The Screening And Analysis Of The Protein Signatures Associated With Chemoresistance Of Gastic Cancer And The Effects Of PLK1Inhibitor On Gastric Cancer Cells

Objection:Identification of the protein signatures associated with signal pathway in differentchemosensitivity gastic cancer through Protein Pathway Array, establishes thechemoresistance predictive model of gastric cancerafter radical surgery and studies the effectsof PLK1inhibitor on gastic cancer cells.Methods:In the first part, Protein Pathway Array and286antibodies were used to assess theprotein expression of140clinical staging III of gastric cancer samples after D2radicalgastrectomy, including73chemoresistant tumors (disease-free survival <1year) and67chemosensitive tumors (disease-free survival>3years). The Significant Anaylisis ofMicroarray tool (SAM) analysis software and clustering and discriminant analysis were usedto screen and identify the differentially expressed proteins between chemosensitive andchemoresistant subsets of gastric cancerpatients, and then we established a chemoresistancepredictive model for patients after D2radical gastrectomy using the independentpredictivefactors which were filtered from differentially expressed proteins. We also used the IngenuityPathway Analysis (IPA) software to investigate the siginifigant proteins’ expression inchemoresistance signaling network. In the second part,we determine the effects of BI2536ongastric cancer cell viabilityco-treated with Cispaltin through MTT and Colony formationassay, and then we determine the effect of BI2536on cell invasion ability co-treated withCisplatinthrough Transwell assay. To determine whether the growth inhibitionof gastriccancer cells by BI2536was associated with cell cycle arrest or apoptosis, and the influence ofBI2536on gastric cancer cells when treated together with Cisplatin,flow cytometry wereperformed. We further examined the expression of cellular proteins of the cell cycleregulating components through Westen-blot,we also performed protomic-wide expressionanalysis using Protein Pathway Array and Ingenuity Pathway Analysis(IPA)software to investigate differences in protein expression between BI2536treated and untreatedSGC-7901/DDP.Findings:A total of23proteins were differentially expressed between chemosensitive andchemoresitant tumor tissues by SAM analysis. Of them,18were selected as the bestclassifiers by SVM and weresupposed as a robust set of proteins for classification. Thesensitivity and specificity were94.8%and93%, respectively. Chemosensitive gastric cancersand chemoresistant gastric cancers could beseparated into two categories by hierarchicalclustering analysis and the23proteins could alsobe separated into the upregulated proteinsand downregulated proteins.Based on thedifferentially expressed proteins, IPA system wasproposed to analysis the chemosensitivity signalingtransduction network of gastric cancer.The results showed that theseproteins were closely related to cellular growth and proliferation,apoptosis, cell cycle, invasion, DNA methylation and DNA damage and repair, and alsoinvolved in ERK/MAPK,Wnt/-catenin,PI3K/AKT,apoptosis and p53signaling pathways.Our study also showed that six proteins (PLK1DACH1E-cadherin FKHR ADHandERCC1) were independent risk factors of chemoresistanceof gastric cancer. Using this6proteins, we established a chemoresistance predictive model of gastric cancer, the rate ofcorrect classification, sensitivity and specificity to predict the chemosensitivity were87.9%89.6%and86.3%, respectively.As the expression of PLK1showed significantly differencebetween the gastric cancer cell lines SGC-7901and SGC-7901/DDP, in the second part westudiedthe functionof PLK1inhibitor-BI2536and found it could improve the inhibiting abilityof Cisplatin when co-treated to gastric cancer cells; especially improve the chemosensitivityof SGC-7901/DDP to Cisplatin. We also observed that BI2536induced G2/M cell cycle arrestand cell apoptosis, possibly by regulating G2/M checkpoint regulating components such asp-Cdc2, p-Cdc25Cand Cyclin B1. After treated with BI2536on cell lineSGC-7901/DDP,Protein Pathway Array analysis found68proteins were differentially expressed whencompared with control. IPA analysis identified that BI2536induces a potent alteration of theproteins involved in many cell functions and signal pathways, such as cell death, celldevelopment, tumorgenesis, cell cycle, DNA duplication/recombination/repair and cellularmovment, also involved in Wnt/-catenin and MEK-ERK-RSK1singnaling transduction networks.Conclusion:As the genetic and epigenetic variance in gastric cancer patients, the chemoresistance indifferent patients are various, even showed same histopathology and same clinical stage.These differentiations are related with a broad dysregulation of signaling proteins intumorgenesis and these proteins could be selected as clinically useful biomarkers. Specially,based on the expression of PLK1, DACH1, E-cadherin, FKHR, ADH and ERCC1,weestablished the chemoresistance predictive model of gastric cancer which is an effectivedetection system for predicting the chemosensitivity of gastric cancer patients after D2gastrectomy.PLK1inhibitor-BI2536improved cell growth and invasion inhibitingability ofCisplatin in gastric cancer cell lines and induced G2/M cell cycle arrest, possibly byregulating G2/M checkpoint regulating components. BI2536could induce a potent alterationof the proteins involved in many cell functions and signal pathways.