INTRODUCTION
MATERIALS AND METHODS
Establishment of an O. viverrini-associated hamster CCA cell line
Chromosome analysis
Ham-1 allograft hamster model and anti-tumor activity of BBR in vivo
Immunohistochemistry analysis
Tetrazolium dye methylthiotetrazole assay
Cell cycle analysis
RESULTS
Ham-1 morphology and chromosome analyses
![]() | Fig. 1Ham-1 cell line. (A) A mixture of CCA cells and stromal cells were isolated and cultured for a week. Arrowheads indicate CCA cell clusters surrounded by fibroblasts. (B) Ham-1 cells at the 15th passage. Ham-1 cells exhibited polygonal shapes. Pictures were taken under a phase contrast microscope at ×100 magnification. (C) Ham-1 chromosome. Ham-1 cells were prepared and stained as previously described [21]. Pictures on the left represent diploid chromosomes (2n=44) (upper) and polyploid chromosomes (lower). Pictures were taken under a light microscope using an oil immersion lens (×1,000). A representative diploid karyotype is presented on the right. |
Ham-1 allograft transplantation
![]() | Fig. 2Ham-1 allograft tumors. (A) 2×105 or 5×105 Ham-1 cells were injected intradermally into the hamster skin (n=10/group). Tumors were successfully formed at all injected sites and were collected on day 24. The tumors were weighed. Tumor weights were correlated with the starting cell numbers. Tumors within the hamster skin were demonstrated in the small insets. (B) The allograft transplanted tumors exhibited similar histopathological properties as the primary tumor; strongly positive tumor cell cytoplasmic immunostaining for cytokeratin 19 and positive stromal staining for a myofibroblast marker (α-smooth muscle actin). Pictures were taken under light microscopy (×100). Bar=100 µm. |
Effects of BBR on Ham-1 cells in vitro and in vivo
![]() | Fig. 3BBR inhibited Ham-1 proliferation and induced G1 cell cycle arrest in vitro. (A) Ham-1 cells were treated with various concentrations of BBR for 24 hr and 48 hr. The anti-proliferative effects were determined by the MTT assay. The percent cell numbers relative to untreated cells are presented as the mean±SD of 3 independent experiments. (B) Effects of BBR on cell cycle distribution were checked by propidium iodide staining and flow cytometry analysis. The percentages of cells in each phase are indicated. Data are representative of 2 independent experiments. |
![]() | Fig. 4In vivo anti-tumor effects of BBR on the Ham-1 allograft hamster model. 5×105 cells of Ham-1 were injected intradermally into the dorsal skin of hamster (n=10/group). 10 mg/kg of BBR were given orally every day for 21 days. Equal volumes of drinking water were given to the control group. (A) Average weights of hamsters were compared. (B) Tumor weights were determined and representative tumors from control and BBR groups are shown (small inset). (C) The proliferative index (PCNA) and expressions of cell cycle control proteins (cyclin D1 and cyclin E) of Ham-1 cells in tumor tissues were assessed by immunohistochemistry. Pictures were taken under light microscope (×400). Bar=20 µm. (D) IHC scores were determined by 2 independent assessors and presented as mean±SD. *P<0.05, **P<0.001, vs the control group. |