Expression of Oncogenes and Changes of the Liver and Bile Ducts in Cholangiocarcinoma of Syrian Golden Hamsters Induced by Clonorchis sinensis and N-Nitrosodimethylamine

Abstract

Clonorchis sinensis has been reclassified as Group-I bio-carcinogen for cholangiocarcinoma (CCA) in humans by IARC in 2009, however, the mechanism of carcinogenesis is little known. The present study investigated CCA mechanism in hamster model by means of imaging, histopathology, immunohistochemical analysis, and expression of oncogenesis related genes. The experimental animals were divided into 4 groups: uninfected control (Ctrl.), C. sinensis (Cs), N-Nitrosodimethylamine (NDMA), and C. sinensis with NDMA (Cs+NDMA). The body weight of hamsters in every week and liver and spleen weight at necropsy were measured. Abdominal ultrasonography (USG) of all hamsters and magnetic resonance imaging (MRI) of randomly selected hamsters were performed at every 2 weeks up to 16th week of infection. After 4, 8, 12, and 16 weeks animals were sacrificed and investigated. USG detected first mass-forming lesion (MFL) in the Cs+NDMA group at 6th week and 77.3% of overall prevalence at 16th week of infection. The MFLs were consistent and increased in size during the follow-up examination. MRI recognized same MFLs with USG, but recovered a cyst in the Cs+NDMA group at the 6th week. Findings by both USG and MRI of the Ctrl. and NDMA groups were non-specific throughout the experiment. The Cs+NDMA group hamsters underwent significant loss of body weight (24.8%) and gain of the liver (88.6%) and spleen (255.2%) weight. Thin 2-4 mm slicing of the liver confirmed 100% prevalence of MFLs in the Cs+NDMA group with an intensity of 10.92 ± 5.76. Semi quantitative histopathology (as % of total tissue area) revealed severe inflammation (34.3% ± 3.6%), proliferation of bile ducts (11.4% ± 2.1%), periductal fibrosis (11.2% ± 1.9%), and abscess (8.7% ± 3.13 %) in the Cs group. In the Cs+NDMA group the above histopathological findings were present moderately but showed intrahepatic CCA of well (21.8% ± 1.5 %), moderate (13.3% ± 1.3%), and poorly (10.8% ± 1.3%) differentiated types. The CCA mass was filled or surrounded by collagen fibers, mainly of type I. Immunohistochemisty against proliferative cell nuclear antigen (PCNA) found strong positive reaction in the Cs+NDMA group. Metaplasia of mucin secreting cells was 7 times higher and mitotic figures were significantly increased in the Cs+NDMA group. Real-time PCR and western blot analysis showed dysregulation of a number of genes/proteins. A novel oncogene for CCA called gankyrin was overexpressed, CDK4 and p16INK4 genes were upregulated but, p53 gene and RB protein were downregulated in the tumor tissue. In conclusion, CCA in hamsters may develop after 6 weeks of infection with weight loss and hepato-spleenomegaly. The mass can be monitored by USG and MRI. Upregulation of oncogenes of gankyrin, CDK4, and downregulation of p53 gene and RB protein may play an important role in the process of carcinogenesis of CCA in the C. sinensis-NDMA mediated hamster model. Excessive mRNA expression of p16INK4 and increased PCNA may be marker candidates for CCA in clonorchiasis.