The Relationship Between The Cell Inhibitor Of Apoptosis Protein 2 Expression And Gemcitabine Chemotherapy In Pancreatic Carcinoma

ObjectiveThis study was to observe the expression of C-IAP2 in pancreatic carcinoma and pancreatic cancer cells and to analyze the relationship between its expression and pathological grade, clinical stage,and to further explore the relationship between the expression of C-IAP2 and the pro-apoptotic role of gemcitabine chemotherapy in pancreatic cancer cell,and to investigate the relationship between C-IAP2 expression and gemcitabine chemotherapy resistance in pancreatic cancer. Methods1.32 cases of pancreatic cancer specimens and 18 cases of adjacent tissues from August 2005 to March 2008 in Union Hospital of Fujian Medical University were examined by immunohistochemistry and the relationship of C-IAP2 expression to the clinical stage and pathological grade was analyzed.Using Western blot to further observe the expression of C-IAP2 in the poorly differentiated PANC-1, in the moderately differentiation of AsPC-1, well-differentiated BxPC-3 pancreatic cancer cell lines,the relations between the expression of C-IAP2 levels and the pathological class of pancreatic cancer cells was analyzed.2.To study Gemcitabine in vitro inducing human pancreatic cancer cell PANC-1 apoptosis and its molecular mechanism,cell proliferation activity was analyzed by MTT colorimetric assay;cell morphology was observed by transmission electron microscopy;cell apoptosis rate was observed by PI staining flow cytometry;C-IAP2 mRNA expression was analyzed by RT-PCR;C-IAP2, Smac, Bcl-2 and Bax protein expression was analyzed by Western blot;Caspase-3,Caspase-9 activity were detected by spectrophotometry.3.A gemcitabine-resistant pancreatic cancer cell line(PANC-1)was obtained b y gradient increase of concentration.It's biological properties and activity of C-IAP2 were detected.Results1.C-IAP2 were expressed in pancreatic carcinoma (32/32,100%), adjacent normal pancreatic tissue (8/18, 44.4%).The C-IAP2 expression level of pancreatic cancer significantly stronger than the normal pancreatic tissue adjacent to cancer.The differences of C-IAP2 expression level at different degree of differentiation of tumor tissue were significant (P<0.05).C-IAP2 was highly expressed in poorly differentiated pancreatic cancer cells PANC-1,and expressed lowly in the moderate differentiation and well-differentiated pancreatic cancer cells AsPC-1 and BxPc-3.2.MTT colorimetric assay showed:Gemcitabine inhibited pancreatic cancer cell line PANC-1 proliferation with a dose-dependent manner;its value of IC50 5.79ug/ml. Gemcitabine inducing pancreatic cancer cell line PANC-1 apoptosis was confirmed by FCM analysis of PI staining and transmission electron microscopy.RT-PCR analysis showed that gemcitabine significantly reduced expression of C-IAP2 mRNA in PANC-1 cells,.Western blot analysis showed that gemcitabine significantly reduced C-IAP2,Bcl-2 protein expression level,increased Smac,Bax protein expression level in pancreatic cancer cells PANC-1.Spectrophotometric analysis showed that gemcitabine significantly increased Caspase-3, Caspase-9 activity in pancreatic cancer cells PANC-1.3.Stable drug resistant PANC-1/Gem cell strain was established by culturing with gemcitabine for 3 months.The morphology and growth characteristics of the cell strain was changed remarkably.The cells shrinked and became rounder,granular substance increased;and the doubling-time was prolonged.Resistance of the cell line to gemcitabine,fluorouracil,adriamycin,and mytomycin sinnificantly increased.The C-IAP2 protein expression levels of the drug-resistant cells were significantly increased.Conclusions 1.The expression level of C-IAP2 as well as the positive rate in pancreatic carcinoma were significantly higher than the adjacent normal pancreatic tissue,high expression of C-IAP2 may play a role to some extent in pancreatic cancer development.There was significant difference of C-IAP2 expression in different differentiation of pancreatic cancer.Expression of C-IAP2 may predict the degree of malignancy in pancreatic cancer tissues.2.The apoptosis of human pancreatic cancer PANC-1 cell could be induced by gemcitabine.Gemcitabine pro-apoptotic role may be due to decrease of C-IAP2 and Bcl-2 expression levels,increase of Smac,Bax expression levels,and activation of Caspase-3, Caspase-9.3.Stable drug resistant PANC-1/Gem cell strain could be obtained by intermittent incremental concentrations of gemcitabine.PANC-1/Gem have multiple drug resistance situation.C-IAP2 may play a role to some extent in the process of drug resistance.