Study On Adenoviruses Ad.rms For Targeting Pancreatic Cancer Therapy

Pancreatic cancer is the leading cause of cancer deaths in both men and women in the world and has the worst prognosis among common gastrointestinal cancers. Despite advances in surgery, chemotherapy, and radiation therapy in recent decades, the 5-year survival rate less than 5%. Therefore, the outcome of patients with this lethal malignancy depends on novel significant treatment strategies. Scientists now focus on gene therapy.It has been demonstrated that its carcinogenesis is a multistep process,often including oncogene mutated, function of tumor-suppressor gene inactivated, cell surface receptor protein absent. In order to develop effective anti-cancer therapies, it is essential to know which of these events is happened in the process of pancreatic carcinoma progression. Accumulating evidences suggest that 85% cases of pancreatic carcinoma K-ras gene was mutated, and the gene somatostatin receptor subtype 2 (SSTR2) was frequently lost in human pancreatic adenocarcinomas Loss of SSTR2 expression in pancreatic cancer may lead to a deficiency in negative regulation of cell growth by its ligand somatostatin (SST), and thus contribute to cancer cell growth and proliferation. In our study, K-ras^val12 and SSTR2 wereselected as therapeutic genes. In the other hand, it's important to select a proper gene transferring vector. In cancer gene therapy, approaches based on recombinant adenoviruses have provided promising results, suggesting successful gene transfer and therapeutic effect. But how to construct a proper recombinant adenovinis vector carrying the favorite gene that is specifically expressed only in cancer cells has become a bottle neck which restricts the application in clinical gene therapy for tumors. In order to solve this problem, we employed a tumor-specific promoter MUC1. Accumulative studies showed that the MUC1 expression increased more than 10 times in pancreatic cancer., On the basis, of these findings, we constructed adenoviruses which named AdMUCl-hSSTR2, AdHl-K-rasval12 and Ad.rms, and employed them to infect Panc-1 cell, respectively.We amplified a 786-base pair (bp) MUC1 promoter by two-step nest PCR, and identified the tumor-specificity using enhanced green fluorescent protein (EGFP) gene as a reporter gene. It has been demonstrated that EGFP regulated by MUC1 promoter was selectively expressed in Panc-1 by fluorescence microscopy and flow cytometry analysis. Subsequently, the gene hSSTR2 driven by MUC1 promoter was cloned into the pAdTrack. After homologous recombination with pAdEasy in bacteria and co-transfection into a mammalian packaging cell line 293, we obtained recombinant adenovinis AdMUCl-hSSTR2. After infection with AdMUCl-hSSTR2 in Panc-1, PNPC and HeLa cell respectively, the anticancer effect of AdMUCl-hSSTR2 was determined in Panc-1. RT-PCR analysis and western blot examination revealed that hSSTR2 protein was over-expressed in Panc-1, not expressed in HeLa, and no significant change in PNPC. Furthermore, the results demonstrated that there was a significant cell proliferation inhibition, involved in the up-regulation of the cyclin-dependent kinase (CDK) inhibitors p21 and p27.