title: Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo.
abstract: Cholangiocarcinoma is a virtually incurable tumor, resistant to current surgical, chemotherapy, and radiotherapy interventions. We applied the gene therapy strategy of toxin gene conversion of nontoxic prodrug to chemotherapeutic drug in combination with radiation therapy to the treatment of cholangiocarcinoma. In this regard, 5-fluorouracil (5-FU) is an accepted radiosensitizing and chemotherapeutic agent presently used in cancer therapy. The Escherichia coli enzyme cytosine deaminase (CD) converts the prodrug 5-fluorocytosine (5-FC) to 5-FU. Therefore, our goal was to express the CD gene in the human cholangiocarcinoma cell line, SK-ChA-1, assess the cytotoxicity of intracellular production of 5-FU, and determine any enhanced cell killing by the addition of external beam radiation. The susceptibility of SK-ChA-1 cells to recombinant adenoviral infection was determined by fluorescence-activated cell sorting analysis. We used the recombinant adenoviral vector AdCMVLacZ, encoding the E. coli beta-galactosidase reporter gene under control of the human cytomegalovirus (CMV) promoter, to infect SK-ChA-1 and HeLa cells at 10 and 100 plaque forming units (pfu)/cell, followed by FACS analysis. To evaluate CD-mediated conversion of 5-FC to 5-FU and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes CD. Cells were then plated in 96-well microtiter plates and exposed to varying concentrations of 5-FC. Cell proliferation assays (tetrazolium salt conversion to formazan colorimetric assay) were performed beginning 2-8 days after plating. We evaluated the effects of external beam radiation using a single 8 Gy 60Co dose to AdCMVCD infected cells, with prior exposure to 5-FC for 2-3 days. MTS assays were performed following radiation treatment. Radiation dose-response analysis, via clonogenic assay, was used as a more sensitive assay to confirm the interaction of the treatment conditions. s.c. SK-ChA-1 tumors in athymic nude mice were established, which then received three intratumoral injections of 1 x 10(9) pfu AdCMVCD. Mice received i.p. injections of 400 mg/kg of 5-FC twice daily for 7 days beginning the day of initial AdCMVCD injection (day -2). The radiation treatment group received 10 Gy of 60Co exposure to their tumor on day 0. SK-ChA-1 cells were efficiently transduced (48.7 and 99.2%) by 10 and 100 pfu/cell of AdCMVLacZ, respectively. From 37.9 to 84.4% of SK-ChA-1 cells were killed following infection with 10 pfu/cell AdCMVCD and 8 days of exposure to various concentrations of 5-FC (5, 10, 30, 50, and 100 microg/ml). Higher 5-FC concentrations and longer duration of exposure resulted in greater cell killing. Radiation treatment (8 Gy) enhanced cell killing by greater than 70% when combined with 10 or 20 microg/ml of 5-FC. Radiation dose-response analysis with clonogenic assay confirmed enhanced SK-ChA-1 cell cytotoxicity as a result of radiation treatment following AdCMVCD infection and 5-FC exposure, with radiobiological parameters alpha = 0.44 and D0 = 0.96. Combined treatment of SK-ChA-1 tumors with AdCMVCD, 5-FC, and radiation in animals resulted in significantly greater survival, time to tumor regrowth, and doubling time compared to the nonradiation treatment group (P = 0.03, 0.015, and 0.002, respectively). Significantly greater change in tumor size, smaller ratio of final tumor size to original tumor size, and smaller final tumor size were observed in the radiation treatment group compared to the no radiation treatment group (P = 0.02, 0.03, and 0.03, respectively). Human cholangiocarcinoma cells were transduced with a recombinant adenovirus in vitro at high efficiency and were susceptible to CD-mediated intracellular 5-FU production. Radiobiological survival curve parameters confirmed an interactive cytotoxic effect when viral infection and prodrug therapy were combined with external beam radiation exposure. (ABSTRACT TRUNCATED)
authors.affiliation: Department of Surgery, University of Alabama at Birmingham, 35294, USA. Lagan@uab.edu
authors.forename: L C
authors.initials: LC
authors.lastname: Pederson
authors.forename: D J
authors.initials: DJ
authors.lastname: Buchsbaum
authors.forename: S M
authors.initials: SM
authors.lastname: Vickers
authors.forename: S R
authors.initials: SR
authors.lastname: Kancharla
authors.forename: M S
authors.initials: MS
authors.lastname: Mayo
authors.forename: D T
authors.initials: DT
authors.lastname: Curiel
authors.forename: M A
authors.initials: MA
authors.lastname: Stackhouse
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use*; D001650:Bile Duct Neoplasms / Q000532:radiotherapy / Q000628:therapy*; D001653:Bile Ducts, Intrahepatic / Q000187:drug effects / Q000528:radiation effects; D018281:Cholangiocarcinoma / Q000532:radiotherapy / Q000628:therapy*; D003131:Combined Modality Therapy; D003587:Cytomegalovirus / Q000235:genetics; D043525:Cytosine Deaminase; D005472:Fluorouracil / Q000494:pharmacology / Q000627:therapeutic use*; D005720:Gamma Rays; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009700:Nucleoside Deaminases / Q000235:genetics / Q000627:therapeutic use*; D011355:Prodrugs / Q000378:metabolism / Q000493:pharmacokinetics*; D011401:Promoter Regions, Genetic; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D000964:Antimetabolites, Antineoplastic; D011355:Prodrugs; D011838:Radiation-Sensitizing Agents; D011993:Recombinant Fusion Proteins; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil