Expression Of Copper Transporter Ctr1 In Esophageal Squamous Cell Carcinoma And Its Effect On The Sensitivity Of Cisplatin

Background and ObjectiveEsophageal cancer(EC)is one of the most common digestive tract tumors and the sixth leading cause of death worldwide.Esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC)are the most common histological types of EC,and ESCC is a master histologic type in China.At present,the clinical treatment of ESCC patients mainly includes surgery,chemotherapy and radiotherapy.However,the prognosis and 5-year survival rate of ESCC patients remain still very poor.For advanced and metastatic patients with ESCC,it is often treated by local chemotherapy and systemic chemotherapy.Cisplatin,as one of the most common tumor chemotherapy drug,can be used in the treatment of many malignant tumors such as testicular cancer,colon cancer,lung cancer,breast cancer,etc.However,inherent or acquired resistance of tumor itself will lead to therapeutic failure during therapy process.Therefore,it is very important to elucidate the molecular mechanism of drug resistance to further improve the chemotherapeutic efficacy in clinic.The major cause of drug resistance to cisplatin includes decreased drug accumulation,improved DNA repair efficiency,cisplatin failure triggered by small molecule or other proteins,etc.Among these,reduced cellular drug accumulation is the most cause of cisplatin resistance.Copper transporter 1(Ctr1)is an ATP-independent transmembrane transporter,which is mainly localized in cell membrane.Ctr1 is the main channel that copper ions enter cells,and it is also the main way for cells to uptake cisplatin,which controls the sensitivity of cells to cisplatin.Some studies demonstrated that Ctr1 overexpression increased the sensitivity of tumor cells on cisplatin,conversely,Ctr1 knowdown led to the decrease of cisplation uptake,further promotes drug resistance of cells.These data suggest that Ctr1 level may play a pivotal role in evaluating cisplatin efficacy,and thus may be an important molecular marker of assessment of cisplatin chemotherapy.To explore the role of Ctr1 in occurrence and development of ESCC and its correlation with cisplatin sensitivity,and two parts were performed in this study.In part I,Ctr1 expression was investigated using The Cancer Genome Atlas(TCGA),and semi-quantitative RT-PCR,in situ hybridization and immunohistochemistry were employed to detect the expressions of Ctr1 mRNA and protein,and the association of Ctr1 expression with clinicopathological features of ESCC patients was analyzed by SPSS21.0 software.The purpose of this study was to explore the role of Ctr1 in the occurrence and development of ESCC.In part II,Ctr1 expression were upregulated or downregulated,and the role of its expression change in mediation of the sensitivity of ESCC cells on cisplatin were investigated,which will provide the experimental evidence for ancillary evaluation of cisplatin chemotherapeutic efficacy of ESCC patients using Ctr1 gene.Part ? Expression of copper transporter Ctr1 in human esophageal squamous cell carcinoma tissues and its correlations withclinicopathological featuresMethods1.The expression profile of Ctr1 gene and its associations with clinicopathological features and prognosis were obtained from the dataset of esophageal cancer patients in TCGA database.2.The expression of Ctr1 mRNA was detected by semi-quantitative RT-PCR in10 cases of ESCC tissues and corresponding para-carcinoma tissues.In situ hybridization and immunohistochemistry were used to analyze the expressions of Ctr1 mRNA and protein in 108 cases of ESCC tissues and paired para-carcinoma tissues.The correlations of Ctr1 mRNA and protein expression with clinicopathological features of ESCC patients were investigated using SPSS21.0software.3.Statistical assay:The experimental data were analyzed by SPSS21.0 software.The result of semi-quantitative RT-PCR was investigated using t test and the results of in situ hybridization and immunohistochemistry was examined by chi-square test(?~2).The relationship between Ctr1 expression and prognosis of the patients with EC was evaluated by Log-rank test.A P value less than 0.05 was considered to be statistically significant.Results1.The results of TCGA database showed that the expression of Ctr1 in EC tissues was significantly higher than that in normal tissues,and the differences were statistical significance(P<0.05).There was no statistical difference between the survival time of EC patients with high Ctr1 level and medium/low Ctr1 level(P>0.05).2.The results of semi-quantitative RT-PCR showed that the relative expression level of Ctr1 mRNA in 10 cases of ESCC tissues was significantly higher than that in paired para-carcinoma tissues(P<0.05).The results of in situ hybridization and immunohistochemistry showed that the positive rates of Ctr1 mRNA and protein in ESCC tissues(65.7%and 60.2%,respectively)were markedly higher than those in para-carcinoma tissues(32.4%and 28.7%,respectively),and the differences were statistical significance(P<0.0001).3.The results The Ctr1 expression was not related to patients'age and gender(P>0.05),but tightly associated with histological grade,invasive depth,TNM staging and lymph node metastasis(P<0.05).Part ? The role of copper transporter Ctr1 in mediating the sensitivity of ESCC cells on cisplatinMethods1.The expressions of Ctr1 mRNA and protein were detected by qRT-PCR and Western blot in ESCC cells Kyse70,EC9706,Eca109,Kyse450,EC1,TE1 and normal esophageal epithelial cell Het-1A.2.The expression of Ctr1 protein was investigated by Western blot 48h after treatment with different concentration cisplatin(0.5?g/ml,2.0?g/ml and 4.0?g/ml)in ESCC cells Kyse70,EC9706,Eca109 and Kyse450.3.The Ctr1 overexpression vector pcDNA3.1-Ctr1 was constructed.Control siRNA(si-Con)and Ctr1 siRNA(si-Ctr1)as well as pcDNA3.1 empty vector and pcDNA3.1-Ctr1 were utilized to transfect into Kyse70,EC9706,Eca109 and Kyse450,and the expressions of Ctr1 mRNA and protein were detected using qRT-PCR and Western blot.4.The effects of si-Ctr1 or pcDNA3.1-Ctr1 combined with cisplatin on the survival rate of ESCC cells Kyse70,EC9706,Eca109 and Kyse450 were detected by CCK-8 assay.5.The effects of si-Ctr1 or pcDNA3.1-Ctr1 combined with cisplatin on cell apoptosis of ESCC cells Kyse70,EC9706,Eca109 and Kyse450 were investigated using FITC/PI double staining.6.The effects of si-Ctr1 or pcDNA3.1-Ctr1 combined with cisplatin on the migration and invasion of ESCC cells Kyse70,EC9706,Eca109 and Kyse450 were analyzed using Transwell chamber.7.Statistical assay:Experimental data were performed by SPSS21.0 software.One-way ANOVA was employed for comparison of multiple samples and t test was utilized for comparison of two groups.A P value less than 0.05 was considered to be statistically significant.Results1.The expressions of Ctr1 mRNA and protein in ESCC cells Kyse70,EC9706,Eca109 and Kyse450 were significantly higher than those in normal esophageal epithelial cells Het-1A(P<0.01).2.The expression of Ctr1 protein was significantly downregulated in ESCC cells treated with different concentration cisplatin,which exhibited in a dose-dependent manner.3.Compared with si-Con group,the expressions of Ctr1 mRNA and protein in si-Ctr1 group was markedly downregulated in ESCC cells Kyse70,EC9706,Eca109and Kyse450(P<0.01).4.The results of CCK-8 showed that survival ratio in si-Ctr1 combined with cisplatin group was dramatically higher than that in si-Con combined with cisplatin group(P<0.01).5.Cell apoptosis assay demonstrated that apoptotic ratio in si-Ctr1 combined with cisplatin group was dramatically lower than that in si-Con combined with cisplatin group(P<0.01).6.The results of Transwell revealed that migratory and invasive cell numbers in si-Ctr1 combined with cisplatin group was dramatically higher than that in si-Con combined with cisplatin group(P<0.01).7.Compared with pcDNA3.1 combined with cisplatin group,cell survival ratio was decreased,apoptotic ratio was increased and migratory and invasive cell numbers was obviously reduced in pcDNA3.1-Ctr1 group in ESCC cells Kyse70,EC9706,Eca109 and Kyse450,and the differences exhibited statistical difference(P<0.01).Conclusions1.Copper transporter Ctr1 displayed high expression in ESCC tissues,and its high expression was tightly correlated with histological grade,invasive depth,TNM staging and lymph node metastasis,but not related to the patients'age and gender,suggesting that Ctr1 may be implicated in the occurrence and development of ESCC.2.The Ctr1 downregulation combined with cisplatin promotes cell proliferation,suppresses cell apoptosis and increases cell migration and invasion abilities in ESCC cells.3.The Ctr1 upregulation combined with cisplatin suppresses cell proliferation,induces cell apoptosis and inhibits cell migration and invasion abilities in ESCC cells.