Adenoviraler p53-Gentransfer (Adp53) und 5-FU-Chemotherapie wirken synergistisch in vitro und in vivo beim experimentellen Pankreaskarzinom

Abstract

Background: In preclinical models adenovirus-mediated p53 gene transfer (Ad-p53) demonstrated therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53, both in vitro and in vivo. Alterations in the p53 function are present in 50% – 75% of pancreatic cancer patients. The traditional therapeutic approach for advanced pancreatic cancer is still chemotherapy. Before starting clinical trails it is important to study possible interactions between Ad-p53 gene transfer and chemotherapeutic drugs. Methods: Transfection of two human (DANG, Capan-1) and one murine (DSL6A) pancreatic cancer cell lines with a replication-deficient adenoviral vector expressing wt p53 (Ad-p53) were performed in vitro. Determination of the p53 status of pancreatic cancer cell lines and Ad-p53 gene transfer were evaluated by Western blot analysis and immunofluorescence. Proliferation of tumor cells and apoptosis were quantitated after incubation with different ratios of Ad-p53 particles and cell numbers (multiplicities of infection, MOI 1–100) in combination with various 5-FU doses by cell proliferation assay (WST-1) and FACS (PI staining). In vivo 1 x 106 DSL6A syngenic pancreatic tumor cells were inoculated subcutanously in Lewis rats and 8 weeks later treatment started. The animals were separated into the following groups: (1) Control, no treatment, n = 8; (2) 5-fluorouracil (5 mg/kg/BW i.p.), n = 8; (3) Ad-p53 (1 x 108 infectious particles i.t.), n = 8; and (4) Ad- P53 + 5-FU, n = 8. The treatment was performed twice a week for 1 month. Our evaluation included tumor size, weight and survival of the animals. Results: Capan-1, DANG and DSL6A were very efficiently transduced at MOI of 1–10 by Ad-p53 and revealed a significant inhibitory effect on tumor growth. The pancreatic tumor cell lines Capan-1, DANG and DSL6A were sensitive to the cytotoxic action of 5-FU. The combined application of Ad-p53 and 5-FU chemotherapy resulted in a more reduced number of viable tumor cells (40% vs. 70%) and higher apoptotic rate (30% vs. 10%) compared with Ad-p53 or 5-FU therapy alone. In vivo experiments showed the most potential tumor regression in animals treated with Ad-p53+5-FU (p < 0.05) and prolonged survival time. In contrast, 5-FU single therapy had no effect on tumor growth and survival rate. Ad-p53 gene therapy alone inhibited tumor progression for the first 2 weeks of treatment, but then tumor growth started again rapidly. Conclusions: Our results demonstrated a greater anticancer efficacy of the combination Ad-p53 plus 5-FU chemotherapy and support this approach for advanced pancreatic cancer patients in clinical trails.