10.1245/s10434-006-9191-9
Annals of Surgical Oncology 14:1374-1380 (2007)
© 2007 Society of Surgical Oncology
Antitumor Effect of Gemcitabine on Orthotopically Inoculated Human Gallbladder Cancer Cells in Nude Mice
Yoshiyasu Mita, MD1,
Tetsuo Ajiki, MD1,
Takashi Kamigaki, MD1,
Taro Okazaki, MD1,
Hiroshige Hori, MD1,
Hideki Horiuchi, MD1,
Kenro Hirata, MD1,
Tsunenori Fujita, MD1,
Takahiro Fujimori, MD2 and
Yoshikazu Kuroda, MD1
1 Department of Gastroenterological Surgery, Kobe University Graduate School of Medical Sciences, Kobe, Japan
2 Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Tochigi, Japan
Correspondence: Address correspondence and reprint requests to: Tetsuo Ajiki, MD; Department of Gastroenterological Surgery, Kobe University Graduate School of Medical Sciences, 7-5-1 Kusunoki-cho, Chuo-ku Kobe 650-0017, Japan; E-mail: ajiki{at}med.kobe-u.ac.jp
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ABSTRACT
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Background: The prognosis of gallbladder carcinoma is poor; therefore, investigating the efficacy of new chemotherapy agents is essential for the treatments for this tumor. Recently, several studies have reported clinical trials using gemcitabine as treatment for advanced gallbladder cancers. However, the antitumor effects of gemcitabine on gallbladder carcinoma have not been examined in in vitro and in vivo model systems.
Methods: We examined the cytotoxicity of gemcitabine in four biliary tract cancer cell lines using the WST-1 assay. In addition, we examined the effect of gemcitabine on gallbladder cancers resulting from orthotopic inoculation of NOZ gallbladder tumor cells into nude mice. One week after transplantation, the mice were randomized into two groups: In Group A, the mice were treated by an intra-peritoneal injection of 0.9% sodium chloride for three weeks after inoculation (control). In Group B, the mice were treated by an intra-peritoneal injection of gemcitabine (125 mg/kg) for three weeks. All mice were sacrificed one week after the end of treatment, and macroscopic and histological findings were evaluated. The expression levels of proliferating-cell nuclear antigen (PCNA) were examined to investigate cellular proliferation activity, and Tunnel assays were performed to determine apoptotic status. Survival duration of the mice after gemcitabine treatment was compared to that of untreated mice.
Results: The gemcitabine sensitivity of the four biliary tract cancer cell lines was similar in a dose dependent manner. In the in vivo models, the Group A mice showed huge tumors of the gallbladder, with liver invasion and lymph node metastases. However, there were no abdominal tumors in the Group B mice, and microscopic gallbladder cancer could only be detected from histological findings. The mean percent of PCNA-positive tumor cells was significantly higher in tumors from mice in Group A (71.9%) compared to those of Group B (34.7%). The mean percent of Tunnel-positive tumor cells was significantly lower in mice from Group A (2.0%) than those from Group B (5.7%). Survival duration was prolonged significantly in the gemcitabine-treated mice relative to untreated mice.
Conclusions: Gemcitabine treatment may inhibit tumor progression and prolong survival in gallbladder cancer by inhibiting cell proliferation and inducing apoptosis.
Key Words: Gallbladder cancer • Gemcitabine • Survival • Apoptosis • PCNA
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INTRODUCTION
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Using a newly-devised model of nude mice inoculated orthotopically with gallbladder cancer, we found that gemcitabine treatment of gallbladder cancer may inhibit tumor progression and prolongs survival by inhibiting cell proliferation and inducing apoptosis.
Gallbladder cancer is the most common malignancy in the biliary tract.1 Since symptoms from gallbladder cancer manifest themselves late in the disease course, tumors often are detected only at an advanced stage, when patients may not qualify for surgical treatment. For this reason, successful chemotherapy is especially important for cases of advanced gallbladder cancer.
Recently, several reports have described gemcitabine treatment as an effective new regimen for treating gallbladder cancers.2–7 Gemcitabine is a novel nucleoside analogue that requires phosphorylation to become an active metabolite, gemcitabine triphosphate. Since gemcitabine triphosphate is a competitor of deoxycytidine triphosphate for incorporation into DNA, its presence inhibits DNA synthesis. Gemcitabine activity has been shown to function broadly in a variety of tumors and is currently used to treat non-small-cell lung cancer and pancreatic cancer in Japan.8,9 Phase II studies in Western countries, as well as in Japan, of single-agent gemcitabine treatment of patients with biliary tract cancer proved efficacious to some degree, with manageable toxicity.2–7,10
The murine orthotopic model is useful for determining the effectiveness of a chemotherapeutic agent and the most efficient mechanism of administration.11,12 Previously, no animal model for examining the effects of chemotherapeutics on biliary tract cancers existed. However, our group has recently established an orthotopic gallbladder-cancer model useful for drug tests.13,14 In this paper, we examined the effect of gemcitabine in vivo using this gallbladder cancer model. Our results showed that decreased cell proliferation and increased apoptosis correlated with prolonged survival.
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MATERIALS AND METHODS
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Cell Lines and Cell Culture Conditions
Four biliary tract carcinoma cell lines, 1 bile duct carcinoma cell line (SK-ChA-1), and 3 gallbladder carcinomas cell lines (NOZ, TGBC-1, MZ-1), were used in this study.15–17 Cells were cultured at 37° C in DMEM (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS, Sigma, St.Louis, Mo) under a humidified atmosphere containing 5% CO2.
Comparative Cytotoxicity Assays for Gemcitabine and 5–FU
The cytotoxicity for gemcitabine was assessed in the 4 biliary tract cancer cell lines using a WST-1 Cell Counting Kit (Wako, Osaka, Japan). These cell lines were cultured at a density of 1500–3000 cells/well in 96-well micro-plates with 100 µ l conditioned medium and grown overnight at 37° C in an atmosphere containing 5% CO2. Twenty-four hours later, gemcitabine was dissolved in medium at nine different concentrations (100 µ M-0.256 nM) and added to the plates at a volume of 100 µ l per well. The plates were incubated for 72 h with the chemotherapeutic agent. Then, 10 µ l of the Cell Counting Kit (WST-1, HE-PES, and 1-Methoxy PMS) was added to each well and incubated for another 2 or 3h at 37 ° C. The absorbance was read at 450 nm on a plate-spectrophotometer (BIO-RAD model 450). Absorbance values were expressed as a percentage of untreated controls and an inhibition concentration 50 (IC50) was calculated. The IC50 values represent the means of at least four independent experiments.
Animals and Orthotopic Implantation of Tumor Cells
Two-week-old athymic BALB/c male nude mice obtained from CLEA Japan Inc. (Tokyo, Japan) were used in this study. For orthotopic inoculation, we used an NOZ cell line among the 4 cell lines in the present in vivo study because NOZ cells yield a steady high frequency of tumor formation at the gallbladder and have a high potential of tumor growth and metastasis. NOZ cells were isolated from ascites derived from a 48-year-old female patient with gall-bladder cancer.15 The method used for inducing orthotopic gallbladder cancers in the mice has been described previously.13,14 Briefly, NOZ cells were suspended in 50 µ l of serum-free medium. Nude mice were anesthetized and laparotomy was done. After the cystic duct was ligated on its proximal side with 7–0 Prolene (Ethicon, Inc., Somerville, NJ), the fundus of gallbladder was ligated to support. After removing the bile juice, NOZ cells were injected with a 27-gauge Myjectoer syringe (Termo Corporation, Tokyo, Japan) into the lumen of the gallbladder. Immediately after injection, ligation was performed distally to the injection site.
Mice were kept at the Animal Care and Use Facilities at Kobe University Graduate School of Medical Sciences under specific pathogen-free conditions. All experiments were approved by the Animal Care and Ethics Committee of the Kobe University Graduate School of Medical Sciences.
Experimental Conditions for Gemcitabine Therapy for Established Gallbladder Carcinoma
Gemcitabine was supplied by Eli Lilly Japan (Kobe, Japan). Seven days after implantation of NOZ cells into the gallbladder, five mice were killed, and the presence of cancer lesions was determined and confirmed histologically. Mice were randomized into four groups as follows: (a) Group A (n = 10), beginning on the seventh day after implantation of NOZ cells, twice weekly intra-peritoneal injections of 0.9% sodium chloride were continued for three weeks, and animals were then sacrificed on the 28th day after implantation, (b) Group B (n = 10), beginning on the seventh day after implantation of NOZ cells, twice weekly intra-peritoneal injections of 125 mg/kg gemcitabine were continued for three weeks, and animals were then sacrificed on the 28th day after implantation, (c) Group C (n = 10), beginning on the seventh day after implantation of NOZ cells, twice weekly intra-peritoneal injection of 125 mg/kg gemcitabine were continued for three weeks, and the survival duration was measured, and (d) Group D (n = 6), no treatment was administered after implantation of NOZ cells and survival duration was measured.
Histological Studies
When the mice were sacrificed, tumor status, the presence or absence of liver, lung and lymph node metastases, and the presence or absence of peritoneal dissemination were recorded. Histopathology using H & E staining confirmed the identity of the disease.
Immunohistochemical Determination of PCNA and TUNEL (Apoptotic Cells)
For immunohistochemistry procedures, the tumors were fixed in phosphate-buffered formalin, embedded in paraffin, cut in 4-µ m thickness, and stained. Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) was performed using a labeled streptavidinbiotin technique described previously.18 Anti-PCNA monoclonal antibody PC 10 (DAKO, Carpenteria, CA), which reacts exclusively with nuclei, was used at a dilution of 1:200. The number of PCNA-positive cells was counted in five high-power fields (0.135 mm2 fields at x 200 magnification) selected at random, and the PCNA labeling index for each field was calculated as the percent of PCNA-positive cells (relative to the total). Apoptosis in tumor cells was detected using the terminal de-oxynucleotidyl-transferase-mediated d-UTP-biotin nick end-labeling (TUNEL) assay, as described previously.19 In the same manner as PCNA, five fields (0.135 mm2 fields at x 200 magnification) were selected at random, and the apoptotic index of each field was calculated as the percent of TUNEL-positive cells.
Statistical Analysis
Statistical analyses were performed using StatView (Abacus Concepts Inc, Berkeley, Calif). Survival curves were computed using the Kaplan-Meier method and compared using the log-rank test. PCNA-labeling indices and apoptotic indices were assessed using unpaired Student’s t-test. The incidence of metastasis was compared using Fisher’s exact test. P < 0.05 was considered statistically significant.
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RESULTS
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Gemcitabine Sensitivity in Biliary Tract Cancer Cell Lines
Gemcitabine IC50 of the biliary tract cancer cell lines, SK-ChA-1, NOZ, MZ-1 and TGBC-1, were 0.0052 µ M, 0.021 µ M, 0.16 µ M, and 0.037 µ M, respectively. SK-ChA-1 showed the highest sensitivity (5.2 nM). The gemcitabine sensitivity of the four biliary tract cancer cell lines was similar in a dose dependent manner (Fig. 1
).
Received for publication August 10, 2006.
Accepted for publication August 10, 2006.
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ABSTRACT
INTRODUCTION
) and D (thick line with (
). Mice treated with gemcitabine show a significantly better long-term survival than those with no treatment.
