Sanguinarine inhibits melanoma invasion and migration by targeting the FAK/PI3K/AKT/mTOR signalling pathway

Abstract

Context: Sanguinarine (SAG) is the most abundant constituent of Macleaya cordata (Willd.) R. Br. (Popaceae). SAG has shown antimammary and colorectal metastatic effects in mice in vivo, suggesting its potential for cancer chemotherapy.

Objective: To determine the antimetastatic effect and underlying molecular mechanisms of SAG on melanoma.

Materials and methods: CCK8 assay was used to determine the inhibition of SAG on the proliferation of A375 and A2058 cells. Network pharmacology analysis was applied to construct a compound-target network and select potential therapeutic targets of SAG against melanoma. Molecular docking simulation was conducted for further analysis of the selected targets. In vitro migration/invasion/western blot assay with 1, 1.5, 2 μM SAG and in vivo effect of 2, 4, 8 mg/kg SAG in xenotransplantation model in nude mice.

Results: The key targets of SAG treatment for melanoma were mainly enriched in PI3K-AKT pathway, and the binding energy of SAG to PI3K, AKT, and mTOR were -6.33, -6.31, and -6.07 kcal/mol, respectively. SAG treatment inhibited the proliferation, migration, and invasion ability of A375 and A2058 cells (p < 0.05) with IC₅₀ values of 2.378 μM and 2.719 μM, respectively. It also decreased the phosphorylation levels of FAK, PI3K, AKT, mTOR and protein expression levels of MMP2 and ICAM-2. In the nude mouse xenograft model, 2, 4, 8 mg/kg SAG was shown to be effective in inhibiting tumour growth.

Conclusions: Our research offered a theoretical foundation for the clinical antitumor properties of SAG, further suggesting its potential application in the clinic.

Keywords: Network pharmacology; adhesion; metastasis; molecular docking.

Figure 1.

(A) Chemical structure of SAG. (B and C) Effect of SAG on cell viability in A375 and A2058 cells. A375 and A2058 cells were treated with SAG for 48 h, and the 0.5% DMSO group was used as a control. (D and E) A375 and A2058 cells were treated with different concentrations of SAG or DMSO, and cell viability and the IC₅₀ at 48 h were examined by CCK8 assay. (F-H) The results of flow cytometry showed there was no significant difference in the viability of the cells at each concentration of SAG. The data are shown as the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, as compared to the controls).

Figure 2.

Analysis of the melanoma-SAG-related targets and construction of the SAG-melanoma-target network: (A) The Venn diagram of SAG and melanoma intersection targets. (B-D) GO enrichment analysis of core target for SAG against melanoma. For (B) Cellular Components, (C) Molecular Function and (D) Biological Process, was shown in histograms. (E) Whole 20 enriched KEGG pathways of core targets for SAG against melanoma. The colour scales indicate the different thresholds of adjusted p-values, and the sizes of the dots represent the gene count of each term.

Figure 3.

(A) The Protein-protein interaction (PPI) network of 71 common targets of SAG and Melanoma. (B, Table 1) According to the screening threshold values of degree >2, betweenness-centrality >0.00212038 and closeness-centrality >0.425, 18 key targets were screened out. (C) The PPI network of 18 core targets was then constructed including 18 nodes and 62 edges.

Figure 4.

Molecular docking of SAG with PI3K (A), AKT (C) and mTOR (E) receptors. Map of sites where SAG binds to the residues of (B) PI3K, (D) AKT and (F) mTOR receptors.

Figure 5.

(A-D) Effect of SAG on the migration of A375 and A2058 cells. The cells were cultured with different concentrations of SAG (0, 1, 1.5, 2 μM) for 48 h. Each scratch was photographed at 0 h, 24 h and 48 h. The histogram shows the closure rate of the scratches after 24 h and 48 h (*p < 0.05, ** p < 0.01, *** p < 0.001 as compared to the controls).

Figure 6.

Effect of SAG on melanoma cell adhesion and invasion. (A) Effect of different concentrations of SAG on the adhesion ability of A375 and A2058 cells. (B and C) Effect of SAG on MMP2 and ICAM-1 protein expression in A375 cells. (D–F) SAG significantly reduced the numbers of A375 and A2058 cells that invaded through the membrane as compared to the controls. The results shown here are representative of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001 as compared to the controls).

Figure 7.

SAG reduces A375 cell metastasis by down-regulating the FAK/PI3K/AKT/mTOR signalling pathways. (A-C) Western blotting assays were used to assess the effect of SAG on FAK and p-FAK (Tyr397). (D) The whole cell extract was harvested to detect p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR proteins by Western blot. (E-G) The histogram shows the relative protein expression of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR in different groups quantified and normalized against β-actin bars, standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001, as compared to the controls).

Figure 8.

SAG inhibited the growth of A375 cells in vivo. (A) The timeline for the mouse in vivo experimental details. (B and C) Nude mice were injected with different concentrations of SAG and tumour volumes were measured daily. Compared with the control group, the SAG-treated group significantly inhibited tumour growth. (D) The body weight of the mice was weighed daily, and there was no significant difference between the body weight of the SAG-treated group and the control group. (E and F) p-FAK, p-PI3K, p-AKT, p-mTOR, MMP2 and ICAM-1 were detected by immunohistochemistry, the expression levels were downregulated with the increasing SAG levels (*p < 0.05, **p < 0.01, ***p < 0.001, as compared to the controls).

Figure 9.

Mice with tumours were treated with SAG, and heart, liver, spleen, lung, and kidney tissue were stained with haematoxylin-eosin (HE). In both groups, there were no significant abnormalities in HE staining, and there was no difference between the treatment and control groups.

Figure 10.

Schematic diagram of the mechanism of SAG inhibition of melanoma migration and invasion.