induced cell cycle arrest at the G1/S phase in H1975 and PC9 cells. Conclusion: These results delineated that DMDD holds therapeutic potential that blocks tumorigenesis by cell cycle regulation in lung cancer, and may provide potential therapies for lung cancer.

Keywords: DMDD; TCGA; cell cycle arrest; lung cancer; network pharmacology.

FIGURE 1

PPI, GO and KEGG analysis of DMDD potential target proteins. (A) PPI network of 60 DMDD potential targets which consists of 59 nodes and 47 edges. The node color changes from light to dark red in according to the MCODE score. (B, C) Cellular component, molecular function, biological process, and KEGG analysis of 60 DMDD potential target proteins.

FIGURE 2

Differentially expressed genes in lung cancer and normal lung tissues. (A) Enrichment of KEGG pathways for up regulated genes in lung cancer. (B) Enrichment of KEGG pathways for down regulated genes in lung cancer. (C) 8 overlapping genes were found between 60 potential therapeutic target proteins of DMDD and lung cancer related DEGs. (D, E) FUT8, CCNE1, E2F1, ARHGAP11A were upregulated and ARHGEF7, RUNX1T1, ACADVL, FES were down regulated in lung cancer tissues compared with normal lung.

FIGURE 3

CCNE1, E2F1, ARHGAP11A, RUNX1T1 and FES were significantly associated with patient survival in lung cancer. Kaplan-Meier curves and log-rank test of CCNE1 (A), E2F1 (B) and ARHGAP11A (C) in lung cancer data sets indicating higher CCNE1, E2F1 and ARHGAP11A expression were associated to poorer patient survival. Kaplan–Meier curves and log-rank test of RUNX1T1 (D) and FES (E) in lung cancer data sets indicating lower RUNX1T1 and FES expression were associated to poorer patient survival.

FIGURE 4

Docking model of DMDD with CCNE1 and E2F1. (A) DMDD binding with the pocket of CCNE1 is composed of hydrogen bonds. And the interaction pattern of DMDD with the residues. (B) 2D diagram between the CCNE1 and residues. (C) DMDD binding with the pocket of E2F1 is composed of hydrogen bonds. And the interaction pattern of DMDD with the residues. (B) 2D diagram between the E2F1 and residues.

FIGURE 5

DMDD Inhibits proliferation, migration and colony formation in lung cancer Cells. (A, B) Dose-dependent curves of DMDD in PC9 and H1975 cell lines. (C) Cell proliferation decreased after 60 µM DMDD treatment in PC9 and H1975 cell lines at indicated time. (D) Colony formation in PC9 and H1975 cells after treatment with DMDD at 0, 10, 20 and 40 µM. (E) Bar chart shows the colony formation rate calculated from Figure 5D. (F) Cell migration was determined using a wound-healing assay. Images of the wound areas were shown at 0 and 24 h in PC9 and H1975 cell lines (scale bar 200 μm). (G) Bar chart shows the wound area calculated from Figure 5F. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

FIGURE 6

DMDD induced cell cycle arrest at the G1/S phase and potentially through CCNE1 and E2F1 regulation. (A, B) CCNE1, E2F1 and CDK2 were decreased after DMDD treatment with indicated doses for 48 h measured by Western blot in PC9 and H1975 cells. (C) DMDD treatment with indicated doses caused G1/S arrest in both PC9 and H1975 lung cells by flow cytometer.