Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo

• Published in: Cancer research (Chicago, Ill.) Ill.), 1997-10, Vol.57 (19), p.4325-4332
• Main Authors: PEDERSON, L. C, BUCHSBAUM, D. J, VICKERS, S. M, KANCHARLA, S. R, MAYO, M. S, CURIEL, D. T, STACKHOUSE, M. A
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; Animals; Antimetabolites, Antineoplastic - pharmacology; Antimetabolites, Antineoplastic - therapeutic use; Antineoplastic agents; Bile Duct Neoplasms - radiotherapy; Bile Duct Neoplasms - therapy; Bile Ducts, Intrahepatic - drug effects; Bile Ducts, Intrahepatic - radiation effects; Biological and medical sciences; Cholangiocarcinoma - radiotherapy; Cholangiocarcinoma - therapy; Combined Modality Therapy; Combined treatments (chemotherapy of immunotherapy associated with an other treatment); Cytomegalovirus - genetics; Cytosine Deaminase; Fluorouracil - pharmacology; Fluorouracil - therapeutic use; Gamma Rays; Genetic Therapy; Genetic Vectors - genetics; Humans; Medical sciences; Mice; Mice, Nude; Nucleoside Deaminases - genetics; Nucleoside Deaminases - therapeutic use; Pharmacology. Drug treatments; Prodrugs - metabolism; Prodrugs - pharmacokinetics; Promoter Regions, Genetic; Radiation-Sensitizing Agents - pharmacology; Radiation-Sensitizing Agents - therapeutic use; Recombinant Fusion Proteins - metabolism; Transfection; Transplantation, Heterologous
• ISSN: 0008-5472; 1538-7445

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 wi