title: 5-Fluorouracil or gemcitabine combined with adenoviral-mediated reintroduction of p16INK4A greatly enhanced cytotoxicity in Panc-1 pancreatic adenocarcinoma cells.
abstract: BACKGROUND
  Pancreatic cancer is one of the most lethal of all the common gastrointestinal malignancies. Although surgery offers the best chance for survival, it is not appropriate for all cases. The only adjuvant treatment to show promise is chemotherapy. Hence new treatments are urgently sought. We previously reported that adenoviral (Ad)-mediated delivery of p53 (Adp53) and p16(INK4A) (Adp16) significantly inhibited the growth of pancreatic cancer cell lines and established subcutaneous pancreatic tumours in nude mice (Ghaneh P, et al. Adenovirus mediated transfer of p53 and p16INK4A results in pancreatic cancer regression in vitro and in vivo. Gene Ther 2001; 8: 199-208). In this study we examine whether combining Ad-mediated delivery of p53 or p16(INK4A) with clinically relevant chemotherapeutic drugs has therapeutic potential for pancreatic cancer.
  
  
  RESULTS
  Four pancreatic adenocarcinoma cell lines were evaluated for their sensitivity to 5-fluorouracil (5-FU) and gemcitabine and two of these, Suit-2 and Panc-1, were chosen for combination experiments because they showed moderate and poor sensitivity, respectively, to 5-FU and gemcitabine. We found no evidence for enhanced cytotoxicity when either cell line was transduced with Adp53 before or after incubation with chemotherapeutic drugs. In contrast, incubation of Panc-1 cells with either 5-FU or gemcitabine followed by Ad-mediated overexpression of p16(INK4A) resulted in a substantial reduction in cell viability under conditions where the drugs alone had minimal cytotoxicity. Incubation of Suit-2 cells with 5-FU followed by Ad-mediated overexpression of p16(INK4A) also resulted in a significant reduction in cell viability. This, however, was observed only with higher concentrations of 5-FU and viral vector. Cell cycle analysis of Panc-1 cells showed that the combination of cytotoxic drugs and Adp16 resulted in an increase in the sub-G1 population suggesting an increase in apoptosis. Dual labelling of these cells with annexin V and propidium iodide (PI) confirmed that the combination of 5-FU and Adp16 resulted in a significant increase in early apoptotic cells (annexin V positive and PI negative) compared with controls. Moreover, overexpression of p16(INK4A) was associated with a reduction in pRb levels in these cells-high levels of pRb have been proposed to contribute to chemoresistance in pancreatic cancer cells.
  
  
  CONCLUSIONS
  We have shown that the currently used chemotherapeutic drugs for pancreatic adenocarcinoma combined with restoration of p16(INK4A) expression hold promise for the adjuvant treatment of this disease. Importantly, the combination facilitated the use of chemotherapeutic drugs at lower concentrations than would otherwise be effective.
authors.affiliation: Department of Surgery, Royal Liverpool University Hospital, 5th Floor, UCD Building, Daulby Street, University of Liverpool, Liverpool, UK.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Halloran
authors.forename: Paula
authors.initials: P
authors.lastname: Ghaneh
authors.forename: Susannah
authors.initials: S
authors.lastname: Shore
authors.forename: William
authors.initials: W
authors.lastname: Greenhalf
authors.forename: Lou
authors.initials: L
authors.lastname: Zumstein
authors.forename: Deborah
authors.initials: D
authors.lastname: Wilson
authors.forename: John P
authors.initials: JP
authors.lastname: Neoptolemos
authors.forename: Eithne
authors.initials: E
authors.lastname: Costello
mesh_terms: D000230:Adenocarcinoma; D000256:Adenoviridae / Q000235:genetics*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D010190:Pancreatic Neoplasms; D016160:Retinoblastoma Protein / Q000096:biosynthesis; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D003841:Deoxycytidine; D005472:Fluorouracil; D000093542:Gemcitabine