Table 1.
Summary of observation in NotchICD :: AlbCre :: p53 L/L mice.
Table 2.
Summary of observation in NotchICD :: AlbCre mice.
Figure 1.
GSI IX inhibits cell proliferation in human cholangiocarcinoma cell lines.
TFK1, SZ1 and EGI1 cells showed a different expression of the Notch downstream target Hes1 (A). The cell proliferation of (B) SZ1 and (C) TFK1 cells was measured by cell proliferation assay, GSI (5 µM, 20 µM, 40 µM) inhibited cell proliferation in a dose- and time-dependent manner. Light microscopic pictures (10X magnification) were taken at 96 h to show the effect of GSI on cell proliferation of (D) SZ1 and (E) TFK1. Note that these results reveal the anti-proliferative effects of GSI IX on human cholangiocarcinoma cells.
Figure 2.
Notch plays a pivotal role for the regulation of migration in human cholangiocarcinoma cells.
Treatment with GSI IX suppresses the migration potential of human cholangiocarcinoma cell lines SZ1 and TFK1. Wound healing experiments of (A) SZ1 and (C) TFK cells cultured with GSI (5 µM, 20 µM, 40 µM) or control (DMSO). A scratch was made at (time 0 h) in both SZ1 and TFK1 and maintained for 24 h in conditioned medium with GSI or DMSO. The dotted lines are representing the edges of the wound. Photographs were taken under light microscope (10X magnification). After 24 h (A) SZ1 showed significant inhibition under 5–40 µM GSI and (C) TFK1 with a dose of 20–40 µM GSI treatment. In DMSO treated cells 80% to 90% of the wound healing was observed after 24 hrs. (A,C) The migration index (B,D) was calculated as described in Material and Methods and plotted in bar graphs. P values were calculated with ANOVA analysis of variance along with Bonferroni post test. The error bar represents standard deviation. Differences were considered as statistically significant (*) when the P-value was less <0.05.
Figure 3.
GSI IX attenuate invasion of human cholangiocarcinoma cells.
SZ1 (A) and TFK1 (A) cell lines were treated for 48 h with control (DMSO) and GSI (5 µM, 20 µM, 40 µM) to investigate the effect of GSI on invasiveness of human cholangiocarcinoma cell lines. The number of cells that invaded through the membrane was determined by light microscope (20X magnification) counterstained and invasion index (B,C) was calculated as described in Material and Methods and plotted in bar graphs. Both TFK1 and SZ1 showed significant decrease in number of invading cells by light microscope. P values were calculated with ANOVA analysis of variance along with Bonferroni post test. The error bar represents standard deviation. Differences were considered as statistically significant (*) when the P-value was less <0.05.
Figure 4.
GSI IX treatment significantly inhibited the colony formation ability of cholangiocarcinoma cell lines.
(A) Soft agar assay of GSI-treated human cholangiocarcinoma cell line (SZ1), with quantification. Compared to DMSO (control) GSI inhibits colony formation at a concentration of 20 µM. (B) Soft agar assay of GSI-treated human cholangiocarcinoma cell line (TFK1), with quantification. Compared to DMSO (control) GSI inhibits colony formation at a concentration of 5 µM. P values were calculated with ANOVA analysis of variance along with Bonferroni post test. The error bar represents standard deviation. Differences were considered as statistically significant (*) when the P-value was less <0.05.
Figure 5.
Change in the expression of epithelial and mesenchymal cell markers after GSI IX treatment in human cholangiocarcinoma.
(A) SZ1 and (B) TFK1 cells were treated with control (DMSO) and GSI (5 µM, 20 µM and 40 µM) for 48 h and 96 h. The expression of EMT markers: E-cadherin, N-cadherin, Snail, Beta Catenin and Vimentin were analyzed by Western blot. β-actin was used as a loading control. Both A) SZ1 and (B) TFK1 cells showed an increase of epithelial marker E-cadherin, Beta Catenin and an decrease of Snail and Vimentin. However, N-cadherin expression was unchanged for both cholangiocarcinoma cell lines.
Figure 6.
NICD overexpression and loss of p53 is influencing the tumor development of cholangiocarcinomas in mice.
Macro- und microscopic pictures of mice with expression of AlbCre and Notch-ICD and inactivation of p53 at different timepoints like indicated. Each mouse was dissected at the indicated timepoint. H&E staining (20X magnification) was performed and analysis was accordingly done in collaboration with an independent pathologist. Note, there was no tumor either macroscopically or microscopically detected with an age of 3 month. Starting with an age of 6 month, mice with expression of AlbCre, Notch-ICD and loss of p53 developed cholangiocarcinomas.