Drug repurposing of ticagrelor suppresses renal cell carcinoma growth by blockading the EGFR/PI3K/AKT axis.

Figures

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  Figure 1.. Ticagrelor inhibited the proliferation of renal cancer cell lines and promotes their apoptosis. (A) The dose-response curve of ticagrelor on inhibiting renal cancer cell lines for 48 h (786-O, Caki-1, ACHN and OS-RC-2). (B-C) Ticagrelor inhibited the proliferation of the 786-O renal cancer cell line for 24 h, which was detected by the Edu staining method, *p < 0.05, **p < 0.01. (D-E) Ticagrelor inhibited the proliferation of the ACHN renal cancer cell line for 24h, which was detected by the Edu staining method, ns > 0.05, ****p < 0.0001. (F-G) Ticagrelor promoted apoptosis in the 786-O renal cancer cell line for 48 h, which was detected by AO/PI staining, *p < 0.05, ***p < 0.001. (H-I) Ticagrelor promoted apoptosis in the ACHN renal cancer cell line for 48 h, which was detected by AO/PI staining, *p < 0.05, ****p < 0.0001.

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  Figure 2.. Ticagrelor inhibits the invasion, migration and adhesion of the 786-O and ACHN renal cancer cell line. (A-C) Ticagrelor, at concentrations of 10 μM and 20 μM, inhibited the invasion of 786-O and ACHN cells for 24 h and 48 h respectively detected by wound healing assay, *p < 0.05, ***p < 0.001, ****p < 0.0001. (D-F) Ticagrelor, at concentrations of 10 μM and 20 μM, inhibited the migration of the 786-O and ACHN cell line for 24 h detected by Transwell assay, **p < 0.01. (G-H) After 10 min and 20 min of cell quiescence, the adhesion ability of 786-O cells significantly decreased 48 h after ticagrelor administration., *p < 0.05, ****p < 0.0001. (I-J) After 10 min and 20 min of cell quiescence, the adhesion ability of ACHN cells significantly decreased 48 h after ticagrelor administration, ****p < 0.0001.

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  Figure 3.. Ticagrelor inhibited the tumor growth in 786-O CDX model. (A) Tumor mass image of 786-O cells treated with ticagrelor for 24 days. (B) Ticagrelor inhibited the growth of tumor volume in 786-O CDX model, ***p < 0.001. (C) Ticagrelor inhibited the tumor weight for 24 days treatment in 786-O CDX model, *p < 0.05. (D) Ticagrelor did not affect the body weight of mice in the 786-O CDX model.

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  Figure 4.. The network pharmacology of ticagrelor in treating renal cancer has been analyzed. (A) Venn diagram illustrated the overlapping targets of ticagrelor and RCC. (B) PPI network was constructed for common targets of ticagrelor and renal cancer. (C) GO Analysis. (D) KEGG Analysis. (E) Molecular docking of ticagrelor with EGFR, PI3KCB, PI3KCD and AKT. (F) Molecular Dynamics Simulation of Ticagrelor and AKT, the time-dependent RMSD values, Rg values, SASA values, HBonds values and RMSF values of AKT- ticagrelor.

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  Figure 5.. Ticagrelor inhibited EGFR/PI3K/AKT Pathway in 786-O renal carcinoma cell line. (A) The dose-response curve of gefitinib on inhibiting 786-O renal cancer cell line for 48 h. (B) Ticagrelor (5 μM or 10 μM) and gefitinib (5 μM or μM) significantly enhanced the inhibition rate of 786-O cells. (C) Ticagrelor (10 μM) decreased the expression of p-EGFR, p-PI3K and p-AKT, and Ticagrelor (10 μM)+ gefitinib (5 μM) increased inhibition of EGFR/PI3K/AKT pathway by WB test. (D-I) Statistical analysis of p-EGFR, EGFR, p-PI3K, PI3K, p-AKT and AKT protein expression, ns > 0.05, *p < 0.05, ** p < 0.01.