. The Pathophysiology Department, School of Basic Medical Sciences, College of Medicine, Zhengzhou University, Zhengzhou 450001 China . China-US (Henan) Hormel Cancer Institute, Zhengzhou 450003, China . Basic Medicine Sciences Research Center, Zhengzhou University, Zhengzhou 450052, China . State Key Laboratory of Esophageal Cancer Prevention and Treatment, Zhengzhou 450001, China . Provincial Cooperative Innovation Center for Cancer Chemoprevention, Zhengzhou University, Zhengzhou 450001, China . Cancer Chemoprevention International Collaboration Laboratory, Zhengzhou 450001, China
Treatment options for patients with esophageal squamous cell carcinoma (ESCC) often result in poor prognosis and declining health-related quality of life. Screening FDA-approved drugs for cancer chemoprevention is a promising and cost-efficient strategy. Here, we found that dronedarone, an antiarrhythmic drug, could inhibit the proliferation of ESCC cells. Moreover, we conducted phosphorylomics analysis to investigate the mechanism of dronedarone-treated ESCC cells. Through computational docking models and pull-down assays, we demonstrated that dronedarone could directly bind to CDK4 and CDK6 kinases. We also proved that dronedarone effectively inhibited ESCC proliferation by targeting CDK4/CDK6 and blocking the G0/G1 phase through RB1 phosphorylation inhibition by in vitro kinase assays and cell cycle assays. Subsequently, we found that knocking out CDK4 and CDK6 decreased the susceptibility of ESCC cells to dronedarone. Furthermore, dronedarone suppressed the growth of ESCC in patient-derived tumor xenograft models in vivo. Thus, our study demonstrated that dronedarone could be repurposed as a CDK4/6 inhibitor for ESCC chemoprevention.
Just Accepted Date: 25 July 2024Online First Date: 14 September 2024Issue Date: 29 October 2024
Cite this article:
Bo Li,Jing Zhang,Yin Yu, et al. Dronedarone inhibits the proliferation of esophageal squamous cell carcinoma through the CDK4/CDK6-RB1 axis in vitro and in vivo[J]. Front. Med.,
2024, 18(5): 896-910.
Fig.1 Dronedarone inhibited ESCC cell proliferation. (A) Chemical structure of dronedarone. (B, C) SHEE, KYSE150, and KYSE450 cells were treated with dronedarone. Cell viability was measured at 24 (B) and 48 h (C). (D–F) Effect of dronedarone inhibited the cell proliferation of ESCC cells. SHEE (D), KYSE150 (E), and KYSE450 (F) cells were treated with dronedarone (0, 1, 1.5, 2, and 2.5 µmol/L). Cell numbers were measured at 0, 24, 48, 72, and 96 h. Data were analyzed by homogeneity of variance and one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (G) Dronedarone inhibited the anchorage-independent growth of ESCC cells. KYSE150 and KYSE450 were treated with dronedarone (0, 1, 1.5, 2, and 2.5 µmol/L). Clone numbers were measured at 7 days. Data were analyzed by homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 500 µm. (H) Dronedarone inhibited colony formation of ESCC cells. KYSE150 and KYSE450 were treated with dronedarone (0, 1, 1.5, 2, and 2.5 µmol/L). Clone numbers were measured at 12 days. Data were analyzed by homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
IC50 (μmol/L)
24 h
48 h
SHEE
24.654
14.921
KYSE150
12.498
5.914
KYSE450
5.188
3.974
Tab.1 IC50 of cytotoxicity assay
Fig.2 Dronedarone inhibited the expression of p-RB1 S807, p-RB1 S811, and p-RB1 T826. (A) Histogram showed differentially expressed protein and phosphorylated protein sites after treatment with DMSO or dronedarone (2.5 μmol/L) in KYSE150 cells. (B) The heat map displayed the differentially expressed protein in KYSE150 cells treated with DMSO or dronedarone for 24 h. (C) The volcano map indicated 350 upregulated phosphorylated and 910 downregulated phosphorylated protein sites. (D) Top ten downregulated protein phosphorylation sites. Data are shown as the ratio value. (E) GPS5.0 software predicted the upstream protein kinase of RB1. Data are shown as the E-ratio value and P value. (F, G) Spearman correlation analysis was performed to investigate the relationship between CDK4 (F) or CDK6 (G) and RB1 gene expression in ESCC using data from the TCGA database. (H) After DMSO and dronedarone treatment, p-RB1 S807, p-RB1 S811, and p-RB1 T826 were tested by Western blot in KYSE150 cells.
Fig.3 Dronedarone bound to CDK4/CDK6. (A, B) Schrödinger software simulated the binding of dronedarone to CDK4 (A) or CDK6 (B) at the ATP binding pocket, and the binding sites were labeled. (C) Pull-down assay indicated that dronedarone could bind to CDK4 in vivo and ex vivo. (D) Pull-down assay indicated that dronedarone could bind to CDK6 in vivo and ex vivo. (E) Mutation experiment showed that CDK4 (WT) could bind with dronedarone, whereas the mutant CDK4 (D97A, D158A, and T177A) could not. (F) Mutation experiment showed that CDK6 (WT) could bind with dronedarone, whereas the mutant CDK6 (E21A, N150A, and D163A) could not.
Fig.4 Dronedarone blocked ESCC at the G0/G1 phase by inhibiting the CDK4/CDK6-RB1 axis. (A, B) Protein levels of p-RB1 S807, p-RB1 S811, p-RB1 T826, T-RB1, CDK4, CDK6, cyclin A2, and cyclin B1 were measured in KYSE150 (A) and KYSE450 (B) cells treated with various concentrations of dronedarone (0, 1, 1.5, 2, and 2.5 µmol/L). (C, D) In vitro kinase experiment assay showed that treatment with dronedarone (0, 1, 1.5, 2, and 2.5 µmol/L) inhibited the activation of p-RB1 S807 and p-RB1 S811 by CDK4 (C) and CDK6 (D) in a dose-dependent manner. (E) After being treated with dronedarone (0, 1, 1.5, 2, and 2.5 µmol/L) and palbociclib (10 µmol/L) for 24 h, the cell cycle of KYSE150 and KYSE450 cells was analyzed using homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig.5 Depletion of CDK4/CDK6 inhibited the proliferation of ESCC. (A) UALCAN and GEPIA analyzed CDK4 and CDK6 mRNA expression in normal and esophageal cancer tissues. (B) Expression levels of CDK4, CDK6, p-RB1 S807, and p-RB1 S81 were detected after depletion of CDK4 (sgCDK4) or CDK6 (sgCDK6) in KYSE150 and KYSE450 cells by Western blot. (C) Cell growth of sgCDK4 and sgCDK6 was analyzed in KYSE150 and KYSE450 cells by measuring OD values at 0, 24, 48, 72, and 96 h in MTT assay. The data were analyzed using homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Colony numbers were measured after 12 days of colony formation using sgCDK4 and sgCDK6 KYSE150 cells and KYSE450 cells. Data were analyzed by homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (E) After treatment with dronedarone, cell proliferation was measured in sgCDK4 and sgCDK6 cells from KYSE150 and KYSE450 by MTT assay at 72 h. Data were analyzed by homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (F) Cell cycle of sgCDK4 and sgCDK6 in KYSE150 and KYSE450 cells was analyzed at 24 h. Data were analyzed by homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig.6 Dronedarone inhibited ESCC growth in vivo via the CDK4/CDK6-RB1 axis. (A) The images of xenografts in three cases (EG20 with 8 mice/group, LEG34 with 11 mice/group, and LEG110 with 10 mice/group) after dronedarone treatment were displayed. (B, C) Growth curves (B) and tumor weights (C) of xenografts in three cases after dronedarone treatment. The data were analyzed using homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Immunohistochemistry was used to analyze Ki67 levels in tumor tissues EG20, LEG34, and LEG110. Data were analyzed using homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 100 µm. (E) Expression levels of p-RB1 S807, p-RB1 S811, and p-RB1 T826 in tumor tissues from case LEG34 were analyzed by IHC. Data were analyzed using homogeneity of variance and ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 100 µm.
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