Study On The Therapeutic Effect Of ZD55-lipocalin 2 For Pancreatic Cancer

Part One Reconstruction and identification of the replication-defective /replication-competent virus expressing lipocalin 2 geneObjective To reconstruct replication-selective oncolytic adenovirus and replication-defective adenovirus,which can both express lipocalin 2 gene.Methods Lipocalin 2 gene was cloned into pCA13 plasmid to reconstruct pCA13-1ipocalin 2.PCA13-1ipocalin 2 was digested with BglⅡrestriction enzyme to obtain the expression cassette including CMV promoter and Lipocalin 2' CDS.This cassette was subcloned into pZD55 plasmid to produce pZD55-1ipocalin 2.PZD55-1ipocalin 2 and pBHGE3 plasmids were cotransfected and recombinated in 293 cells to obtain oncolytic adenovirus ZD55-1ipocalin 2.PCA13-1ipocalin 2 and pBHGE3 plasmids were cotransfected and recombinated in 293 cells to Obtain replication-defective adenovirus Ad-lipocalin 2.Results ZD55 lipocalin-2 retains the E1A gene sequence with E1B55 missing,and Ad-lipocalin-2had no E1A sequence.Lipocalin 2 gene was amplificated by PCR from the corresponding viral DNA.The novel adenovirus can express Lipocalin 2 proteins in PANC-1 cells by wester blot.Conclusions ZD55-1ipocalin 2 and Ad-lipocalin 2 had been successfully constructed, which would provide a platform for the further research on pancreatic cancer.Part Two ZD55-1ipocalin 2 can induce apoptosis and inhibit the growth of pancreatic cancer cells in vitroObjective To study the anti-pancreatic cancer effects and mechanisms of ZD55-lipocalin 2.Methods Human pancreatic cancer cell line PANC-1 ce11s were treated with the different virus of different titer. The proliferation inhibition was examined by MTT assay.Morphological examination,Annexin V and PI staining with flow cytometry were used to detect the apoptosis.The wester-blot method was used to detect the expression of apoptosis-related gene caspase-3 and PARP to study the mechanisms of ZD55-lipocalin-2-induced apoptosis in pancreatic cancer.Results The anti-cancer effects of ZD55-lipocalin 2 are better than that of the single virus(ZD55) or of the gene therapy alone(Ad-lipocalin 2), whose anti-cancer effects are time-dependent and dose-dependent. ZD55-lipocalin 2 can induce apoptosis of pancreatic cancer cells by activating Caspase-3.Caspase-3′s activation induced by ZD55-lipocalin 2 was also time-dependent and dose-dependent.Conclusions The anti-cancer effects of ZD55-lipocalin 2 were performed through caspase-dependent apoptosis pathway in pancreatic cancer cells.Part Three Study on the anti-migratory and anti-invasive effects of Lipocalin 2 in human pancreatic carcinoma cell in vitroObjective To investigate the anti-migratory and anti-invasive effects of lipocalin 2 in human pancreatic carcinoma cells.Methods Human pancreatic cancer cell line PANC-1 cells were treated with the Ad-lipocalin 2 of different titer.The ability of movement was examined by migration assay.The ability of invasion was performed by matrigel invasion assay.Results The higher was Ad-lipocalin 2 infection titer,the fewer would be PANC-1 cells through the basement membrane of Transwell chambers with(or without) coated matrigel within 48 hours. This Phenomenon had been shown in migration assay and matrigel invasion assay.Conclusions Lipocalin 2 protein exerts markedly anti-migratory and anti-invasive effects on pancreatic cancer cells. Part Four Study on anti-tumor effects of ZD55-1ipocalin 2 on the transplantation tumor models of the human pancreatic cancer in nude miceObjective To investigate anti-tumor effects of ZD55-lipocalin 2 on the transplantation tumor models of human pancreatic cancer in nude mice.Methods Transplantation tumor models of human pancreatic cancer were established on nude mice.Thirty models were randomly divided into 3 groups(group PBS,group ZD55-EGFP and group ZD55-lipocalin 2,n=10 in each group).Viruses were injected intratumorally into the 2 groups at doses of 1×109 PFU/day.PBS group was injected with equal volume of PBS as above.The tumor volume was examined and the survival time in each group was also observed.Apoptosis in tumor tissue was examined by TUNEL staining.Results The tumor volume in each group(group ZD55-EGFP or group ZD55-lipocalin 2)was reduced significantly(compared with group PBS,P＜0.05),and group ZD55-lipocalin 2 was reduced significantly(compared with group ZD55-EGFP, P＜0.05).The survival time prolonged in group ZD55-EGFP and group ZD55-lipocalin 2(compared with group PBS,P＜0.05);The apoptosis index in group ZD55-lipocalin 2 was significantly higher than that of other groups.Conclusions ZD55-lipocalin 2 has significant anti-tumor effects on the transplantation tumor model of the human pancreatic cancer in nude mice.Induction of apoptosis of tumor cells was one of the anti-tumor mechanisms.