| Background & Objective:Pancreatic cancer,which prognosis is poor,remains a difficult disease to be treated.Most patients present with locally advanced or metastatic disease and therefore,only a small proportion of patients are suitable for surgical resection.Conventional chemotherapy and radiation therapy remain ineffective. Therefore,new modalities in the treatment of this disease are required.Epidermal growth factor receptor(EGFR) overexpresses on the surface of a group of tumor cells,including pancreatic cancer,breast cancer and colorectal cancer.It has been implicated in neoplastic traits of mitogenesis,inhibition of apoptosis,cell migration,metastases, angiogenesis,and resistance to standard cytotoxic therapies.Therefore,EGFR has become a promising target of cancer treatment.We tried to challenge pancreatic cancer with anti-EGFR gene therapy.We sought to construct a recombinant adeno-associated virus(rAAV) vector containing a human anti-EGFR single chain variable fragment antibody gene,and to observe the effects on pancreatic cancer cell lines and nude mouse pancreatic cancer xenograft model.This will provide a basis of clinical study in the future.Methods:1.Construction of a recombinant adeno-associated virus(rAAV) vector containing a human anti-EGFR single chain variable fragment antibody gene.2. Observation of the expression of anti-EGFR single chain variable fragment antibody gene in 293 cell,pancreatic cancer cell lines(Panc-1,SW1990,Aspc-1,Capan-1,PCT-3 and MiaPaCa-2) and nude mouse pancreatic cancer xenograft tissues with Western blot. 3.In vitro experiments:including(1) Cell proliferation analysis:WST-8 assay;(2) Cell apoptosis analysis:Annexin V-FITC/PI apoptosis detection.4.In vivo experiments: (1) comparison of the volume of nude mouse pancreatic cancer xenografts;(2) extraction of total RNA from tumor tissues.The transcription levels of vascular endothelial growth factor(VEGF) mRNA was analyzed with RT-PCR;(3) Micro vessel density was examined with immunohistochemistry. Results:1.We successfully constructed a recombinant adeno-associated virus(rAAV) vector containing a human anti-EGFR single chain variable fragment antibody gene.2. Western blot assay demonstrated the expression of anti-EGFR antibody in 293cell, pancreatic cancer cell lines and nude mouse pancreatic cancer xenograft tissues.3. Anti-EGFR antibody inhibited proliferation of ASPC-1 pancreatic cancer cells by 15.1%. 4.Anti-EGFR antibody remarkably enhanced apoptosis in all 6 pancreatic cancer cell lines compared with control group(Panc-1:9.1%vs.0.4%;SW1990:17.2%vs.0.8%; Aspc-1 50.9%vs.0.8%;Capan-1:19.2%vs.0.8%;PCT-3:10.2%vs.0.5%;MiaPaCa-2: 28.6%vs.3.0%).5.In vivo,anti-EGFR antibody reduced the volume of nude mouse pancreatic cancer xenografts(1.38±0.83cm~3 vs.4.60±2.17cm~3,p＜0.05).6.Anti-EGFR antibody did not supresse the transcription of VEGF mRNA in nude mouse pancreatic cancer xenografts remarkably(0.10±0.06 v s.0.35±0.24,p＞0.05).7.Anti-EGFR antibody resulted in reduced angiogenesis as measured by micro vessel density(41.4±5.4 vs.58.2±6.1,p＜0.05).Conclusions:Our results of in vitro and in vivo assays demonstrated that successfully constructed recombinant adeno-associated virus(rAAV) vector containing a human anti-EGFR single chain variable fragment antibody gene that expresses Anti-EGFR antibody exhibited therapeutic efficiency in both pancreatic cancer cell lines and nude mouse pancreatic cancer xenografts model. |
