Gemcitabine Resistance Affected By Abnormal Expression Of ATF-2 In Human Pancreatic Adenocarcinoma Cells

Introduction: Pancreatic cancer is a lethal tumor,approximately 90% are pancreatic ductal adenocarcinoma(PDAC).In the United States,about 33000 people are diagnosed each year.The mortality rate remains higher in China,Europe and America.The occurrence,development,proliferation,apoptosis and invasion of PDAC are related to the activation of various oncogenic and inactivation of anti-oncogenic genes as well as the regulation of some growth factors.There is an evidence that the activated transcription factor(ATF)family can be activated into phosphorylated ATF-2.ATF-2transforms into homodimer or heterodimer,which can binds to specific DNA sequences,and regulates the expression of target genes.ATF-2 was reported in various tumor tissues,and played a key role in signaling pathways and tumor related factors.ATF-2 was described as an oncogenic agent in some tumors,but in others,it was characteristic of tumor suppression.Oncogenesis or anti-oncogenesis both existed in the progression,while the mechanism is related to its subcellular localization.Oncogenesis appears in nucleus,but the contrary in cytoplasm.Although there are numerous reports on the relationship between ATF-2 and other tumors,but the research of the relationship between ATF-2 and PDAC is rare.Gemcitabine is recognized as the first-line treatment of PDAC,it can inhibit the invasion of tumor cells.Epithelial-to-mesenchymal transition(EMT)is the procedure of epithelial cells differ into the phenotype of mesenchymal cell.An enormous number of studies suggested that EMT is related to the occurrence,development and invasion of tumor.The E-cadherin expression decreased while the Vimentin,Snail were over expressed.In the typical EMT model,cells showed strong invasiveness by losing of intercellular polarization and cell-cell interaction.Objective: To detect the expression of ATF-2 in PDAC and paracancerous tissue.To measure the expression of ATF-2 in PDAC cell line.Cell line with the highest expression level of ATF-2 was selected.On this basis,the cell models were constructed with different levels of ATF-2.The differences of gemcitabine resistance in the cell line for each expression level of ATF-2 were observed.The differences of invation in the cell line for each expression level of ATF-2 after adding gemcitabine.EMT model of PDAC cellswere constructed,and the changes of invasiveness of ATF-2 under EMT condition was observed.Methods: In this pre-experiment,we selected 42 cases of PDAC in the First Affiliated Hospital of China Medical University from January 2010 to December 2012.Different expression of ATF-2 protein and mRNA in cancer and paracancerous tissue were tested by western blot.If the different expression level of ATF-2 was confirmed,the PDAC cell lines(sw1990/capan-2/ Panc-1/BxPC-3)were cultured.And the expression levels of ATF-2 in the cell lines were obtained by Western blot andqRT-PCR.In the trial of human PDAC BxPC-3 expression to gemcitabine resistance in different ATF-2 levels,we selected one group from four cell lines(Panc-1/BxPC-3/sw1990/capan 2),which showed the highest level of BxPC-3 expression.The concentration of IC50 in BxPC-3 cells was measured by MTT maneuver.We constructed the BxPC-3model with low ATF-2 expression by using the transfection of siRNA-ATF-2 into BxPC-3 cells.Certain gemcitabine(10ug/ml)calculated by the different concentration of IC50 were added to BxPC-3 cells with normal and lower expression of ATF-2.The dynamic changes of BxPC-3 cells in each group were observed under the inverted microscope To verify the invasiveness in the procedure of EMT with different expression of ATF-2 in human PDAC cell lines,we selected Panc-1,the classic human PDAC cells.To construct Panc-1 cells with high expression of ATF-2 by transfection of PGEX-HA-ATF2 plasmid.TGF-β1 was added into Panc-1 cells with noramal ATF-2 expression to construct the EMT model of Panc-1 cell line.The expression levels of ATF-2,Snail and E-cadherin were detected by RT-PCR and Western blot maneuvers in each Panc-1 cell line.The difference of cell invasion ability was tested by the use of Transwell chamber invasion assay.Results: We confirmed the expression of ATF-2 in PDAC is higher than that in paracancerous tissues.The highest expression exists in the BxPC-3 cell lines.Gemcitabine can inhibit human PDAC cells,BxPC-3 transfected siRNA-ATF-2 are more sensitive to gemcitabine and less invasive.More invasivenss,higher level of Snail and lower level of E-cadherin were found in the Panc-1 cell line with high ATF-2concentration in the EMT condition..Conclusion: The expression of ATF-2 in PDAC is higher than that in paracancerous tissues.BxPC-3 cells with lower expression of ATF-2 have lower drug resistance to gemcitabine and invasiveness.More invasivenss were detected in the Panc-1 cell line with high ATF-2 in the EMT condition.