Simultaneous Tissue-specific Promoter Amy-2 And Tumor-targeted Promoter HTERT Control Of The EPNP/MePdR For Pancreatic Cancer Therapy

Background :Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. The reasons for the poor prognosis include: the difficulty of early diagnosis due to lack of specific early symptoms; the frequent occurrence of distant metastasis; the poor response to existing chemo-, radio-therapy, therefore it is necessary to establish gene therapy method. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coli purine nucleoside phosphorylase/6-methylpurine eoxyribose (ePNP/MePdR) is a suicide gene/prodrug system where ePNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine (MeP) with high bystander activity. In this study we examined the efficacy of ePNP under the control of simultaneous tissue-specific promoter Amy-2 and tumor-targeted promoter hTERT and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors.Methods Recombinant vector pET-ePNP was constructed. The protein of E. coli PNP was expressed with recombinant bacteria BL21(pET-ePNP) and an antibody to E.coli PNP was prepared. Transcriptional activities of hTERT and Amy-2 promoter sequences were analyzed using a luciferase reporter gene. The recombinant phTPNPamyCre vector was constructed with PBS302 and PBS185 vector. The ePNP/MePdR system's efficacy on the transiently trancfected Capan-1,SW1990,Panc-1,BxPC-3 and MIAPaCa-2 human pancreatic cancer cell lines and stable transfectants BxPC-3·PNP cells was measured by the MTT assay in vitro.Results The hTERT and Amy-2 promoter had high transcriptional activity in transfected tumor cell-lines and pancreas derived cells respectively. The antibody to E. coliPNP was demonstrated to be specific for the ePNP protein. Expression of ePNP gene with phTPNPamyCre vector system was proofed to be simultaneous tissue-specific promoter Amy-2 and tumor-targeted promoter hTERT regulated. The MePdR treatment induced a high in vitro cytotoxicity on the sole ePNP-producing BxPC-3 pancreatic cancer cell lines and affected five pancreatic cancer cells in a dose-dependent manner .The ePNP/MePdR system has a strong bystander killing effect.Conclusions The simultaneous tissue-specific promoter Amy-2 and tumor- targeted promoter hTERT control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cells.