Cloperastine Inhibits Esophageal Squamous Cell Carcinoma Proliferation In Vivo And In Vitro By Suppressing Mitochondrial Oxidative Phosphorylation

Background and purposeEsophageal squamous cell carcinoma(ESCC)is a major type of esophageal cancer.The prognosis of patients with ESCC remains poor because of the high morbidity and mortality of the disease.One strategy for drug discovery for ESCC treatment or prevention is screening FDA-approved drugs.In the present study,we found that the antitussive agent cloperastine can inhibit the proliferation of ESCC cells.However,the underlying mechanism was unclear.To determine the mechanism of this inhibitory effect,we performed proteomic analysis using KYSE150 cells treated with cloperastine and DMSO.The results identified several down-regulated signaling pathways included those of three key proteins:NADH dehydrogenase [ubiquinone] 1alpha subcomplex 1(NDUFA1),NADH ubiquinone oxidoreductase subunit S5(NDUFS5),and cytochrome C oxidase subunit 6B1(COX6B1)involved in oxidative phosphorylation.Meanwhile,we observed that oxidative phosphorylation in mitochondria was inhibited by the drug.Importantly,cloperastine suppressed ESCC growth in a xenograft mouse model in vivo.Our findings revealed that cloperastine inhibits the proliferation of ESCC in vivo and in vitro by suppressing mitochondrial oxidative phosphorylation.Methods1.Explore the inhibitory effect of cloperastine hydrochloride on the proliferation of esophageal squamous cell carcinoma in vitroCytotoxicity experiments was used to screen drugs that inhibit esophageal squamous cell carcinoma KYSE450 cells from the FDA-approved drug library;cytotoxicity test was used to detect the toxicity of cloperastine hydrochloride on esophageal squamous cell carcinoma cell lines;use cell proliferation test to detect the ability of cloperastine hydrochloride to inhibit the proliferation of esophageal squamous cell carcinoma cells;the soft agar clone formation experiment was used to detect the ability of cloperastine hydrochloride to inhibit the anchorage-independent growth of esophageal squamous cell carcinoma cells;the plate cloning experiment was used to detect the ability of cloperastine hydrochloride to inhibit the anchorage-dependent growth of esophageal squamous cell carcinoma cells.2.Proteomics explores the mechanism of cloperastine hydrochloride on esophageal squamous cell carcinomaProteomics was used to explore the changes of signal pathway and key molecules induced by cloperastine hydrochloride;combined with bioinformatics and gene enrichment analysis to explore the mechanism of cloperastine hydrochloride inhibiting the proliferation of esophageal squamous cell carcinoma;western blot was used to verify the authenticity of proteomics results.3.Explore the inhibitory of cloperastine hydrochloride on esophageal squamous cell carcinoma through the oxidative phosphorylation processJC-1 probe and DCFH-DA probe was used to observe the effect of cloperastine hydrochloride on the changes of mitochondrial membrane potential and the changes of reactive oxygen species in esophageal squamous cell carcinoma cell lines;western blot was used to observe the effect of different concentrations of cloperastine hydrochloride on the oxidative phosphorylation of key proteins at the protein level;RT-PCR was used to observe the effect of cloperastine hydrochloride on the oxidative phosphorylation of key proteins at the m RNA level.4.Explore the relationship between key oxidative phosphorylation proteins and the prognosis of patients with esophageal squamous cell carcinomaTCGA database was used to analyze the expression of NDUFA1,NDUFS5 and COX6B1 in esophageal carcinoma and pan carcinoma and their relationship with prognosis;immunotissue microarray analysis of NDUFA1 expression in esophageal squamous cell carcinoma and its prognostic correlation.5.Explore the inhibitory effect of consumption of NDUFA1 on the proliferation of esophageal squamous cell carcinomaConstruct an esophageal squamous cell carcinoma cell line that stably knocks down NDUFA1,and use cell proliferation experiments to detect the inhibitory effect of NDUFA1 depletion on the ability to inhibit the proliferation of esophageal squamous cell carcinoma cells;soft agar clone formation assay was used to detect the inhibitory effect of NDUFA1 consumption on anchorage-independent growth of esophageal squamous cell carcinoma cells;plate cloning experiment was used to detect the inhibitory effect of NDUFA1 consumption on the anchorage-dependent growth of esophageal squamous cell carcinoma cells.6.Explore the inhibitory effect of cloperastine hydrochloride on patient-derived esophageal squamous cell carcinoma xenograft model(PDX model)in vivoEstablish a PDX mouse model of esophageal squamous cell carcinoma,and give the mice an oral route of administration to observe the anti-tumor effect of cloperastine hydrochloride in vivo;observe the protein expression levels of NDUFA1,NDUFS5,and COX6B1 in tumor tissues treated with cloperastine hydrochloride by immunohistochemical staining.Results1.Cloperastine inhibits ESCC cell proliferationDrug screening revealed that cloperastine hydrochloride has the effect of inhibiting the proliferation of esophageal squamous cell carcinoma cells;with the increase in the concentration of cloperastine hydrochloride,its inhibitory effect on the proliferation,anchorage-independent and dependent growth ability and clonal formation ability of esophageal cancer cells is significantly increased and is concentration-dependent2.Cloperastine suppresses NADH dehydrogenase(ubiquinone)1 alpha subcomplex 1(NDUFA1),NADH ubiquinone oxidoreductase subunit S5(NDUFS5),and cytochrome C oxidase subunit 6B1(COX6B1)expressionThe proteomics results combined with the biometric analysis showed that compared with the control group,there were 6472 differential proteins after drug treatment,of which 5155 had quantitative information;GO function enrichment and KEGG pathway analysis were performed for differential proteins greater than 1.5times;cloperastine hydrochloride inhibits Oxidative phosphorylation,Alzheimer disease,Alzheimer disease,Retrograde endocannabinoid signaling protein levels of NDUFA1,NDUFS5,COX6B1;western Blot verified that NDUFA1,NDUFS5,COX6B1 protein levels were down-regulated after esophageal cancer cell lines were treated with cloperastine hydrochloride.3.Cloperastine suppresses oxidative phosphorylationDifferent concentrations of cloperastine hydrochloride decrease mitochondrial membrane potential and increase ROS in esophageal squamous cell carcinoma cell line;western blot showed that the protein levels of NDUFA1、NDUFS5、COX6B1 were down regulated in a concentration dependent manner;RT-PCR showed that the m RNA levels of NDUFA1、NDUFS5、COX6B1 were down regulated.4.The protein level of NDUFA1 is associated with the prognosis of patients with ESCCThe expressions of NDUFA1 、 NDUFS5 、 COX6B1 were up-regulated in esophageal cancer,and the expression of ndufa1 was related to the prognosis of patients;NDUFA1 is highly expressed in esophageal carcinoma,and the higher the expression of NDUFA1,the worse the prognosis.5.Depletion of NDUFA1 inhibits ESCC cell proliferationKnockdown of NDUFA1 in ESCC cells showed that the proliferation,anchorage independent and dependent growth ability and clone formation ability of ESCC cells were inhibited,and the drug sensitivity of ESCC cells was decreased.It indicated that NDUFA1 was the key protein of cloperastine hydrochloride on ESCC cells.6.Cloperastine inhibits patient-derived xenograft(PDX)tumor growth in vivoIn PDX model mice,after treated with cloperastine hydrochloride,the tumor volume and weight of the drug treatment group were significantly smaller than those of the control group;immunohistochemical results showed that the expression of Ki67,NDUFA1 、 NDUFS5 、 COX6B1 in tumor tissue of mice were significantly decreased.ConclusionCloperastine hydrochloride inhibits the proliferation of esophageal squamous cell carcinoma in vitro and in vivo by inhibiting oxidative phosphorylation.