Involvement of PSMD10, CDK4, and Tumor Suppressors in Development of Intrahepatic Cholangiocarcinoma of Syrian Golden Hamsters Induced by Clonorchis sinensis and N-Nitrosodimethylamine

Abstract

Background: Clonorchis sinensis is a group-I bio-carcinogen for cholangiocarcinoma (CCA). Although the epidemiological evidence links clonorchiasis and CCA, the underlying molecular mechanism involved in this process is poorly understood. In the present study, we investigated expression of oncogenes and tumor suppressors, including PSMD10, CDK4, p53 and RB in C. sinensis induced hamster CCA model.

Methods: Different histochemical/immunohistochemical techniques were performed to detect CCA in 4 groups of hamsters: uninfected control (Ctrl.), infected with C. sinensis (Cs), ingested N-nitrosodimethylamine (NDMA), and both Cs infected and NDMA introduced (Cs+NDMA). The liver tissues from all groups were analyzed for gene/protein expressions by quantitative PCR (qPCR) and western blotting.

Principal findings: CCA was observed in all hamsters of Cs+NDMA group with well, moderate, and poorly differentiated types measured in 21.8% ± 1.5%, 13.3% ± 1.3%, and 10.8% ± 1.3% of total tissue section areas respectively. All CCA differentiations progressed in a time dependent manner, starting from the 8th week of infection. CCA stroma was characterized with increased collagen type I, mucin, and proliferative cell nuclear antigen (PCNA). The qPCR analysis showed PSMD10, CDK4 and p16INK4 were over-expressed, whereas p53 was under-expressed in the Cs+NDMA group. We observed no change in RB1 at mRNA level but found significant down-regulation of RB protein. The apoptosis related genes, BAX and caspase 9 were found downregulated in the CCA tissue. Gene/protein expressions were matched well with the pathological changes of different groups except the NDMA group. Though the hamsters in the NDMA group showed no marked pathological lesions, we observed over-expression of Akt/PKB and p53 genes proposing molecular interplay in this group which might be related to the CCA initiation in this animal model.

Conclusions/significance: The present findings suggest that oncogenes, PSMD10 and CDK4, and tumor suppressors, p53 and RB, are involved in the carcinogenesis process of C. sinensis induced CCA in hamsters.

Fig 1. Gross view and histopathology of representative hamster liver by the groups.

(A) Photographs of the liver after 16 weeks of infection (a-d represents Ctrl., Cs, NDMA, and Cs+NDMA group respectively). (B) H&E stained liver sections in different groups of hamsters (original magnification ×200) (a-d represents Ctrl., Cs, NDMA, and Cs+NDMA group respectively). Normal hepatic cells and biliary triad are observed in Ctrl. Group; proliferated bile duct, periductal fibrosis, and inflammatory cells are observed in the Cs group (yellow arrow head indicates an egg of C. sinensis enlarged below left); normal findings in NDMA group; highly proliferated bile duct epithelial cells in a fibrous stroma leading to CCA in Cs+NDMA group.

Fig 2. Semiquantitative neoplastic histopathological changes in different groups of hamsters.

(A) Bar diagram showing well differentiated CCA (WDC), moderately differentiated CCA (MDC), and poorly differentiated CCA (PDC) in Cs+NDMA group. One hamster of NDMA group showed WDC and MDC in a tiny fraction of the liver. (B) Bar diagram showing progressive development of WDC, MDC, and PDC from 8^th weeks of infection. (C-E) Histopathological views of CCA (original magnification ×200): C, WDC; D, MDC; and E, PDC. Nuclear polymorphism of bile cells noted in C.

Fig 3. Collagen, mucin, and PCNA staining of liver tissue after 16 weeks of infection.

(A) The livers of Ctrl. and NDMA groups show little collagen fibers (blue in Masson’s trichrome and red in Sirius red staining) at the portal triad. Hamsters in C. sinensis infected group (Cs) have collagen fibers around the bile duct, and dense collagen fibers are observed in Cs+NDMA group. (B) No stained cells or tissues are in the Ctrl. and NDMA group, but scanty or abundant mucin substances are detected in the Cs or Cs+NDMA groups (positive: blue by Alcian blue and red to pink in rapid mucin staining). (C) PCNA staining is strongly positive (red-brown) in Cs+NDMA group, demonstrating a higher number of proliferative cells (yellow arrowheads). Both large and small sized (enlarged) bile cells show positive reactions. Original magnification ×200.

Fig 4. Immunohistochemistry of collagen type I and IV in Cs+NDMA group by infection duration.

Collagen type I is deposited from 8^th to 16^th week and type IV at the 16^th week of infection (positive: red color). W = C. sinensis worm. Original magnification ×100.

Fig 5. Relative mRNA expression of oncogenesis related genes by real-time PCR.

The significant increase in the expression of PSMD10, CDK4, p16INK4, and decrease of p53, BAX, caspase 9 genes are observed in the CCA tumor tissues (Cs+NDMA-T). P < 0.05 was considered as statistically significant. P < 0.05 and P < 0.001 indicated as asterisk and dagger respectively.

Fig 6. Relative expression of oncogenesis related proteins by western blot analysis by NIH—ImageJ software.

(A) Protein bands representative of three independent experiments. (B) Increased expression of oncogene CDK4 in Cs and Cs+NDMA groups, and decreased expression of p53 and RB proteins in Cs+NDMA group are shown. P < 0.05 was considered as statistically significant. P < 0.05 and P < 0.001 indicated as asterisk and dagger respectively.