Cepharanthine inhibits migration, invasion, and EMT of bladder cancer cells by activating the Rap1 signaling pathway in vitro

Abstract

Background: Cepharanthine, a bioactive constituent of Stephania japonica (Thunb.) Miers, is known for its potent anti-tumor properties. Nevertheless, the precise impact of this substance on bladder cancer remains poorly comprehended. The aim of this study was to demonstrate the effect and mechanism of cepharanthine on the metastasis of human bladder cancer cells.

Methods: The application of network pharmacology was utilized to ascertain the possible targets and signaling pathways of cepharanthine in the treatment of bladder cancer. The antiproliferative effects of cepharanthine were evaluated using Cell Counting Kit-8 and colony formation assays. The migration and invasion capabilities were assessed using Transwell assays and wound healing experiments. Proteins related to the Rap1 signaling pathway, cellular migration, cellular invasion, and Epithelial-Mesenchymal Transition (EMT) were quantified by western blotting.

Results: Through database screening, 313 cepharanthine-acting targets, 277 candidate disease targets in bladder cancer, 22 intersecting targets, and 12 core targets were confirmed. The involvement of the Rap1 signaling system was revealed by the Kyoto Encyclopedia of Genes and Genomes' pathway enrichment study. Cepharanthine was shown to decrease bladder cancer cell proliferation, migration, and invasion in vitro. Cepharanthine activated the Rap1 signaling pathway by upregulating Epac1 and downregulating E-cadherin and C3G protein expression, leading to increased expression of Rap1 GTP protein and decreased expression of protein kinase D1 and integrin α5. Rap1 signalling pathway activation resulted in the downregulation of migration and invasion-related proteins, matrix metallopeptidase MMP2, MMP9, as well as EMT-related proteins, N-cadherin and Snail, without affecting vimentin expression.

Conclusion: Cepharanthine inhibits migration, invasion, and EMT of bladder cancer cells by activating the Rap1 signalling pathway. The results offer helpful insights regarding the possible therapeutic use of cepharanthine for treating bladder cancer.

Keywords: Cepharanthine; Rap1 signaling pathway; bladder cancer; cell invasion; cell migration; network pharmacology.

Figure 1

Molecular structure of cepharanthine, potential therapeutic targets of cepharanthine on bladder cancer, PPI network visualization, core targets, GO, KEGG enrichment analysis, and drug-disease-target-pathway visualization of cepharanthine. A. Molecular structure of cepharanthine. B. Overlapping cepharanthine-related and bladder cancer-related targets on Venn diagram. C. Visualization of PPI network of potential therapeutic targets. D. Top 10 ranking of Degree and MCC values. E. Twelve core targets from the intersection of Degree Top10 and MCC Top10. F. GO enrichment analysis. The top 10 functional categories of biological processes, cellular components, and molecular functions were selected for display. G. KEGG pathway enrichment analysis of the potential therapeutic targets. The 20 pathways with the highest count values were selected for display. H. Results of KEGG pathway enrichment analysis of core targets. I. Visual network diagram of drug-disease-target-pathway of cepharanthine for bladder cancer treatment.

Figure 2

Inhibitory effects of cepharanthine on the viability of bladder cancer cells. Cell viability of HT1376 (A) and 5637 (B) bladder cancer cells treated with different concentrations of cepharanthine for 24 h, was detected by the CCK-8 method. Values are expressed as mean ± SD, n = 3 (experiments were repeated in triplicate independently), *P < 0.05 vs. control group (concentration: 0 μM).

Figure 3

Effect of cepharanthine on colony forming ability of HT1376 and 5637 cells. Cells were treated with cepharanthine and then cultured in fresh medium for 10 days to form colonies. Values are expressed as mean ± SD, n = 3 (experiments were repeated in triplicate independently), *P < 0.05 vs. untreated group (concentration: 0 μM), #P < 0.05 vs. treatment group (concentration: 10 μM). Note: CEP: cepharanthine.

Figure 4

Cepharanthine inhibited migration of HT1375 and 5637 cells. The effect of cepharanthine on migration (wound healing assay) of HT1376 (A) and 5637 (B) cells after 24 h treatment. (C) The statistical results are expressed as mean ± SD, n = 3 (experiments were repeated in triplicate independently), Scale bar = 200 μm; magnification power: 100×. *P < 0.05 vs. untreated group (concentration: 0 μM), #P < 0.05 vs. treatment group (concentration: 10 μM). Note: CEP: cepharanthine.

Figure 5

The effect of cepharanthine on migration and invasion (transwell assay) of HT1376 and 5637 cells after 24 h treatment. Migration (A, B) and invasion (C, D) abilities were observed by transwell assay. Values are expressed as mean ± SD, n = 3 (experiments were repeated in triplicate independently), Scale bar = 100 μm; magnification power: 200×. *P < 0.05 vs. untreated group (concentration: 0 μM), #P < 0.05 vs. treatment group (concentration: 10 μM). Note: CEP: cepharanthine.

Figure 6

Cepharanthine inhibited the expression of migration and invasion related proteins in HT1376 and 5637 cells. The effect of cepharanthine on the expression levels of migration and invasion related proteins MMP2, MMP9, and EMT-related proteins, detected by western blotting, in HT1376 (A, C) and 5637 (B, D) cells after 24 h treatment. Values are expressed as mean ± SD, n = 3 (experiments were repeated in triplicate independently), *P < 0.05 vs. untreated group (concentration: 0 μM), #P < 0.05 vs. treatment group (concentration: 10 μM). Note: CEP: cepharanthine; MMP2: matrix metalloproteinase 2; MMP9: matrix metalloproteinase 9; EMT: epithelial-mesenchymal transition.

Figure 7

Rap1 signaling pathway. The highlighted part is the related pathway we focused on.

Figure 8

Cepharanthine activate Rap1 and affect the expression of related signaling pathway molecules. The effect of cepharanthine on the expression levels of Rap1, Epac1, E-cadherin, C3G, Rap1 GTP, PKD1, ITGA5, detected by western blotting, in HT1376 (A, C) and 5637 (B, D) cells after 24 h treatment. Values are expressed as mean ± SD, n = 3 (experiments were repeated in triplicate independently), *P < 0.05 vs. untreated group (concentration: 0 μM), #P < 0.05 vs. treatment group (concentration: 10 μM). Note: CEP: cepharanthine; Rap1: Ras-proximate-1; Epac1: Exchange protein directly activated by cAMP 1; E-cadherin: epithelial cadherin; C3G: Crk SH3-domain-binding guanine-nucleotide-releasing factor; PKD1: protein kinase D1; ITGA5: integrin alpha-5.

Figure 9

The flow chart for the network pharmacology analysis and in vitro validation.