Involvement of bile acids in cholangiocarcinoma progression via the Hippo-yes-associated protein signaling pathway

Figure 1 Hippo-yes-associated protein signaling pathway activation by glycochenodeoxycholic acid and deoxycholic acid. A: Western blot was used to detect the protein expression levels of mammalian STE20-like protein kinase 1, phosphorylated-mammalian STE20-like protein kinase 1, large tumor suppressor kinase 1, phosphorylated-large tumor suppressor kinase 1, yes-associated protein (YAP), and phosphorylated-YAP in HuCCT1 cells in the control, glycochenodeoxycholic acid (GDCA), deoxycholic acid (DCA), GDCA + YI, and DCA + YA groups; B: Reverse transcription quantitative real-time polymerase chain reaction was used to detect the expression level of YAP in HuCCT1 cells in the control, GDCA, DCA, GDCA + YI, and DCA + YA groups. Statistical analysis was performed using one-way ANOVA followed by Tuke’s post hoc test. Statistical significance is indicated as: ^bP < 0.01 vs control; ^dP < 0.01 vs GDCA; ^fP < 0.01 vs DCA. Data are presented as mean ± SD (n = 3 independent experiments). MST: Mammalian STE20-like protein kinase; p: Phosphorylated; LAT: Large tumor suppressor kinase; YAP: Yes-associated protein; GDCA: Glycochenodeoxycholic acid; DCA: Deoxycholic acid.

Figure 2 Effects of glycochenodeoxycholic acid and deoxycholic acid on cholangiocarcinoma cell proliferation. A: Cell Counting Kit-8 was used to detect the viability of HuCCT1 cells in the control, glycochenodeoxycholic acid (GDCA), deoxycholic acid (DCA), GDCA + YI, and DCA + YI groups; B: 5-ethynyl-2’-deoxyuridine staining was used to detect the DNA replication ability of HuCCT1 cells in the control, GDCA, DCA, GDCA + YI, and DCA + YA groups. Statistical analysis was conducted using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: ^bP < 0.01 vs control; ^dP < 0.01 vs GDCA; ^fP < 0.01 vs DCA. Data are presented as mean ± SD (n = 3 independent experiments). OD: Optical density; DAPI: 4’,6-diamidino-2-phenylindole; EDU: 5-ethynyl-2’-deoxyuridine; GDCA: Glycochenodeoxycholic acid; DCA: Deoxycholic acid.

Figure 3 Effects of glycochenodeoxycholic acid and deoxycholic acid on cholangiocarcinoma cell migration and invasion. A: Transwell assay for invasion ability of HuCCT1 cells in control, glycochenodeoxycholic acid (GDCA), deoxycholic acid (DCA), GDCA + YI, and DCA + YA groups; B: Transwell assay for migration ability of HuCCT1 cells in control, GDCA, DCA, GDCA + YI, and DCA + YA groups. Statistical analysis was conducted using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: ^bP < 0.01 vs control; ^dP < 0.01 vs GDCA; ^fP < 0.01 vs DCA. Data are presented as mean ± SD (n = 3 independent experiments). GDCA: Glycochenodeoxycholic acid; DCA: Deoxycholic acid.

Figure 4 Effects of glycochenodeoxycholic acid and deoxycholic acid on cell apoptosis. A: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling detection of apoptosis levels of HuCCT1 cells in control, glycochenodeoxycholic acid (GDCA), deoxycholic acid (DCA), GDCA + YI, and DCA + YA groups; B: Western blot detection of expression levels of apoptotic proteins caspase-3, B cell lymphoma-2, and B cell lymphoma-2 associated X protein in HuCCT1 cells in control, GDCA, DCA, GDCA + YI, and DCA + YA groups. Statistical analysis was conducted using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: ^bP < 0.01 vs control; ^dP < 0.01 vs GDCA; ^fP < 0.01 vs DCA. Data are presented as mean ± SD (n = 3 independent experiments). DAPI: 4’,6-diamidino-2-phenylindole; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling; GDCA: Glycochenodeoxycholic acid; DCA: Deoxycholic acid; Bcl-2: B cell lymphoma-2; Bax: B cell lymphoma-2 associated X protein.

Figure 5 Effects of glycochenodeoxycholic acid and deoxycholic acid on tumor growth and molecular signaling in vivo. A and B: Record and measure the size of the tumors of mice in the control, glycochenodeoxycholic acid (GDCA), deoxycholic acid (DCA), GDCA + YI, and DCA + YA groups every week, and calculate the volume, volume = (length × width²)/2; C: Weigh the mass of the tumors of mice in the control, GDCA, DCA, GDCA + YI, and DCA + YA groups. Statistical analysis was performed using two-way repeated-measures ANOVA for tumor growth curves and one-way ANOVA for tumor weights. Significance is indicated as: ^aP < 0.05, ^bP < 0.01 vs control; ^dP < 0.01 vs GDCA; ^fP < 0.01 vs DCA. Data are presented as mean ± SD (n = 3 independent experiments). GDCA: Glycochenodeoxycholic acid; DCA: Deoxycholic acid.

Figure 6 Differential effects of glycochenodeoxycholic acid and deoxycholic acid on cholangiocarcinoma progression through the Hippo-yes-associated protein signaling pathway. Glycochenodeoxycholic acid inhibits mammalian STE20-like protein kinase 1/Large tumor suppressor kinase 1 phosphorylation, activates yes-associated protein nuclear translocation, and promotes tumor growth and invasion, whereas deoxycholic acid enhances mammalian STE20-like protein kinase 1/Large tumor suppressor kinase 1 phosphorylation, retains yes-associated protein in the cytoplasm, and inhibits tumor progression and induces apoptosis. CCA: Cholangiocarcinoma; GDCA: Glycochenodeoxycholic acid; DCA: Deoxycholic acid; P: Phosphorylated; MST: Mammalian STE20-like protein kinase; LAT: Large tumor suppressor kinase; YAP: Yes-associated protein; TEAD: Transcriptional enhanced associate domain.