CB-5083 Combined With Cisplatin Enhances The Anti-tumor Effect Of Esophageal Squamous Cell Carcinoma By Regulating Endoplasmic Reticulum Stress

Esophageal cancer,a complex malignant tumor of digestive tract,is the sixth leading cause of cancer death in the world.There are two pathological types of esophageal cancer: esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).ESCC is the main type of esophageal cancer in China.It has a high incidence,diverse etiology,poor prognosis and prone to metastasis.Most of the first-line chemotherapy for ESCC is platinum-based or fluorouracil-based drugs.However,the toxicity of traditional chemotherapy drugs leads to seriously side effects,which strongly affect the quality of life of patients.Meanwhile,drug resistance often occurs after chemotherapy.Once treatment failure,the median survival time of patients is less than 1 year,and the prognosis is very poor.The exact efficacy of targeted drug therapy has been well verified in some digestive tract tumors(such as colorectal cancer and gastric cancer),but there is no effective molecular targeted drug for ESCC,and related studies are still under the exploratory stage.Therefore,it is hopeful to provide new ideas for the treatment of ESCC by finding new effective targeted drugs,exploring underlying signaling pathways,and analyzing the potential value of combination of targeted therapy and traditional chemotherapeutic drugs.p97,a member of the adenosine triphosphate superfamily,is widely present in cells and is involved in a variety of important cellular biological processes,including protein homeostasis,autophagy,chromatin remodeling,transcriptional activity regulation,and immune signaling.Studies have shown that p97 can participate in the regulation of intracellular protein quality by assisting in the degradation of a largenumber of misfolded proteins in cells,ensuring the survival of cells under protein toxic stress.As a key component of protein quantity control,p97 has become a research hotspot of anti-cancer targeted therapy.Studies have shown that p97 is highly expressed in many cancers,such as non-small cell lung cancer,pancreatic cancer,prostate cancer,breast cancer and other tumors.Moreover,its expression is closely related to tumor invasion,histological type,pathological stage and so on.Inhibition of p97 protein activity can significantly inhibit the proliferation of various tumors such as non-small cell lung cancer,colorectal cancer,and plasma cell carcinoma.In recent years,breakthroughs have been made in the screening of p97 inhibitors,and inhibitors such as DBeQ,NMS-873 and CB-5083 have been reported.However,the role of p97 in ESCC has not attracted attention,and there are few reports.Accordingly,the aim of this study was to investigate the anti-tumor effect of p97 inhibitor CB-5083 alone or in combination with cisplatin on ESCC from in vitro and in vivo.Moreover,combination with clinical tissue analysis,we aim to demonstrate the potential value of p97 as a therapeutic target for ESCC,which will provide new ideas and scientific basis for the chemotherapy of ESCC.Methods:Part I: Effect of CB-5083 on phenotype and endoplasmic reticulum stress pathway of ESCC1.Expression of p97 in ESCC tissues and cellsThe clinically resected ESCC tissues and adjacent normal tissues were collected.qRT-PCR and immunohistochemistry were used to detect the expression of p97 mRNA and protein in clinical specimens.Then,qRT-PCR and Western blot were used to detect p97 mRNA and protein expression in esophageal squamous carcinoma cells.2.Influence of p97 inhibitor on phenotype and endoplasmic reticulum stress pathway of ESCCCCK-8 method was used to screen the most effective p97 inhibitor among three p97 inhibitors(DBeQ 、 NMS-873 and CB-5083),and the optimal time and concentration of CB-5083 on ESCC cells were determined.The effect of CB-5083 on the proliferation of esophageal squamous carcinoma cells was detected by EdU fluorescence method.The effect of CB-5083 on the apoptosis of ESCC was detected by FITC/PI double staining.The effect of CB-5083 on the cell cycle of ESCC was detected by flow cytometry.The effect of CB-5083 on the migration of esophageal squamous carcinoma cells was detected by Transwell.The effect of CB-5083 on the expression of the proteins related to endoplasmic reticulum stress in ESCC was detected by Western blot.Part II: Effect of CB-5083 combined with cisplatin on phenotype of ESCCThe effect of CB-5083 combined with cisplatin on the proliferation of esophageal squamous carcinoma cells was detected by EdU fluorescence method.The effect of combination therapy on the apoptosis of ESCC was detected by FITC/PI double staining.Flow cytometry was used to detect the cell cycle of ESCC.The migration of esophageal squamous carcinoma cells was detected by Transwell.And the expression of the proteins related to endoplasmic reticulum stress in ESCC was detected by Western blot.Part III: In vivo study on the anti-tumor effect of CB-5083 combined with cisplatin on ESCCNude mice were randomly divided into four groups,then Eca109 cells were injected subcutaneously to construct a nude mouse xenograft model.When the tumor grew to a suitable volume,the tumor-bearing nude mice were randomly divided into four groups(NC control group,CB-5083 group,cisplatin group,CB-5083 + cisplatin group).The long and short diameters of xenografts and the body weight of nude mice were measured every other day,then the tumor growth curve was drawn.After4 weeks,the nude mice were sacrificed,the transplanted tumors were weighed and the volume and inhibition rate of the transplanted tumors were calculated.The apoptosis of the transplanted tumor tissues was detected by TUNEL method,and the expression of Bcl-2 and Bax protein in the transplanted tumor tissues was detected by immunofluorescence.ResultsPart I: Effect of CB-5083 on phenotype and endoplasmic reticulum stress pathway of ESCC1.Expression of p97 in ESCC tissues and cellsAmong 32 samples of ESCC detected by qRT-PCR,22 samples of esophageal squamous cell carcinoma showed significantly higher expression of p97 Mrna than those of adjacent normal tissues.The immunohistochemical results of another 30 samples of ESCC showed that the overexpression of p97 was positively correlated with the pathological grade of ESCC.In addition,compared with normal esophageal epithelial cells Het-1A,TE13,TE1,Eca109,Kyse70 and EC9706 cells had higher p97 expression,and p97 expression in EC1 cell expression was not significantly different with Het-1A.2.Influence of p97 inhibitor on phenotype and endoplasmic reticulum stress pathway of ESCCThe CCK-8 assay showed that CB-5083 inhibited the growth of esophageal squamous carcinoma cells most significantly among the p97 inhibitors DBeQ,NMS-873 and CB-5083.Three concentrations of CB-5083 were used to treat esophageal squamous carcinoma cells Eca109,Kyse70 and TE1,respectively.It was found that with the increase of drug concentration,the proliferation and migration of esophageal squamous carcinoma cells was significantly inhibited,the apoptosis rate gradually increased,and the cell cycle was rested at G0/G1 phase.In addition,after CB-5083 treatment,proteins related to endoplasmic reticulum stress p-EIF2α,PERK,Bip,and Chop were significantly up-regulated in esophageal squamous carcinoma cells.Part II: Effect of CB-5083 combined with cisplatin on phenotype of ESCCCCK-8 results showed that CB-5083 combined with cisplatin had a synergistic effect on the inhibition of ESCC growth.Compared with CB-5083 or cisplatin alone,the combination group showed more significant effect on the proliferation,migration,apoptosis and the cell cycle of ESCC.In addition,changes of endoplasmic reticulum stress-related protein expression were more significant in the combination group.Part III: In vivo study on the anti-tumor effect of CB-5083 combined with cisplatin on ESCCThe in vivo results showed that CB-5083 could inhibit the growth of xenografts in nude mice compared with the control group.Compared with the CB-5083 orcisplatin group,the volume of transplanted tumors in the combination group was significantly smaller,and the apoptosis-related protein Bcl-2/Bax ratio was downregulated more significantly;and the number of apoptotic cells was more.Conclusion1.Compared with normal tissues adjacent to the cancer,the expression of p97 mRNA and protein was up-regulated in ESCC tissues,and the expression of p97 protein was positively correlated with the pathological grade of ESCC.2.p97 inhibitor CB-5083 can significantly inhibit the proliferation and migration of esophageal squamous carcinoma cells;promote the apoptosis of esophageal squamous carcinoma cells;and repress the cell cycle in G0/G1 phase.CB-5083 can significantly upregulate the expression of proteins related to endoplasmic reticulum stress.The results showed that CB-5083 can inhibit the proliferation of esophageal squamous carcinoma cells and promote its apoptosis by promoting endoplasmic reticulum stress pathway.3.Compared with the single-drug group,the combination of CB-5083 and cisplatin can significantly inhibit the proliferation and migration of ESCC Eca109 cells,promote the apoptosis of esophageal squamous carcinoma cells,and lead to cell cycle disorder.4.In vivo studies showed that the transplanted tumor of nude mice in CB-5083 treatment group was slightly smaller than that of the control group,and the growth inhibition effect of CB-5083 and cisplatin combination group was more significant.The results showed that CB-5083 can exert synergistic anti-tumor effect with cisplatin.