Double Inhibition Of NF-κB And XIAP Via RNAi Affects The Sensitivity Of Pancreatic Cancer Cells To Gemcitabine And Molecular Mechanism

Objective:Pancreatic cancer is the fourth leading cause of cancer-related mortality in Western countries. The medial survival rate for patients with pancreatic cancer is approximately five months and the five-year survival rate is approximately5%for all stages of the disease. Chemotherapy is the main treatment regimen for pancreatic cancer. Gemcitabine monotherapy is currently the first-line therapy recommended by the National Comprehensive Cancer Network (NCCN) panel. However, the majority of patients are resistant to gemcitabine. One of the potential mechanisms involved is insensitivity to gemcitabine-induced apoptosis. Nuclear factor-kB (NF-kB) and X-linked inhibitor of apoptosis protein (XIAP) are two important factors in the apoptotic pathway which render them promising targets in reversing the chemoresistance of pancreatic cancer cells. But targeting NF-kB or XIAP alone is not so sufficient as expected to improve the chemosensitivity of pancreatic cancer.NF-KB is a ubiquitous transcript factor with the ability of modulating XIAP expression,correspondingly, XIAP is able to upregulate NF-kB in endothelia cells and help them with apoptosis evasiono We hypothesized that NF-kB in conjunction with XIAP may confer the chemoresistance of pancreatic cancer cells; simultaneously targeting NF-kB and XIAP by RNAi may enhance chemosusceptibility to gemcitabine.Methods:①Mia PaCa-1and Panc-1cells were treated with varying concentrations (0.01-100μM) of gemcitabine for72h. The number of viabilities was detected by the CCK-8assay.The survival curve was made.Basal and gemctabine induced NF-kB DNA binding ability and XIAP protein were detected by EMS A or Western blot.②P65-shRNA、 XIAP-shRNA plasmid and negative control plasmid were designed and synthetised.The transfection effectivity in both Mia PaCa-2and Panc-1cells were detected. The inhibition rates of p65-shRNA、XIAP-shRNA plasmid were tested.Real time RT-PCR was used to detect p65and XIAP mRNA.Western blot was used to detect p65protein and XIAP protein.③Mia PaCa-2and Panc-1cells were treated as follows:negative plasmid、gemcitabine、65-shRNA plasmid、65-shRNA plasmid together with gemcitabine,then apoptosis rates were detected.The cells were treated as before,NF-kB DNA binding ability and XIAP protein were tested.④Mia PaCa-2and Panc-1cells were treated as follows:negative plasmid、gemcitabine、XIAP-shRNA plasmid、XIAP-shRNA plasmid together with gemcitabine,then apoptosis rates were detected.The cells were treated as before, and XIAP protein and NF-kB DNA binding ability were tested.Western blot was used to detect IKKβ、IkBα protein.⑤Mia PaCa-2and Panc-1cells were treated as follows:gemcitabine、 XIAP-shRNA plasmid together with gemcitabine、p65-shRNA plasmid together with gemcitabine、 XIAP-shRNA plasmid and p65-shRNA plasmid together with gemctabine,apopotosis rates were detected by Annexin V.Cell were treated as before,NF-KB DNA binding ability and XIAP protein were detected.Results:①LD50of gemcitabine for Mia PaCa-2was about1μM and over50μM for Panc-1.Mia PaCa-2was relative more sensitive than Panc-1to gemcitabine and had a much lower basal level of NF-kB and XIAP compared to PANC-1cells.Gemcitabine could induce NF-kB activity and XIAP protein level in both pancreatic cancer cells.②XIAP-shRNA、p65-shRNA plasmid and negative plasmid were effective in transfecting MiaPaCa-2and Panc-lcells,transfection rates were more than80%.Both XIAP-shRNA and p65-shRNA plasmid were effectively in inhibiting corresponding mRNA and protein level in the two pancreatic cancer cells.The inhibiton rates were about80%in mRNA (P<0.05) and higher than80%in protein(P<0.05),while the negative plasmid had no affection in mRNA or protein level((P<0.05).③P65-shRNA plasmid together with gemcitabine could increase apoptosis rates in both Mia PaCa-2and Panc-1cells compared to gemcatabine(P<0.05), but the apoptosis rates were lower than expected which were only (19.7±1.9)%in Mia PaCa-2and (14.9±1.6)%in Panc-1. P65-shRNA plasmid mediated RNAi effectively inhibited the gemcitabine induced NF-kB DNA binding ability in both Mia PaCa-2and Panc-1cells accompanied by downregulation of XIAP(P<0.05).④IAP-shRNA plasmid together with gemcitabine could increase apoptosis rates in both Mia PaCa-2and Panc-lcells compared to gemcatabine(P<0.05), but the apoptosis rates were lower than expected which were only (18.6±2.0)%in Mia PaCa-2and (11.6±1.3)%in Panc-1cells. XIAP-shRNA plasmid mediated RNAi effectively inhibited the gemcitabine induced XIAP protein in both Mia PaCa-2and Panc-1cells accompanied by downregulation of NF-kB DNA binding ability and IKKβ、IkBa protein level(P<0.05).⑤In both Mia PaCa-2and Panc-1cells,XIAP-shRNA and p65-shRNA plasmid together with gemcitabine could remarkablely increase apoptosis rates compared with either XIAP-shRNA plasmid together with gemcitabine or p65-shRNA plasmid together with gemcitabine(P<0.05) and reached as high as (43.7±4.4)%in Mia PaCa-2and (39.2±3.1)%in Panc-lcells.Correspodingly,NF-kB DNA binding ability and XIAP protein were remarkablely reduced(P<0.05)Conclusion:①NF-KB and XIAP together confer chemoresisitance of pancreatic cancer cells.②ouble inhibition of XIAP and NF-kB via RNAi can enhance the chemosensitivity of pancreatic cancer cells to gemcitabine.