Enhancement of Chemosensitivity to Fluoropyrimidines by Retroviral ...

Bulletin of the Osaka Medical College 52（1）：19-27, 2006

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〈Original Article〉

Enhancement of Chemosensitivity to Fluoropyrimidines by Retroviral Transduction of Thymidine Phosphorylase cDNA: an in vitro and in vivo Study

Hitoshi INOUE 1, Nobuhiko TANIGAWA 1, Tetsuhisa YAMAMOTO 1, Yoshinobu MANOME 2

1 Department of General and Gastroenterological Surgery, Osaka Medical College, Osaka, Japan 2 Department of Molecular Cell Biology, Institute of DNA Medicine, Research Center for Medical Science, Jikei University School of Medicine, Tokyo, Japan

Key Words：thymidine phospholyrase, retroviral vector, gene therapy,

chemotherapy, fluoropyrimidines

ABSTRACT Thymidine phospholyrase (TP) is an essential enzyme in activating 5’-deoxy-5-fluorocytidine (5’DFUR) into 5-fluorouracil (5-FU) and for the conversion of 5-FU into 5-fluoro-2’-deoxyuridine. The purpose of this study was to examine the therapeutic efficacy of the retroviral vector-mediated TP gene in the sensitivity to fluoropyrimidines, 5-FU and its prodrugs, 5’-DFUR and capecitabine, in MC38 murine colon adenocarcinoma cells in vitro and in vivo. After retroviral infection with or without human TP cDNA, we obtained MC38 cells having the stable expression of TP (MC38-TP) and control-vector transfected cells (MC38-Neo). There was no significant difference in the doubling time in vitro and tumor growth rate in vivo among parental MC38 cells (MC38-P), MC38Neo and MC38-TP, demonstrating that the TP gene was not directly toxic. The in vitro study showed significant increases in sensitivities to 5-FU, 5’-DFUR and capecitabine in MC38-TP cells. The 50% growth inhibitory concentration (IC 50 ) of MC38-TP cells to 5-FU, 5’-DFUR and capecitabine, respectively, was about 10-fold, 800-fold and 40-fold higher than that of MC38-P cells and MC38-Neo cells. The in vivo study showed significant increases in sensitivities to 5-FU, 5’DFUR and capecitabine in MC38-TP tumors. There was no significant difference in the sensitivities to 5-FU, 5’-DFUR and capecitabine between MC38 and MC38-Neo tumors. The tumor-cure rate in MC38-TP tumors treated with capecitabine was 100% and that treated with 5’-DFUR was 63%. In conclusion, our results demonstrate that the stable expression of TP gene by using recombinant retroviral vector could dramatically increase the anticancer effect of fluoropyrimidines.

Address correspondence to: Nobuhiko Tanigawa, M.D., Department of General and Gastroenterological Surgery, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki-city, Osaka 569-8686, Japan Phone: +81-72-683-1221, Fax +81-72-684-6513 E-mail: [email protected]

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INTRODUCTION

MATERIALS and METHODS

Gene transfer offers the possibility of effective approaches to cancer treatment. One gene transduction strategy for the treatment of cancer involves the introduction of a drug sensitivity gene, which encodes an enzyme that can intratumorally activate prodrug or conventional chemotherapeutic agents. A representative example of this strategy is the herpes-simplex virus thymidine kinase (HSV-TK) gene, which encodes the protein that activates ganciclovir, a nucleoside analog [1-3]. Capecitabine, a novel fluoropyrimidine carbamate, is an orally administered, tumorselective cytotoxic agent that is converted to 5-FU in three distinct steps. It is sequentially converted first to 5’-deoxy-5-fluorocytidine (5’-DFCR) by carboxylesterase located in the liver, then to 5’deoxy-5-fluorouridine (5’-DFUR) by cytidine deaminase, also with high activity in the liver and various solid tumors, and finally to 5-fluorouracil (5-FU) by thymidine phosphorylase (TP, dThdPase, EC 2.4.2.4) with high activity in many type of tumors. Enhanced TP expression at tumor sites therefore would selectively increase the 5-FU level in situ [4]. TP expression increases in various cancers, such as esophageal, gastric, pancreatic, colorectal, lung, bladder, ovarian, breast and kidney, as compared with their normal surrounding tissues [5-13]. A number of studies have shown that TP can be upregulated after exposure to several chemical agents, such as paclitaxcel, docetaxel, cyclophosphamide, vinblastine and gemcitabine [14-16], and cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-12 (IL-12) and interferon-gamma (IFN-γ) [17, 18] in various human cancer cell lines and human tumor xenografts. On the other hand, transfection of TP cDNA into cancer cell lines using plasmid vectors has been reported to increase the sensitivity to fluorouridines [19-22]. In the present study, we used a retroviral expression system, because the gene transfer method employing retroviruses can effectively introduce genes into various types of cancer cells. We cloned the TP gene into the retroviral vector and examined the efficacy of retroviral gene transduction in vitro and in vivo.

Tumor cell line and animals MC38 murine colon adenocarcinoma cells [23] were grown in Dulbecco’s modified Eagle’s medium (DMEN) supplemented with 10% fetal bovine serum. Six-week-old female C57BL/6J-Jcl mice were purchased from Clea Japan (Tokyo, Japan). MC38 tumor cells were syngeneic with the C57BL mice. All animal procedures were carried out under guidelines approved by the Animal Care and Use Committee in Osaka Medical College. Chemicals 5-FU was provided by Kyowa Hakko Kogyo (Tokyo, Japan). 5’-DFUR and capecitabine were provided by Nippon Roche (Tokyo, Japan). Retroviral transfection of thymidine phosphorylase cDNA A 1.6-kilobase (kb) fragment of PD-ECGF cDNA from pcDNA3-PD-ECGF (kindly provided by Dr. Kato Y, Fukui Medical School, Fukui, Japan) was cloned into the EcoRI site of pMV7 retroviral vector (pMV7-TP) [24]. The pMV7 retroviral vector contains a selective neomycin 3’phosphotransferase (neoR) driven by the tk promoter. The amphotropic PA317 retrovirus packaging cells (American Type Culture Collection, Rockville, MD, USA) were transfected with either plasmid pMV7 or pMV7-TP using the standard calcium phosphate co-precipitation technique. After 72 hours, the culture supernatant was filtered (0.45μm) and 1ml of virion-containing medium (titer of 104geneticinresistant colony-forming units per mL) was added to each dish of MC38 target cells (104cell/dish). The cells were incubated for 24 hours and then selected for 2 weeks in the presence of geneticin sulfate 400μg/ml (GIBCOBRL, Gaitherburg, MD, USA). Selected cells of MC38-Neo (infected by pMV7 alone) and MC38-TP (infected by pMV7TP) were further characterized by Western blot analysis, immunohistochemical staining and ELISA assay. Immunoblotting and immunohistochemical staining The anti-human TP monoclonal antibody, 1C6203 [25], was kindly provided by Nippon Roche Research Center, Kamakura, Japan and was used as a primary antibody for either immunoblotting or immunohistochemical staining experiments.


Gene therapy using retroviral vector bearing TP gene For immunoblotting, cells were lysed and the protein concentration was determined with bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL, USA). Then, 50 μg of total protein was separated by 15% sodium dedecyl sulphate (SDS) -polyacrylamid gel electrophoresis and transferred onto Immunobilon-P membrane (Millipore, Co., MA, USA). After incubation in phosphate-buffered saline (PBS) containing 5% non-fat milk, blots were incubated with antihuman dThdPase monoclonal antibody, and reacted with anti-mouse IgG (Amersham Life Science, Arlington Height, IL, USA). Immunolabelled proteins were visualized using Xray film after incubation with chemiluminescence (ECL) regents. For immunohistochemical staining, the cultured cells were plated on coverslips treated with 1% paraformaldehyde. The anti-human TP monoclonal antibody, 1C6-203, was added to the slides after quenching in 3% hydrogen peroxide and blocking, then the slides were incubated overnight. After washing, the histofine avidin-biotin method (Nichirei, Tokyo, Japan) was applied. Color was developed with 3, 3’-diaminobenzidine and hematoxylin was used for counterstaining. Negative control slides in the absence of primary antibody were included for each staining. Measurement of dThdPase Activity Enzyme extraction was performed according to the method of Eda et al [17] and enzyme activity was measured based on the identification of dThdPase with monoclonal antibodies, as described by Nishida et al [6]. Briefly, cultured cells (1×108) were homogenized with 10 mM TrisHCl buffer (pH7.4) containing 15 mM NaCl, 1.5 mM MgCl2, and 50 mM potassium phosphate. The homogenate was centrifuged at 105,000g for 90 min at 4℃ . The supernatant was dialyzed overnight against 20 mM potassium phosphate buffer and 1mM 2-mercaptoethanol, and then subjected to an enzyme-linked immunosorbent assay (ELISA) method using monoclonal antibodies specific for human dThdPase. Data from the ELISA method were presented in U/mg protein, where 1U is equivalent to the amount of TP that generates 1μg 5-FU from 5’-DFUR in 1hr. In vitro cell proliferation assay The cells (3×103 cells/well) were seeded in DMEN with 10% fetal bovine serum (200 μl each) into 96-well tissue culture plates. Cell proliferation was determined at 24 hr intervals by

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the methylene blue staining procedure [26]. All determinations were repeated 3 times. In vitro cytotoxic assay The cells (3×103/well) were seeded in DMEN with 10% fetal bovine serum (100 μl each) into 96-well microtiter plates and cultured at 37C in 5% carbon dioxide. After 24 hr incubation, the cells were treated with varying does of anti-cancer agents (100 μl each). 5-FU and 5’-DFUR were dissolved in PBS. Capecitabine was dissolved in 0.5% dimethyl sulphoxide (DMSO) with PBS since it was suspended in PBS only. In preliminary experiments, the concentration of 0.5% DMSO remained below those that were not toxic to MC38 (data not shown). Five days after the exposure to drugs, cell proliferation was determined using the methylene blue staining procedure. For control experiments, the drug-treatment step was omitted. The 50% inhibitory concentration (IC50) was determined graphically as the dose of drug causing a 50% reduction in the absorbance compared with the control. All determinations were repeated 3 times. In vivo experiments Single cell suspensions of MC38-Neo and MC38-TP (2×10 6 /mouse) in 150 μl PBS were injected s.c. into the backs of 6-week-oldC57BL/6J-Jcl mice, respectively. Subcutaneous tumors were established within 3 weeks after tumor inoculation. The experiments were started when the tumor volume reached 80 to 200 mm3. 5-FU was dissolved in PBS and administered intraperitoneally 5 days per week for 2 weeks. 5’DFUR and capecitabine were dissolved or suspended in 40mM citrate buffer (pH 6.0) containing 5% gum Arabic as the vehicle and were administered orally 5 days per week for 2 weeks. In the control groups, the vehicle alone was administered orally. The fluoropyrimidines were administered at maximum tolerated doses (MTDs); MTDs of 5-FU, 5’-DFUR and capecitabine were 0.21, 1.05 and 2.1 mmol/kg/day, respectively [4] [27]. Tumor size was measured twice a week. Tumor volume was estimated by using the following equation: V = ab2/2, where a and b are tumor length and width, respectively. Gastrointestinal toxicity was estimated by observing the faces. Statistical analysis Statistical significance between different groups was determined by Student’s t-test (two-


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tailed). P values of