Functional And Mechanism Analysis Of Baicalin On Human Cholangiocarcinoma Cells

Objective: To investigate the inhibitory effect of baicalin on human cholangiocarcinoma cells and its molecular mechanisms.Methods:(1)The role of Baicalin on the viability of human cholangiocarcinoma cells: After QBC939 and RBE cells were cultured and treated with different concentrations of Baicalin,cells viability was detected by CCK-8;(2)The mechanism of Baicalin on the viability of cholangiocarcinoma cells: QBC939 and RBE cells were treated with Rapa(rapamycin)or AZD(ATP-competitive mTOR kinase inhibitor),both are mTORC1 signaling pathway inhibitors,cells viability was detected by CCK-8;(3)The role of Baicalin on the proliferation of cholangiocarcinoma cells: After QBC939 and RBE cells were treated with different concentrations of Baicalin,the growth rate of the cells was observed under a microscope,and the cell proliferation was analyzed using plate cell clone formation experiments;(4)The mechanism of Baicalin on the proliferation of cholangiocarcinoma cells: QBC939 and RBE cells were treated with different concentrations of Baicalin,the protein levels of Cyclin D1,cyclin inhibitor kinase p27 and c-Myc were detected by Western blot,and the cells cycle were detected by flow cytometry.After c-Myc was interfered with siRNA in Baicalin-treated cells,Western blot was used to detect p27 and Cyclin D1 protein levels;(5)The role of Baicalin on apoptosis induction in cholangiocarcinoma cells: QBC939 and RBE cells were treated with different concentrations of Baicalin,then,the levels of apoptosis markers PARP and caspase3(c-caspase3)were detected by Western blot;(6)The mechanism of Baicalin–induced apoptosis in cholangiocarcinoma cells: After QBC939 and RBE cells were treated with different concentrations of Baicalin,the levels of P70s6 k,S6,raptor,AMPK and their phosphorylation levels were detected by Western blot.Moreover,QBC939 and RBE cells were treated with Rapa or AZD,the levels of PARP and caspase3(c-caspase3)were detected by Western blot.Subsequently,using AICAR(AMPK activator)to activate AMPK and down-regulating AMPK expression with siRNA,Western blot was used to detect the expression levels of AMPK,p-AMPK,raptor,p-raptor(Ser792 and Ser863),P70s6 k,p-P70s6 k,S6,p-S6,Rheb,PARP and c-caspase3.Results:(1)Baicalin inhibits the viability of human cholangiocarcinoma cells: CCK-8 results showed that Baicalin resulted viability significantly reduced in QBC939 and RBE cells in a dose-and time-dependent manner;(2)Baicalin inhibits the viability of human cholangiocarcinoma cells through the mTORC1 signaling pathway: QBC939 and RBE cells were treated with Rapa and AZD,CCK-8 results showed that cell viability was significantly reduced upon Baicalin treatment;(3)Baicalin inhibits the proliferation of cholangiocarcinoma cells: Both microscope and the plate cell clone formation experiments showed that the cell growth rate of QBC939 and RBE cells were significantly inhibited upon Baicalin treatment;(4)Baicalin inhibits the proliferation of cholangiocarcinoma cells through blocking the cell cycle: QBC939 and RBE cells were treated with Baicalin,and Western blot results showed that the protein level of p27 was significantly increased,while the protein level of Cyclin D1 was significantly reduced compared with control group.The results of flow cytometry showed that the cell cycle of QBC939 cells was arrested in the S phase.Moreover,Western blot results showed that Baicalin resulted the protein levels of c-Myc were significantly reduced in QBC939 and RBE cells in a dose-and time-dependent manner.In addition,using siRNA to interfere with the expression of c-Myc in QBC939 and RBE cells,Western blot results showed that the expression level of p27 protein was significantly increased,while that of Cyclin D1 was significantly reduced compared with control group;(5)Baicalin induces apoptosis of cholangiocarcinoma cells: QBC939 and RBE cells were treated with different concentrations of Baicalin,Western blot results showed that the expression levels of PARP and c-caspase3 were significantly increased in a dose-and time-dependent manner;(6)Baicalin induces apoptosis through the AMPK/raptor/ mTORC1 signal pathway: QBC939 and RBE cells were treated with different concentrations of Baicalin,Western blot results showed that the phosphorylation levels of P70s6 k,S6 and raptor(ser792)were significantly reduced in a dose-dependent manner compared with control group.Moreover,QBC939 and RBE cells were treated with Rapa and AZD,Western blot results showed that the expression levels of PARP and c-caspase3 were significantly increased compared with control group.In addition,QBC939 and RBE cells were treated with different concentrations of Baicalin,Western Blot results showed that the phosphorylation level of AMPK was significantly up-regulated in a dose-and time-dependent manner compared with control group.On the other hand,QBC939 and RBE cells were treated with AICAR,Western blot results showed that AICAR promoted the phosphorylation levels of AMPK and raptor at Ser792(direct phosphorylation),but inhibited raptor phosphorylation at Ser863(indirect phosphorylation)and Rheb expression.Further results showed that the phosphorylation levels of P70s6 k and S6 were significantly reduced in AICAR-treated QBC939 and RBE cells.After siRNA was used to silence AMPK,the phosphorylation levels of P70s6 k and S6 were increased significantly.In addition,after using AICAR to promote AMPK phosphorylation in combination with Baicalin,Western blot results showed that the protein levels of PARP and c-Caspase3 were significantly increased.On the contrary,AMPK knockdown inhibited Baicalin-induced the protein levels of PARP and c-Caspase3 in QBC939 and RBE cells.Conclusion:(1)Baicalin can inhibit the viability and proliferation and induce apoptosis of cholangiocarcinoma cells;(2)Baicalin inhibits the proliferation of cholangiocarcinoma cells through c-Myc inhibition;(3)Baicalin inhibits the cell viability and induces apoptosis of cholangiocarcinoma cells through the AMPK/raptor/mTORC1 signaling pathway.