Domperidone inhibits cell proliferation via targeting MEK and CDK4 in esophageal squamous cell carcinoma

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of digestive system tumor related death in the world. Unfortunately, effective chemopreventive agent is lack for patients with ESCC in clinical practice, which leads to the extremely high mortality rate.

Methods: A library of prescribed drugs was screened for finding critical anti-tumor properties in ESCC cells. The phosphoproteomics, kinase array, pulldown assay and drug affinity responsive target stabilization assay (DARTS) were applied to explore mechanisms and searched for synergistic targets. Established models of PDX in mice were used to determine the therapeutic effect of domperidone.

Results: After screening a library of prescribed drugs, we discovered that domperidone has anti-tumor properties. Domperidone, acting as a gastroprokinetic agent, has been widely used in clinic for gastrointestinal motility disorders. Despite limited research, there are indications that domperidone may have anti-tumor properties. In this study, we determined that domperidone significantly inhibited ESCC proliferation in vitro and in vivo. We employed phosphoproteomics to reveal p-ERK, and p-SMAD3 down-regulation upon domperidone treatment. Then, the results of kinase assay and pulldown assay further validated that domperidone directly combined with MEK1/2 and CDK4, leading to the inhibition of their kinase activity. Furthermore, our results revealed that MEK/ERK and CDK4/SMAD3 signal pathway were major pathways in domperidone against ESCC.

Conclusion: Collectively, these findings suggest that domperidone serves as an effective "multi-target" inhibitor of MEK1/2 and CDK4, offering potential benefits for the chemoprevention of ESCC.

Keywords: CDK4; Domperidone; ESCC; MEK1/2.

Fig. 1

Domperidone effectively inhibits proliferation of ESCC cells (A) Domperidone was identified through soft agar assay from the clinical drug library. (B) The chemical structure of Domperidone is depicted. (C) KYSE150, KYSE450 and SHEE cells were treated with different doses of domperidone (0, 5, 10, 25, 50 100 and 200 µM) for 48 h, and IC 50 calculated by GraphPad Prism 8. (D) The human ESCC cell lines, KYSE150 (left panel) and KYSE450 (right panel), were exposed to various concentrations of domperidone or DMSO as control group for 24 h, 48 h, 72 and 96 h. The cell numbers were calculated and analyzed by IN Cell Analyzer 6000. (E) Anchorage-independent cell growth was assessed using a soft agar assay with different concentrations of domperidone (0, 10, 20, 40 and 60 µM). After incubation for 10 days, colonies were captured and analyzed using In Cell Analyze 6000. Results from three independent experiments were presented by comparing domperidone treatment group with control group. (F) Representative images obtained from the clone formation assay are shown. (G) KYSE150 (Left) and KYSE450 (right) were treated with 40 µM domperidone for 24 h, and then cell proliferation was detected by EdU staining. Data were showed as mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Phosphoproteomics analysis of domperidone inducing molecular changes. (A) Schematic representation of phosphorylated proteomics analysis in KYSE150 cells treated with 40 µM domperidone after 24 h. (B) Quantitative proteins were presented as a volcano plot, where down-regulated proteins are represented by green plots and up-regulated proteins by red plots. (C) KEGG pathway enrichment analysis was performed to identify down-regulated proteins in domperidone treatment group compared to control group. (D) A network illustrating major was presented, with circles representing down-regulated proteins after domperidone treatment. (E) Western blot results confirmed down regulation of phosphorylation sites, including p-SMAD3^T8, p-ERK2^T185/Y187 and p-JUND^S100

Fig. 3

Domperidone directly binds to MEK1/2 and CDK4 (A) Docking model analysis illustrated the interaction between domperidone and MEK1/2 or CDK4. (B) KYSE150 or (C) KYSE450 cell lysate was incubated with domperidone-conjugated Sepharose 4B beads, MEK1/2 and CDK4 were pulled down through visualization using Western blot. Immunoblot analysis of pronase-digested KYSE150 (D) and KYSE450 (E) cell lysate showed the presence of MEK1/2 and CDK4. (F) Pull down assay was used to evaluate the binding affinity between domperidone and mutant MEK1. (G) Pull down assay was used to evaluate the binding affinity between domperidone and mutant MEK2. (H) Pull down assay was used to evaluate the binding affinity between domperidone and mutation CDK4.

Fig. 4

Domperidone inhibits MEK/ERK pathway and CDK4/SMAD3 pathway (A) MEK1 and MEK2 (B) kinase activity was assessed by an in vitro kinase assay using p-ERK^T185/Y187 antibody. Active MEK1 or MEK2 incubated with inactive ERK2 in kinase reaction buffer together with different dose of domperidone and then followed by Western blot analysis. (C) KYSE150 cells were co-transfected with CDK4-Flag and CylinD1-HA, and then treated with 40 µM or 60 µM domperidone for 24 h. Cell lysates were immunoblotted with anti-p-Smad^T8 antibody. Mutation version MEK1 (D) or MEK2 (E) incubated with inactive ERK2 in kinase reaction buffer, followed by Western blot analysis. (F) KYSE150 cells were co-transfected with either CDK4-Flag or CDK4 K35A-Flag along with CylinD1-HA. Cell lysates were immunoblotted with anti-p-SMAD^T8 antibody. (G) KYSE150 and KYSE450 cells were treated with different concentration of domperidone after 24 h. Protein levels of P-ERK, p-MEK1/2, T-ERK, T-MEK1/2, p-SMAD3, T-SMAD3, CDK4, P21 and c-myc were analyzed by Western blot. (H) Pull down assay was used to evaluate the binding affinity between domperidone and c-myc or P21. (I) KYSE150 cells and CDK4 knock out cells were treated with 40 µM domperidone for 24 h, and then Western blot was performed. (J) KYSE150 was incubated with 40 µM domperidone or domperidone and SMAD3 inhibitor (E)-SIS3 3µM for 24 h simultaneously.

Fig. 5

Reduction of cell proliferation by domperidone is dependent on MEK1/2 and CDK4 (A) Cell lines with knock down of MEK1, MEK2 or CDK4 were established, and the expression levels of MEK1, MEK2 or CDK4 were determined by Western blot. MTT assay was used to evaluate cell proliferation (B, C, D). The MEK1 (E), MEK2 (F) or CDK4 (G) knock down cell lines were treated with domperidone for 48 h, and the inhibitory rates were calculated. Data were showed as mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Domperidone suppresses ESCC tumor growth in PDX model The tumor tissues obtained from patients were subcutaneously injected into SCID mice, and then mice were given with domperidone (5 mg/kg, 20 mg/kg) or vehicle six times per week. (A, E) The mice were sacrificed and tumors mass were isolated from mice. (C, D, G, H) Tumor volume was monitored throughout the study period. Data are showed as mean ± S.D. **, P < 0.01 indicates a significant decrease between tumors from domperidone treatment and vehicle group. (B, F) Upon sacrifice of the mice, the weight of dissected tumor tissue was recorded. Data are showed as mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (I) The expression of p-ERK and p-SMAD3 were examined in PDX model tumor tissue using IHC analysis (400×) (left panel). Quantification analysis of IHC staining results (Right panel). The positive rate was measured by Image J pro and represented as mean ± S.D. *, P < 0.05, **, P < 0.01. (J) Graphical conclusion for the findings of this work: domperidone bindings with MEK1/2 and CDK4, which disturbs their kinase activity in turn leads to suppress MEK/ERK and CDK4/SMAD3 pathway, and consequently inhibits ESCC proliferation.