The Mechanism Of Benzydamine Inhibiting The Growth Of Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer（EC）is histopathologically classified into esophageal squamous cell carcinoma（ESCC）and esophageal adenocarcinoma.It is estimated that 456,000patients with ESCC exist worldwide,with approximately 90%of them being found in Eastern and Central Asia.Esophageal squamous cell carcinoma（ESCC）is among the leading causes of cancer-related deaths worldwide owing to late detection and low survival rate.Currently,a combination therapy of surgical resection,radiotherapy,and chemotherapy is the primary approach for the treatment of EC.However,the overall 5-year survival rate for advanced stage ESCC remains lower than 15%,and the recurrence rate is still very high after treatment.To date,the availability of targeted therapies for ESCC is lacking.Therefore,it is urgent to identify effective drugs for the treatment of ESCC.Currently,chemoprevention is a new strategy which is widely used to reduce the onset of cancer and the relapse rate after treatment.Over the past few years,numerous drugs approved by the U.S.Food and Drug Administration（FDA）have been considered to be chemopreventive agents owing to their safety and pharmacodynamic characteristics.Among them,several non-steroidal anti-inflammatory drugs（NSAIDs）such as aspirin and ibuprofen have been widely reported in cancer chemoprevention.By screening FDA-approved drugs,we found that benzydamine,a NSAID,could suppress the proliferation of ESCC cells.It has been shown to possess local anesthetic and analgesic properties.However,its anti-tumor activity and underlying molecular mechanisms have not yet been elucidated.In this study,we investigated the inhibitory effect of benzydamine on the proliferation of ESCC in vivo and in vitro and found its potential targets and molecular mechanisms.Methods:1.Effect of benzydamine on ESCC cell proliferation.To detect the cytotoxicity of benzydamine on SHEE cells and ESCC cells,KYSE150 and KYSE450 cells by cytotoxicity assay.To evaluate the inhibitory effect of benzydamine on the proliferation of ESCC cells by cell proliferation assay.To investigate the effect of benzydamine on the clone formation ability of KYSE150 and KYSE450 cells by soft agar assay and plate colony assay.2.Phosphorylation profiles revealed the anti-tumor mechanism of benzydamine.To detect the changes of proteins and phosphorylation sites after benzydamine treatment by phosphoproteomic analysis.And combined with bioinformatics analysis to find the inhibitory mechanism of benzydamine on the proliferation of ESCC.To detect the total protein level of CDK2,MCM2 and the phosphorylation level of MCM2^S41by Western blotting assay.3.Benzydamine-induced G1/S phase arrest and inhibited the DNA replication pathway.Cell cycle assay:KYSE150 and KYSE450 cells were treated with various concentrations of benzydamine（0,2.5,5,10 and 20μM）for 24 h or 48 h,and the effect of benzydamine on cell cycle of ESCC cells was detected by flow cytometry.To verify the immunofluorescence intensity of MCM2^S41and the total immunofluorescence intensity of CDK2 and MCM2^S41after benzydamine treatment by immunofluorescence assay.To detect the total protein and phosphorylation levels of MCM2,c-Myc,and Rb by Western blotting assay.The concentrations of the protein of each group were quantified by the BCA protein kit（Beyotime,China）.A same amount of protein of each group were fractionated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes.4.Benzydamine bound directly to CDK2 and inhibited CDK2 activity.To explore the binding mechanism of CDK2-benzydamine interaction by molecular docking.Pull down assay:Sepharose 4B beads bound to benzydamine were incubated with active CDK2,KYSE150 and KYSE450 cell lysate,then the binding between CDK2 kinase and benzydamine was detected by Western blotting.To verify the effect of benzydamine on the phosphorylation of MCM2 by CDK2kinase was detected in vitro by kinase assay.5.Knockdown of CDK2 decreased the sensitivity of ESCC cells to benzydamine.Construct the knock-down CDK2 cells lines:KYSE150 and KYSE450 cells were infected with lentiviral particles.The transduction efficiency was analyzed by Western blotting.The CDK2-knockdown cells treated with or without benzydamine were examined by cell proliferation assay and colony formation assay to verify the function of CDK2 on ESCC cell lines and the effect of benzydamine on ESCC cell proliferation.6.Benzydamine suppressed patient-derived esophageal xenograft tumor growth in vivo.To verify the effect of benzydamine on the tumor growth on ESCC in vivo by patient-derived xenografts models.To determine the expression of Ki67 and the phosphorylation level of MCM2^S41in tumor tissues of mice treated with benzydamine by immunohistochemical（IHC）staining.Results1.Effect of benzydamine on ESCC cell proliferation.Benzydamine inhibited the survival of KYSE150 and KYSE450 cells but didn’t have obvious cytotoxic effect on SHEE cells.With the dose increasing,the inhibitory effect of benzydamine on cell proliferation of KYSE150 and KYSE450 cells was enhanced.Benzydamine inhibited the clone formation ability of ESCC cells,KYSE150 and KYSE450 cells,in a dose-dependent manner.2.Phosphorylation profiles revealed the anti-tumor mechanism of benzydamine.Phosphoproteomic analysis suggested five KEGG pathways were downregulated in KYSE150 cells after benzydamine treatment,that is,RNA transport,DNA replication,spliceosome,ferroptosis,and protein processing in the endoplasmic reticulum.Among these pathways,the phosphorylation level of MCM2^S41in DNA replication pathway was downregulated obviously.Western blotting assay indicated that benzydamine affect the phosphorylation of MCM2^S41in esophageal squamous cell carcinoma cells but didn’t affect the expression of MCM2 and CDK2.3.Benzydamine-induced G1/S phase arrest and inhibited the DNA replication pathway.Immunofluorescence assay showed the colocalization between p-MCM2（Ser41）and CDK2.Benzydamine decreased the fluorescence intensity of MCM2^S41and the total fluorescence intensity of CDK2 and MCM2^S41in KYSE150 and KYSE450 cells.Cell cycle assay showed that benzydamine induced G1/S arrest in KYSE150 and KYSE450 cells.Western blotting assay indicated that benzydamine affect the phosphorylation of MCM2^S41,c-Myc ^S62and Rb ^T826in esophageal squamous cell carcinoma cells.With the dose increasing,the effect was advanced.4.Benzydamine bound directly to CDK2 and inhibited CDK2 activity.Pull down assay showed that benzydamine bound with CDK2 in vitro and in vivo.In addition,benzydamine competed with ATP for binding at the ATP-binding site of CDK2 in vitro and inhibited the kinase activity of CDK2.Benzydamine inhibited the phosphorylation of MCM2 by CDK2 kinase in vitro through inhibiting the kinase activity.5.Knockdown of CDK2 decreased the sensitivity of ESCC cells to benzydamine.CDK2 knockdown suppressed the proliferation and colony formation of KYSE150 and KYSE450 cells and decreased the sensitivity of ESCC cells to benzydamine.Benzydamine suppressed patient-derived esophageal xenograft tumor growth in vivo.Benzydamine suppressed the tumor growth on ESCC in vivo and had no obvious toxicity to mice.Immunohistochemical staining showed that benzydamine decreased the expression of Ki67 and the phosphorylation level of MCM2^S41in tumor tissues of mice treated with benzydamine.ConclusionBenzydamine directly binds with CDK2 and inhibits its kinase activity,down-regulating the phosphorylation levels of MCM2,c-Myc and Rb,induces cell cycle arrest,and exerts the inhibitory effect on ESCC proliferation in vivo and in vitro.This study provides a new strategy for cancer chemoprevention of esophageal squamous cell carcinoma.