The Expremental Study Of Gene Therapy For Pancreatic Cancer By Recombinant Adenovirus Harboring Human PUMA

The incidence of pancreatic cancer is increasing,so far neither an early diagnosis nor a therapeutic strategy for advanced lesions has been developed.It is an urgent mission for both the clinicians and the scientists who are challenging pancreatic cancer to find a breakthrough using new technologies.Apoptosis, also called programmed cell death, is a genetically encoded program that disposes of unwanted cells. Disruptions of this pathway have been implicated as a cause of cancer. Therefore, genetic restoration of the apoptotic pathway or introduction of pro-apoptotic molecules is an attractive approach for treating tumors, including malignant pancreatic cancer. Recently, p53 upregulated modulator of apoptosis (PUMA) was identified as a p53-inducible pro-apoptotic molecule,. PUMA shares homology with Bcl-2 family proteins within a short stretch of amino acids termed the BH3 domain, a region that allows interaction among the family members. Proteins with similar homology, called BH3-only proteins, constitute the third subgroup of the Bcl-2 family and include Bad, Bid, Bik, and Bim .These proteins play an important role in apoptosis induction.It has been reported PUMA is lost or lower expression in many cancer cells,which is significantly related to tumorgenesis.The therapeutic possibility of PUMA as a gene target in pancreatic cancer was evaluated.Pant one The expression and biological function of PUMA in pancreatic carcinomaOBJECTIVE: To investigate the expression of PUMA in pancreatic cancers(PC)tissue and its correlations to clinicopathologic features and carcinogenesisMETHODS:The expression of PUMA ,P53and bcl-2 in 60 specimens of PC were assessed by immunohistochemical staining Envision method ,so was PUMA in 19 speciments of no-cancer pancreatic tissues,PUMA gene was assessed by RT-PCR and Western blot method in 5 cases of newly resected PC tissues and normal PC.Apoptosis index(AI) was determinded with TUNEL method; PUMA correlations to p53,bcl-2,AI and clinicopathologic factors were analyzed .RESULTS: The positive rates of PUMA was significantly lower in PC 30%(18/60)than in no-cancer pancreatic tissues90%(17/19()p<0.01), The results of RT-PCR and Western blot verified the results of PUMA expression by IP analysis.PUMA expression in the tumor was correlated with the tumor size (p<0.05) and lymph node (P<0.05) and distant metastasis (P<0.05). No relationship to any other clinical or pathological features were found. The AI was 17.63%±6.27%, 13.44%±5.86% in cancer tissues of positive and negative expression of PUMA, respectively (P < 0.05). the AI in cancer tissues of negative expression of PUMA was lower than that of in normal pancreatic tissues 16.12 %±5.72 %,( p<0.05 ) .The positive rate of p53 and bcl-2 in PC were 46.7%(28/60) and 41.7%(25/60).Spearman rank correlation coefficient test revealed a significant inverse association for PUMA and bcl-2 , PUMA and p53 expression (P<0.05, respectively)CONCLUSION: The expression of PUMA is lost or lower in pancreatic cancer, which may be involved in the oncogenesis development and lymph node and distant metastasis,PUMA may be as a new target for the therapy of pancreatic cancer.Pant two Relationship between coxsackie adenovirus receptor expression levels and the ad5 transduction eficiency in pancreatic cancer0bjective : To investigate the relationship between coxsackie adenovirus receptor(CAR)expression and the eficiency of adenovirus gene transfer,in an effort to provide evidence for the clinical application of adenovirus-related Biopreparations.Methods:The pancreatic cancer cell lines(AsPC-1,CaPan-1,BxPC-3,Panc-1) were infected with Ad-GFP(100 MOI) labeled by immunofluorescence:0-96h later we measured the Ad-GFP transduction eficiencies in the above cell lines by flow cytometric analysis,and Ad-GFP(0-1000MOI) infected cells obove for 24h, transduction eficiencies was measured.CAR expression levels in vitro were assayed by Western-blotting and IP.CaPan-1,BxPC-3(5×106)cells were implanted subcutaneously into nude mice.When the tumor sizes reached 10 mm in diameter,the xenografts were injected with Ad-GFP(1×108pfu).During 1-14 days, GFP wsa observed by DCC-3 camara in the first groups,and in another group ,after 72h the mice were killed and the GFP expression were observed by fluorescence microscopy to determine the transduction efficiency.CAR expression was detected by Western blotting in the xenograft tissues.Results:We found that Ad transduction efficiencies varied greatly in the pancreatic ce11s of the same tissue 0rigin.The transduction efficiencies in AsPC-1,CaPan-1 (100%,50 MOI), Panc-1(100%,MOI200), BxPC-3(60%,1000MOI) was different.Consistently,GFP expression was much higher in exografts sections resulting from CaPan-1 than thatfrom BxPC-3.Increased CAR expression was predictive for more efficient gene transfer in vitro an d in vivo.Conclusion:CAR expression level is closely correlated to Ad transduction efficiency,which suggests that measurement of CAR expression in tumor tissues is useful for individualized adenovirus based gene therapy in clinic.Part three Construction and Identification of Recombinant Adenovirus for Expression of PUMA geneObjective To construct a recombinant adenovirus for expression of PUMA gene.Methods Amplify gene fragment by PCR using the PUMA gene extracted from recombinant plasmid pHA-PUMA as a template insert into shuttle plasmid pShuttle-CMV and transform to E coli DH 5a.Screen recombinant plasmid pShuttle-CMV-PUMA and transform to competent BJ5183 cells previously transformed witll pAdEasy-1.Screen recombinant plasmid pAdEasy-PUMA and transfect to 293 cels in mediation of Lipofectamine to prepare replication-deficient adenovirus Ad-PUMA carrying PUMA gene.Purify the preparedAd-PUMA virus particles by density gradient centrifugation with cesium chloride and determine the titer.Results Recombinants were selected by kanamycin resistence and confirmed by PacI.The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid Was constructed(30 kb and 4.5 kb fragments were dotained after PacI digestion). Twenty four hours after transfection,the fluorescence Was observed in 293 cels,and the titer of purified Ad-PUMA was 2.032×1010pfu/mlConclusion Recombinant adenovirus Ad-PUMA for expression of PUMA was successfully constructed ,which provided a basis for gene therapy of tumors.Part four Expremental study of Ad-PUMA on pancreatic cancer in vitroObjective To study the effect of recombinant adnenovirus vector mediated PUMA(Ad-PUMA)on pancreatic cancer cells (PC)Methods AsPC-1,CaPan-1,BxPC-3,Panc-1 cells were infected with Ad-PUMA, respectively. The inhibition rate of PC were examined by MTT,the apoptosis was checked by FCM,PI staining and DNA Ladder. Western blotting was used to examine the expression of PUMA and its downstreaming Bax,Bcl-2,cytochrome c,Caspases-9,3, FCM was used to examine the activity of caspases. Results: The Proliferation rate of PC cell lines were suppressed significantly by Ad-PUMA infection in a viral dose-dependent and time-dependent manner,and significant cell apoptosis was checked through DNA Ladder,FCM,PI staining. The expression of PUMA protein increased with titer of Ad-PUMA, they were all does-dependant and time-dependant.With the PUMA upregulation, Bax,Bcl-2,cytochrome c,Caspases-9,3 protein in cells changed too.The activity of caspase-9,3 increased significantly.Conclusions Recombimant adenoviral-mediated PUMA gene increase the cell-killing effect by improve the cell apoptosis,which is through the way of mitochondrion.Part five Experimental study of Ad-PUMA on pancreatic cancer in vitoObjective To study the effect of Ad-PUMA in vitoMethods To establish tumors for model system,5×106AsPC-1 cells were injected subcutaneously onto the flanks of the femals SCID-Bg mice.After approximately 21-28ds,palpable tumors developed.These animals were then treated with Ad-PUMA,Ad-GFP and injection of PBS.At 0,3,6 days,BPS(50ul), Ad-GFP and Ad-PUMA plasmids(3×108PUF/ml/50ul)was injected to the tumor. Tumor growth at different time points was record in 21 days,and after 21d,and the apoptosis index and ki67 were examined by TUNEL and IP,the expression of PUMA and PUMA mRNA protein were examine by western blotting and RT-PCR method.Results Ad-PUMA had a dramatic effect on tumor volume in the first 14days following injection,this effect diminished over time ,but still resulted in a 44.2 %(P<0.05)decrease in tumor after 21 days.Treatment with the Ad-GFP control virus did not substantially alter tumor in this model system(p>0.05).The apoptosis indix in Ad-PUMA groups was higher than than of in control groups,and the ki-67 expression was lower in Ad-PUMA groups.The expression of PUMA and PUMA-mRNA was much higher in Ad-PUMA groups than other groups (p<0.05).Conclusions Recombimant adenoviral-mediated PUMA inhibited growth of tumor in vitro.Part six Experimental study of gemcitabine combined with Ad-PUMA on pancreatic cancer in vitoObjective To study the effect of recombinant adnenovirus vector mediated PUMA combined with gemcitabine on pancreatic cancer in vitro and vivo .Methods:Pancreatic cancer cells AsPC-1 transfected by PUMA(5MOI) or not reseparately treated with serial concentrations of gemcitabine. The sensitivity of cells to gemcitabine was determined by MTT assay. To establish tumors for model system as part five. These animals were then treated with Ad-PUMA (1×109puf/ml/50ul)at0,3,6days,along with viral injection,animals recived a 100ul intraperitoneal injection of gemcitabine(100mg/kg) on days0,3,6,Ad-GFP was as the control. Tumor growth at different points was record in 21dResults Gemcitabine inhibited AsPC-1 cell proliferation in a dose dependent manner,but the cell proliferation was more significant in gemcitabine combined with PUMA expression clones than in parental AsPC-1cells.The IC50(0.0076±0.012)μg/ml in combined groups increased 6.9 times than that of IC50(0.053±0.001)μg/ml in gemcitabine groups. When Ad-PUMA or gemecitabine was injected as a single agent it resultd in reduction in 62.75(P<0.01 vs control) or 42.3% (p<0.05 vs control)tumor growth ,however, the combination of Ad-PUMA with gemcitabine reduced tumor volume by more than 82%(P<0.001 vs control)on day 21.Conclusion PUMA significantly enhances chemosensitivity of AsPC-1 to gemcitabine, and PUMA and gemcitabine exert anadditive anti-tumor effect in vivo.