Experiment On Effect Of Silencing AKT2 Expression By RNA Interference On The Chemosensitivity Of Pancreatic Cancer To Gemcitabine

Partâ… Effects of the Expression of AKT2 on Sensitivity of Pancreatic Cancer Cell LinePanc-1 to GemcitabineObjectiveTo explore the effects of the expression of AKT2 on sensitivity of pancreatic cancercell line Panc-1 to gemcitabine.MethodsIn vitro the expression vector of pAKT2-siRNA was constructed and transferred intoPanc-1 cell by lipofection. The mRNA and protein expression of AKT2 was detected byusing RT-PCR and Western blot. The changes of gemcitabine sensitivity after siRNA wereexamined by MTT assay.ResultsThe mRNA and protein levels of AKT2 in Panc-1 cells were significantly decreasedafter transfection, and the median inhibition concentration of gemcitabine against Panc-1cells was reduced from 1.96±0.22μg / ml to 0.24±0.03μg / ml. The sensitivity of Panc-1 cells to gemcitabine was increased significantly after AKT2 siRNA.ConclusionThe sensitivity of Panc-1 cells to gemcitabine could be enhanced by AKT2-siRNA.Partâ…¡The Effect of Sensitivity of Human Pancreatic Cancer Cell Xenograft in Nude Mice toGemcitabine by RNA Interference Suppressing the Expression of AKT2ObjectiveTo study the effect of sensitivity of human pancreatic cancer cell xenograft in nudemice to gemcitabine by RNA interference suppressing the expression of AKT2.MethodsThe human pancreatic cancer cell implanted tumor model in the nude mice wasestablished. By abdominal cavity administration and intratumoral injection ,the micebearing tumor were treated with gemcitabine in combination with vector ofpAKT2-siRNA. Tumor growth of tumor tissues were observed,the AKT2 mRNAexpression levels by RT-PCR method and, AKT2 protein expression in tumor tissues wasdetected by immunohistochemistry and tumor apoptosis by Tdt-mediated dUTP nick endlabeling(TUNEL).ResultsIn chemotherapy+AKT2-siRNA group the expression of mRNA and protein wassignificantly lower than in control group, chemotherapy group and chemotherapy+ blankplasmid group. The tumor weight and tumor volume in chemotherapy+AKT2-siRNA groupwere significantly lower than those in other three groups. The inhibition rate and apoptosisin chemotherapy+AKT2-siRNA group were significantly higher than those in other three groups.Conclusionsensitivity of human pancreatic cancer cell to gemcitabine could be significantlyimproved by RNA interference suppressing the expression of AKT2.Partâ…¢Development and Characterization of Gemcitabine-resistant Pancreatic Cancer CellLine Pane-1ObjectiveTo establish the resistance to gemcitabine in human pancreatic cancer cell line Panc-1and describe the characteristics of its resistant variant.MethodsResistance of gemcitabine was established by exposing Panc-1 cells to increasingconcentration of gemcitabine, which was designated as Panc1-Gem. The IC₅₀ and resistanceindex (RI) were tested by MTT assay and colony formation test. The growth curve and cellcycle of Panc1 and Panc1-Gem were compared.ResultsThe IC₅₀ increased from 1.96±0.22μg/ml in Pancl to 239.82±35.47μg/ml inPancl-Gem as tested by MTT assay at 72h exposure, the RI was 122. 36(P＜0.05). The RIof colony formation test was 118.93. Double time of Panc1 and Panc1-Gem were 27.1h and35.2h respectively, as evaluated by the growth curve.ConclusionPanc 1-Gem, the gemcitabine resistant phenotype, is stable and suitable for the study ofgemcitabine resistance in pancreatic cancer. Partâ…£The Role of AKT2 in Resistance to Gemcitabine in Pancreatic Cancer CellObjectiveTo investigate the role of AKT2, multidrugresistance-1(mdr-1) and deoxycytidinekinase(dCK) in Gemcitabine (GEM) chemoresistance in the pancreatic cancer cell linePanc-1.MethodsThe GEM-resistant pancreatic cancer cell line model was constructed using a stepwiseincrease in concentration gradient of Gemcitabine. RNA interference of AKT2 was used toreverse the drug resistance in gemcitabine-resistant pancreatic cancer cell line(Panel-GEM), RT-PCR, Western Blot and MTT assay were employed to evaluate therelationship between AKT2 and mdr-1,dCK and the effect of reversing drug resistance byRNA interference of AKT2.ResultsAs compared to the parental Panc-1 cells, Panc1-GEM cells showed increasedexpression of mdr-1 revealed by Western Blot and RT-PCR, while Panc1-GEM cells alsoshowed decreased expression of dCK revealed by Western Blot and RT-PCR. After theAKT2 expression of Panc1-GEM had been interferenced by siRNA, Panc1-GEM showeddecreased expression of mdr-1 revealed by Western Blot and RT-PCR, while Panc1-GEMcells also showed increased expression of dCK revealed by Western Blot and RT-PCR. TheIC50 of Pancl-GEM to gemcitabine was 239.82±35.47μg/ml and RI of Panc1-GEM was 121. 94. After the AKT2 expression of Panc1-GEM had been interferenced by siRNA, theIC50 of Panc1-GEM to gemcitabine was 113.45±21.89μg/ml and RI of Pancl-GEM was57.88.ConclusionThe resistance to gemcitabine of pancreatic cancer cell line is due to decreasedexpression of dCK and increased expression of mdr-1. The AKT2 participates in theregulation of the expression of mdr-1 and dCK, most likely contributing to the GEMchemoresistance in the Panc-1 pancreatic cancer cell line.