Magnolol and 5-fluorouracil synergy inhibition of metastasis of cervical cancer cells by targeting PI3K/AKT/mTOR and EMT pathways

Abstract

Objective: This study is designed to investigate the mode of action of the synergistic effect of 5-fluorouracil (5-FU) and magnolol against cervical cancer.

Methods: Network pharmacological approach was applied to predict the molecular mechanism of 5-FU combined with magnolol against cervical cancer. CCK-8 assay, colony formation assay, immunofluorescence staining, adhesion assay, wound healing mobility assay, cell migration and invasion assay and Western blot analysis were conducted to validate the results of in silico study.

Results: Phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was identified as the key pathway in silico study. The experimental results showed that 5-FU combined with magnolol strongly inhibited cervical cancer cell proliferation, induced the morphological change of HeLa cells by down-regulating the expression of α-actinin, tensin-2 and vinculin. Moreover, magnolol enhanced inhibitory effect of 5-FU on the cell adhesion, migration and invasion. The phosphorylation of AKT and PI3K and the expression of mTOR were strongly inhibited by the combination of 5-FU and magnolol. Moreover, the expression of E-cadherin and β-catenin was upregulated and the expression of Snail, Slug and vimentin was down-regulated by the 5-FU together with magnolol.

Conclusion: Taken together, this study suggests that 5-FU combined with magnolol exerts a synergistic anti-cervical cancer effect by regulating the PI3K/AKT/mTOR and epithelial-mesenchymal transition (EMT) signaling pathways.

Keywords: 5-fluorouracil; EMT pathway; PI3K/AKT/mTOR pathway; cervical cancer; magnolol; network pharmacology.

Fig. 1

Venn diagram of the targets of 5-FU combined with magnolol against cervical cancer. Three different color circles represent magnolol targets (purple), 5-FU targets (yellow) and cervical cancer targets (green), respectively. The overlapped 66 targets were the potential targets of 5-FU combined with magnolol against cervical cancer.

Fig. 2

PPI network diagram (A, node represents target and size and color of nodes reflect degree value); Analysis of top 10 Hub targets of 5-FU combined with Magnolol in the treatment of cervical cancer ranked by MCC method (B, node size from big to small and the color from deep to shallow represent the decreasing importance).

Fig. 3

GO enrichment analysis of anti-cervical cancer targets of 5-FU combined with magnolol.

Fig. 4

KEGG pathway analysis of 5-FU combined with magnolol against cervical cancer.

Fig. 5

Effects of 5-FU combined with magnolol on the activity of cervical cancer cells. (A) CCK-8 assay of different concentrations of magnolol on HeLa cells. (B) CCK-8 assay of 5-FU combined with magnolol on HeLa cells. (C) CCK-8 assay of 5-FU combined with magnolol on SiHa cells. (D) The colony formation assay of 5-FU combined with magnolol on HeLa cells. (E) The colony formation assay of 5-FU combined with magnolol on SiHa cells. The data displayed is the average of three repeated experiments. *P < 0.05, ^**P < 0.01 vs DMSO control group. The scale bar is 200 μm.

Fig. 6

Effects of 5-FU combined with magnolol on morphology and F-actin cytoskeleton structure of cervical cancer cells.

Fig. 7

5-FU combined with magnolol inhibited adhesion of HeLa (A) and SiHa cells (B) for 24 h.

Fig. 8

5-FU combined with magnolol inhibited migration of HeLa and SiHa cells. The wound area was photographed at specified time (0, 8 and 24 h). Results of HeLa (A) and SiHa (B) cellswound healing mobility assay. The mobility of HeLa (C) and SiHa (D) cells at 0, 8 and 24 h (mean ± SD, n = 3). *P < 0.05, ^**P < 0.01 vs DMSO group.

Fig. 9

5-FU combined with magnolol inhibited migration and invasion of HeLa and SiHa cells in vitro. (A) HeLa cell migration assay. (B) SiHa cell migration assay. (C) HeLa cell invasion assay. (D) SiHa cell migration assay.

Fig. 10

Effects of 5-FU combined with magnolol on PI3K/AKT/mTOR pathway and EMT related proteins in HeLa cells. (A) After blotting, the PVDF membranes were treated with p-AKT, AKT, p-PI3K, PI3K and GAPDH antibodies. (B − C) Comprehensive band intensity was determined by Image J software. (D) After blotting, the PVDF membranes were treated with EMT pathway antibodies and mTOR, α-actinin, tensin-2, vinculin and GAPDH antibodies. (E − N) Comprehensive band intensity was determined by Image J software. *P < 0.05, ^**P < 0.01 vs DMSO group.