Molecular Mechanism Of Peptidyl-prolyl Cis/trans Isomerase On Esophageal Squamous Cell Carcinoma

Esophageal carcinoma is one of the most frequently diagnosed cancers. Esophageal squamous cell cancer (ESCC) is the main histological type of esophageal cancer in China. Although the therapy is improved with the development of surgery,radiotherapy and chemotherapy, the survival rate is still 25%～40%. Therefore how to find a new anticancer target and improve the efficiency of chemtherapy become more and more urgent.Recent report shows that Pinl is overexpressed in many cancers and can act as prognosis index. c-Jun,P53,NF-κB,CyclinD1 and (3-catenin corresponding with esophageal carcinoma were regulated by Pin1. Pin1 play important roles in cell differentiation, vascularization and tumor metastasis. Inhibition of Pinl induced cell apoptosis. Post phosphorylation regulation by Pin1 provide a new way for anti-cancer therapy. Pinl knock-down mice can grow normally, which showed inhibitor of Pinl perhaps has low non-specificity toxicity.We studied the expression level of Pinl in tissue and cell and test the cell biological alteration after using siRNA and Juglone to inhibit Pinl expression level. By Mammalian Two-Hybrid System we get a new transcription factor ATF1. Our study provide a platform to study Pinl function in esophageal squamous cell carcinoma; the increase of sensitivity of EC1 to CDDP by inhibiting Pin1 provide new insight to the therapy of esophageal squamous cell carcinoma.PartⅠExpression of Pin1 in esophageal squamous cell carcinoma tissue and cellsMethods1. Investigating the expression of Pinl in esophageal carcinoma tissue by immunity histochemistry.2. Investigating the expression of Pinl in esophageal carcinoma cells by cell histochemistry and Western-blot.ResultsPin1 is overexpressed in esophageal carcinoma tissue and cell lines.PartⅡScreening stable knockdown cell line of Pinl and biological characterization on esophageal squamous cell carcinoma cells lineMethods1. After transfection of plasmid pmU6-Pinl into EC1 cells, the cell lines with stable expression of Pinl siRNA were selected under G418 and identified by Western-blot. Stable knockdown cell lines were generated.2. Cells proliferation was detected by MTT assay and inducing cell apoptosis were detected by flow cytometry.3. Sensitivity to chemotherapy was detected by MTT assay and flow cytometry. Results1. Immunoblotting showed that the EC1/siPinl cell line could effectively inhibit expression of Pin1 protein.2. Knockdown Pinl inhibited cells proliferation and induced cell apoptosis.3. Inhibition of Pinl can significantly increase the sensitivity of EC1 to cisplatin (CDDP).PartⅢInhibitory effects of the inhibitor of Pinl (Juglone)on proliferation of esophageal squamous cell carcinoma cell lineMethodsAfter the secondary culture of EC1 cell, inhibition of EC1 cell growth induced by Juglone at different concentrations was detected by methylthiazolyl tetrazolium (MTT). The cell cycle distribution and cell apoptosis were measured by flow cytometry(FCM).ResultsJuglone treatment decreased the proliferation capacity of cervical cancer cells in a dose-and time-dependent manner and cause cell cycle arrest at G2/M phase.PartⅣFunctional study of ATF1 interacting with Pinl in esophageal squamous cell carcinomaMethods1. Screening transcription factor which can interact with Pinl by Mammalian Two-Hybrid System.2. Confirming the interaction between ATF1 and Pin1 by GST pull down. 3. Effect of ATF1 on cell proliferation by flow cytometry.ResultsATF1 can interact with Pinl, overexpression of ATF1 blocked cell cycles in the S phase. Conclusion1. Pinl is overexpressed in esophageal carcinoma tissue and cells.2. Establish stable Pinl knock down cell line.3. Inhibit the expression of Pinl by si-RNA or Juglone can significantly inhibit the proliferation of Esophageal Squamous Cell Carcinoma' Cell(EC1) and induce the apoptosis of EC1 cell in vitro and increase the sensitivity of EC 1 to cisplatin (CDDP).4. ATF1 can interact with Pin1, overexpression of ATF1 cause S phase cell cycle arrest.