Repurposed benzydamine targeting CDK2 suppresses the growth of esophageal squamous cell carcinoma

doi:10.1007/s11684-022-0956-8

Front. Med.

2023, Vol. 17

Issue (2) : 290-303 https://doi.org/10.1007/s11684-022-0956-8

RESEARCH ARTICLE

Repurposed benzydamine targeting CDK2 suppresses the growth of esophageal squamous cell carcinoma

Yubing Zhou^1,², Xinyu He^1,², Yanan Jiang^1,^2,^3,⁴, Zitong Wang¹, Yin Yu^1,², Wenjie Wu^1,², Chenyang Zhang¹, Jincheng Li¹, Yaping Guo^1,³, Xinhuan Chen^1,³, Zhicai Liu^4,⁶, Jimin Zhao^1,^3,^4,⁵, Kangdong Liu^1,^2,^3,^4,⁵(

), Zigang Dong^1,^2,^3,⁵(

)

¹. The Pathophysiology Department, School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450000, China
². The China-US (Henan) Hormel Cancer Institute, Zhengzhou 450000, China
³. State Key Laboratory of Esophageal Cancer Prevention and Treatment, Zhengzhou 450000, China
⁴. Provincial Cooperative Innovation Center for Cancer Chemoprevention, Zhengzhou University, Zhengzhou 450000, China
⁵. Cancer Chemoprevention International Collaboration Laboratory, Zhengzhou 450000, China
⁶. Oncology Department, The Tumor Hospital of Linzhou City, Linzhou 456500, China

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Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death worldwide. It is urgent to develop new drugs to improve the prognosis of ESCC patients. Here, we found benzydamine, a locally acting non-steroidal anti-inflammatory drug, had potent cytotoxic effect on ESCC cells. Benzydamine could suppress ESCC proliferation in vivo and in vitro. In terms of mechanism, CDK2 was identified as a target of benzydamine by molecular docking, pull-down assay and in vitro kinase assay. Specifically, benzydamine inhibited the growth of ESCC cells by inhibiting CDK2 activity and affecting downstream phosphorylation of MCM2, c-Myc and Rb, resulting in cell cycle arrest. Our study illustrates that benzydamine inhibits the growth of ESCC cells by downregulating the CDK2 pathway.

Keywords benzydamine cyclin-dependent kinase 2 patient-derived xenograft esophageal squamous cell carcinoma

Corresponding Author(s): Kangdong Liu,Zigang Dong

Just Accepted Date: 02 November 2022 Online First Date: 27 December 2022 Issue Date: 26 May 2023

Cite this article:

Yubing Zhou,Xinyu He,Yanan Jiang, et al. Repurposed benzydamine targeting CDK2 suppresses the growth of esophageal squamous cell carcinoma[J]. Front. Med., 2023, 17(2): 290-303.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0956-8
https://academic.hep.com.cn/fmd/EN/Y2023/V17/I2/290

Fig.1 Benzydamine suppressed the anchorage-dependent and -independent growth of ESCC cells. (A) Effect of 20 FDA-approved drugs (No. 1?20) on cell proliferation. KYSE450 cells were treated with drugs (50 μM) for 48 h and the cell survival rate was measured. (B) Chemical structure of benzydamine. (C) KYSE150, KYSE450, and SHEE cells treated with benzydamine for 24 and 48 h. Cell viability was measured using the IN Cell Analyzer 6000 software. (D) Effect of benzydamine on the proliferation of SHEE cells and ESCC cells for 24, 48, 72, and 96 h. (E) ESCC cells treated with benzydamine for 2 weeks. Colony numbers were counted using the IN Cell Analyzer 6000 and Image J software. Quantitative analysis was on the right panel. (F) Clone formation assay and quantitative analysis were conducted on KYSE150 and KYSE450 cells. Cells were treated with different concentrations of benzydamine and then stained with crystal violet dye. The data in (D?F) were derived from three independent experiments and are presented as mean ± SD. Asterisks (*, **, ***) indicated a significant decrease (P < 0.05, P < 0.01, P < 0.001, respectively) by one-way ANOVA followed by multiple-comparison tests. Similar results were obtained in three independent experiments.

Fig.2 Phosphoproteomic profiles of KYSE150 cells after benzydamine treatment. (A)Volcano plot of differentially-expressed phosphorylation proteins, blue dots represented downregulated sites, whereas red dots represented upregulated sites. (B) Quantification of differentially-expressed proteins. (C) KEGG enrichment of downregulated phosphorylation sites (top five pathways). (D) Significantly changed phosphorylation sites involved in DNA replication and cell cycle signaling pathways were shown in heatmap. (E) Phosphorylation and expression levels of proteins were assessed by Western blotting. KYSE150 cells were treated with or without benzydamine for 24 h. (F) The schematic for analysis of multi-omics data and prediction of upstream protein kinases. The LC-MS/MS search and database research were first constructed for the proteome and phosphoproteome analyses. Swiss TargetPrediction and SwissDock were subsequently employed to predict upstream kinases. Spearman’s correlation analysis of the expression of CDK2 and MCM2 genes. Datasets used were comprised of mRNA-seq data from TCGA. Quantified protein and phosphorylation sites were subjected to the following criteria (P < 0.05, fold change > 1.5).

Fig.3 Benzydamine induced G1/S phase arrest and inhibited the DNA replication pathway. (A, B) The effects of benzydamine on cell cycle phase were demonstrated using KYSE150 and KYSE450 ESCC cells. Cells were treated with various concentrations of benzydamine (0, 2.5, 5, 10, or 20 μM) and incubated for 24 h (KYSE150) or 48 h (KYSE450) for cell cycle analysis by flow cytometry. Bar graph displayed the distribution of cell cycle in KYSE150 and KYSE450 ESCC cells. (C, D) Immunofluorescence staining of KYSE150 and KYSE450 cells with or without benzydamine (2.5, 5, 10, or 20 μM) treatment for 24 h. Cells were stained with DAPI (blue) and MCM2 S41 (green). Scale bars, 50 μm. (E, F) KYSE150 and KYSE450 cells were with benzydamine (0, 2.5, 5, 10, or 20 μM) and incubated for 24 h. The protein levels of MCM2 S41, c-Myc S62 and Rb T826 after benzydamine treatment. For the data in (A?D), asterisks (**P < 0.01, ***P < 0.001) indicated a significant difference between control and benzydamine-treated KYSE150 and KYSE450 cells. Data were displayed as mean ± SD by one way ANOVA followed by multiple comparisons.

Fig.4 Benzydamine directly bound to CDK2 and inhibited CDK2 kinase activity. (A) Model of benzydamine binding to CDK2 at the ATP binding site. (B, D) Active CDK2 (200 ng) and cell lysates of KYSE150 and KYSE450 were incubated with benzydamine-conjugated Sepharose 4B beads or with Sepharose 4B beads alone. Pull-down proteins were analyzed by Western blotting. (C) CDK2 (WT, F80A, D145A, F146A, D145A and D146A) proteins were incubated with benzydamine-conjugated Sepharose 4B beads or with Sepharose 4B beads alone. Pull-down proteins were analyzed by Western blotting. (E) The specificity of the binding between benzydamine and active CDK2 in the presence of ATP was evaluated. (F, G) Active CDK2 (500 ng) and various doses of benzydamine were incubated with MCM2 (1 μg) as a substrate at 30 °C for 30 min. The levels of p-Serine and MCM2 S41 were detected by Western blotting. CDK2 and MCM2 proteins were detected by Western blotting and Coomassie blue staining (the bottom picture).

Fig.5 Knockdown of CDK2 decreased the sensitivity of ESCC cells to benzydamine. (A) CDK2 was highly expressed in EC tumor tissues compared with normal tissues in TCGA database (P = 1.764 × 10⁻⁷). (B) The levels of MCM2 S41, c-Myc S62 and Rb T826 after CDK2 knockdown in KYSE150 and KYSE450 cells. (C?E) Knockdown CDK2 in KYSE150 and KYSE450 cells reduced the proliferation and colony formation ability of ESCC cells. Proliferation assay (C), plate cloning assay (D, E). (F, G) Knockdown CDK2 in KYSE150 and KYSE450 cells induced G1/S phase arrest. (H) Proliferation of KYSE150 and KYSE450 cells transfected with shMock or shCDK2, with or without treatment with various doses of benzydamine (0, 2.5, 5, 10, or 20 μM). Cell numbers were measured using the IN Cell Analyzer 6000 software. For (C?H), data were shown as the mean ± SD of triplicate values obtained from three independent experiments. Asterisks (*, **, ***) indicated a significant decrease (P < 0.05, P < 0.01, P < 0.001, respectively) by one-way ANOVA followed by multiple-comparison tests.

Fig.6 Benzydamine suppressed patient-derived xenograft tumor growth in vivo. (A) Tumor images in different groups of EG20, LEG34 and LEG110 cases after sacrifice. (B) The changes of average tumor volumes in different group of EG20, LEG34 and LEG110 cases after benzydamine treatment. (C) Tumor weight analysis in different groups of EG20, LEG34 and LEG110 cases after benzydamine treatment. (D) Immunohistochemistry analysis of Ki67, MCM2 S41 and CDK2 in cases EG20, LEG34, and LEG110 after benzydamine treatment. The positive cell rates were evaluated by Histo Quest-shortcut software. All data were shown as the mean ± SD. (E) Schematic illustrating the anti-tumor activity of benzydamine. Asterisks (*, **, ***) indicated a significant decrease (P < 0.05, P < 0.01, P < 0.001, respectively) by one-way ANOVA followed by multiple-comparison tests.

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