Figure 1.
Overexpression of p-mTOR, p-Akt, p-S6, and PI3K in PCK cholangiocytes.
The immunohistochemical expression of p-mTOR (Ser2448) and p-Akt (Ser473) was examined using liver sections of 10-month-old rats. Increased expression of p-mTOR (Ser2448) and p-Akt (Ser473) was observed in bile duct epithelium of the PCK liver compared to that of the normal liver (A). Western blot analysis using protein extracts from cultured cholangiocytes showed that the expression p-mTOR (Ser2448), p-mTOR (Ser2481), p-Akt (Ser473), p-S6, PI3K p110α, and PI3K p85 was increased in PCK cholangiocytes compared to normal cholangiocytes (B). The results of the semiquantitative analysis of Western blotting are shown in C. Arrows indicate interlobular bile ducts of the normal liver. *, p<0.01; **, p<0.05 (vs. normal cholangiocytes). Original magnifications; x400 (A).
Figure 2.
In vitro effects of PI3K and mTOR inhibitors on cholangiocytes.
Normal and PCK cholangiocytes were treated with the PI3K and/or mTOR inhibitors, and the cell proliferative activity was determined using the WST-1 assay as described in the Materials and Methods. Rapamycin, everolimus, NVP-BEZ235 and LY294002 significantly inhibited the cell proliferative activity of normal (A) and PCK (B) cholangiocytes in a dose-dependent fashion, where the most prominent inhibitory effect was observed in both cholangiocytes treated with NVP-BEZ235 for 120 hours. Rictor siRNA significantly reduced the cell proliferative activity of the PCK cholangiocytes when it was used in combination with rapamycin and LY 294002 (C). Western blot analysis showed that the expression of p-Akt (Se473) and p-S6 was reduced following 24 hour-treatment with rapamycin, everolimus and NVP-BEZ235 in a dose-dependent fashion in PCK cholangiocytes (D). The results of the semiquantitative analysis of Western blotting are shown in E. Note that the expression of p-Akt (Ser473) and p-4E-BP1 was markedly decreased in PCK cholangiocytes following the treatment with 500 nM NVP-BEZ235 (D and E). *, p<0.01; **, p<0.05 (vs. untreated control).
Figure 3.
Effects of mTOR inhibitors on cholangiocyte apoptosis in
Apoptosis was determined following the treatment of PCK cholangiocytes with rapamycin (100 ng/ml), everolimus (100 nM) and NVP-BEZ235 (500 nM) as described in the Materials and Methods. Flow cytometric analysis showed that 24-hour treatment with rapamycin and everolimus significantly induced apoptosis in PCK cholangiocytes, while it was significantly inhibited by the treatment with NVP-BEZ235 (A). NVP-BEZ235 significantly reduced the expression of cleaved caspase 3 in PCK cholangiocytes that was determined using Western blot analysis (B). The results of the semiquantitative analysis of Western blotting are shown in C. Flow cytometric analysis showed that silencing of caspase 3 using siRNA in PCK cholangiocytes resulted in the reduction of the percentage of apoptotic cells under normal and H2O2-treated conditions (D). The analysis with quantitative real-time PCR showed that treatment with NVP-BEZ235 significantly increased the expression of bcl-2 mRNA in PCK cholangiocytes (E). *, p<0.01; **, p<0.05 (vs. untreated control).
Figure 4.
Effects of mTOR inhibitors on cholangiocyte autophagy in vitro.
PCK cholangiocytes were treated with rapamycin, everolimus and NVP-BEZ235 for 24 hours, and autophagy of the cells was detected by the conversion of LC3 I to LC3 II using Western blot analysis. NVP-BEZ235 induced a dose-dependent increase in the autophagy-specific LC3 II form, while rapamycin and everolimus did not significantly induce autophagy in PCK cholangiocytes (A). The results of the semiquantitative analysis of Western blotting are shown in B. Immunocytochemistry of LC3 showed the induction of autophagy by NVP-BEZ235 in PCK cholangiocytes (C). Silencing of LC3 was performed using LC3 siRNA in PCK cholangiocytes, and immunocytochemistry (D) and Western blot analysis (E) showed that LC3 siRNA reduced LC3 II formation in the cholangiocytes. When PCK cholangiocytes were treated with NVP-BEZ235 (100 nM) for 48 hours, the cell proliferative activity of the LC3 siRNA-treated cells was significantly high than those without LC3 siRNA treatment that was determined using the WST-1 assay (F). Following 48-hour treatment of PCK cholangiocytes with 3-methyladenine (500 nM) together with NVP-BEZ235 (100 nM), the cell proliferative activity was significantly increased compared to those with NVP-BEZ235 alone (G). Original magnifications, x1000 (C and D). *, p<0.01; **, p<0.05 (vs. untreated control).
Figure 5.
Inhibition of biliary cyst formation by mTOR inhibitors in vitro.
The effects of mTOR inhibitors on biliary cyst formation were examined for normal and PCK cholangiocytes using three-dimensional cell culture system as described in the Materials and Methods. PCK cholangiocytes grew more rapidly in a spheroidal form in the Matrigel compared to normal cholangiocytes under normal condition (A). NVP-BEZ235 significantly inhibited cystic growth of both normal and PCK cholangiocytes, while rapamycin and everolimus did not have significant inhibitory effects on biliary cyst formation of the cholangiocytes (B). Knockdown of LC3 with siRNA of PCK cholangiocytes that were treated with NVP-BEZ235 significantly increased the biliary cyst formation (C). *, p<0.01 (vs. untreated control) (B): *, p<0.01; **, p<0.05 (C).
Figure 6.
Effects of in-BEZ235 on liver and renal diseases of the PCK rat.
Normal and PCK rats were treated with NVP-BEZ235 or vehicle alone daily between 4 and 8 weeks of age. Liver and kidney sections stained with hematoxylin-eosin showed that NVP-BEZ235 improved dilatation of intrahepatic bile ducts of the PCK rat, but no beneficial effects were observed on kidney lesions (A). For the PCK rat, liver and kidney cyst index (B), TUNEL-labeling index of the biliary epithelial cells (C), Ki-67-labeling index of the biliary epithelial cells (D), and liver fibrosis score (E) were determined as described in the Materials and Methods. Treatment with NVP-BEZ235 significantly reduced liver cyst index, TUNEL-labeling index, Ki-67-labeling index and liver fibrosis score of the PCK rat, whereas kidney cyst index was unaffected. Original magnifications: x200 (A, upper panel; E); x400 (D). Bars, 2 mm (A, lower panel). *, p<0.01.
Table 1.
Treatment of normal and PCK rats with NVP-BEZ235.
Figure 7.
Immunohistochemical analysis of the the liver and kidney of the PCK rat treated with NVP-BEZ235.
Treatment with NVP-BEZ235 reduced the expression of p-Akt (Ser473), p-mTOR (Ser2448) and p-S6 in bile duct epithelium of the PCK rat (A). The treatment increased immunohistochemical expression of LC3 in bile duct epithelium of the PCK rat (B). In the kidney, the expression of p-Akt (Ser473), p-mTOR (Ser2448) and p-S6 was increased in collecting tubule-derived cyst epithelium of the PCK rat without NVP-BEZ235 treatment compared to those in the renal tubules of normal rat, and their expression appeared to be reduced in the kidney of the PCK rat following the treatment (C). Arrows indicate interlobular bile ducts of the normal liver. Original magnifications; x400 (A–C).