Dual Role Of Smad4in Cholangiocarcinoma Cells

Objective:To establish stable Smad4knockdown cholangiocellular carcinoma cell lines. Methods:We choosed human cholangiocellular cell lines HuCCTl and RBE as objects, detected the expression of TGBR I, Smad2, Smad3, Smad4and Smad7in these cells, and the phosphorylation of Smad2and Smad3after exogenous TGF-β by wstern blotting. To generate recombinant retroviruses, a pRetroSuper-puro Smad4(Addgene, United States) was transfected into PT67cells. A pRetroSuper-puro vector was transfected as control. Retroviral supernatants were collected for infection of HuCCTl and RBE cells, which were then selected with puromycin (0.2μg/ml and1μg/ml respectively) for2weeks. The expression of Smad4and Smad2/3/4complex were evaluated by western blotting and co-immunoprecipitation. Results:Both of wild type HuCCTl and RBE cells expressed TGBR Ⅰ, Smad2, Smad3, Smad4and Smad7, and exogenous TGF-β induced the phosphorylation of Smad2and Smad3of these cells in a time-dependent manner. Both of shSmad4HuCCTl and RBE cells habored a signifficantly decreased expression level of Smad4compared to wild type and vector control cells. In contrast to vector control cells, exogenous TGF-P could not cause observable Smad2/3/4complexes in shSmad4cells. Conclusion:Stable Smad4knockdown cholangiocellular carcinoma cell lines were successfully established. Objective:To investigate the role of Smad4in the proliferation of cholangiocellular carcinoma cells. Methods:HuCCTl cells were treated with TGF-β1and/or TGBR Ⅰ inhibitor SB431542for0,1,2,3,4,5and6d respectively, and the proliferation of these cells was detected by using the Cell Counting Kit-8. After treatment with TGF-β1and/or SB431542for0,6,12,24,36,48,60and72h respectively, the proliferation of RBE cells was evaluated by using the Cell Counting Kit-8. Cholangiocellular carcinoma cells in wild type, vector control and shSmad4groups were treated with or without TGF-β1for5d (HuCCT1) or48h (RBE), followed by analysis of their proliferation by using the Cell Counting Kit-8. The expressions of cyclin A2and p21in HuCCTl cells and phospho-Rb and p21in RBE cells from different groups were determined by western blotting. HuCCTl cells form wild type, vector control and shSmad4groups were injected subcutanously into nude mice, and subcutanous trasplanted tumors were monitored. Results:TGF-β1inhibited the proliferation of both HuCCTl and RBE cells. In contrast to wild type and vector control cells, shSmad4RBE cells were insensitive to cytostatic effect of TGF-β1. TGF-β1exerted only minimal inhibitory effect on the proliferation of shSmad4HuCCT1cells compared with wild type and vector control cells, and shSmad4HuCCT1cells grew faster than wild type and vector control cells in the absence of TGF-β1. Treated with or without TGF-β1, shSmad4HuCCT1cells expressed higher level of cyclin A2than wild type and vector control cells. The expression of p21induced by TGF-β1in shSmad4HuCCTl cells was significant lower than that in vector control cells. After TGF-β1stimulation, the expression level of p21in shSmad4RBE cells was lower than that in wild type and vector control cells, but the phosphorylation level of Rb was higher than that in wild type and vector control cells. Compared with wild type and vector control HuCCTl cells, shSmad4HuCCTl cells formed greater subcutanous trasplanted tumors in nude mice. Conclusion:Smad4is involved in the inhibition of proliferation of cholangiocellular carcinoma cells, possiblely by decreasing the expression of cyclin A2and the phosphorylation of Rb and by mediating the induction of p21by TGF-β1. Objective:To investigate the role of Smad4in TGF-P-induced apoptosis of cholangiocellular carcinoma cells. Methods:Wild type RBE cells were treated with different concentration of TGF-β1for36h, and with lng/ml TGF-β1for different time periods, followed by staining with PI/Annexin-Ⅴ and analysis of apoptosis by flow cytometer. The expression of Bim and Bcl-2and the activation of caspase cascade and PARP of wild type RBE cells treated with TGF-β1for different time periods were evaluated by western blotting. After stimulated with or without TGF-β1in the presence of pan-caspase inhibitor Z-VAD-fmk for36h, the apoptosis of wild type RBE cells was tested by flow cytometer. Vector control and shSmad4RBE cells were treated with TGF-β1for36h, followed by analysis of apoptosis by flow cytometer. Wild type RBE cells were treated with TGF-β1and/or JNK inhibitor SP600125for36h, followed by analysis of the apoptosis by flow cytometer and evaluation of the expression of Bim and Bcl-2and the activation of caspase cascade and PARP by western blotting. After stimulated with TGF-β1and/or SP600125in the presence of Z-VAD-fmk for36h, the apoptosis rates of wild type RBE cells were determined by flow cytometer. Vector control and shSmad4RBE cells were treated with TGF-β1and/or SP600125for36h, followed by analysis of apoptosis by flow cytometer and evaluation of the expression of Bim and Bcl-2and the activation of caspase cascade and PARP by western blotting. After stimulated with TGF-β1and/or SP600125for6h, the phosphorylation of Smad2and Smad3of wild type RBE cells was tested by western blotting. Wild type RBE cells were co-transfected with p3TP-Lux, which encodes firefly luciferase, and pRL-TK-luc, which encodes Renilla luciferase, and then were treated with TGF-β1and/or SP600125for24h, followed by analysis of the transcriptional activities of reporter by using a dual-luciferase reporter assay system. Vector control and shSmad4RBE cells were co-transfected with p3TP-Lux, which encodes firefly luciferase, and pRL-TK-luc, which encodes Renilla luciferase, and then were treated with TGF-β1and/or SP600125for24h, followed by analysis of the transcriptional activities of reporter. Results:TGF-β1induced the apoptosis of wild type RBE cells in a dose-and time-dependent manner. Compared with vector control cells, shSmad4RBE cells were insensitive to the pro-apoptotic effect of TGF-β1. SP600125enhanced the pro-apoptotic effect of TGF-β1on wild type RBE cells. Unlike the effect on vector control cells, SP600125could not increase the apoptotic rate of shSmad4RBE cells treated with TGF-β1. Z-VAD-fmk blocked the apoptosis of wild type RBE cells treated with TGF-β1or both TGF-β1and SP600125. TGF-β1led to increased expression of Bim, decreased expression of Bcl-2and activation of caspase cascade and PARP in a time-dependent manner. SP600125enhanced the TGF-β1-induced up-regulation of Bim expression, down-regulation of Bcl-2expression and activation of caspase cascade and PARP, which was blocked by knockdown of Smad4expression. SP600125increased the TGF-β1-induced phosphorylation of Smad2and Smad3, and enhanced the TGF-β1-induced transcriptional response. The effect of SP600125on transcriptional response was reduced by knockdown of Smad4expression. Conclusion:Smad4mediated the pro-apoptotic effect of TGF-β1on RBE cells. SP600125enhanced the pro-apoptotic effect of TGF-β1on RBE cells that involves Smad4-dependent mitochondria-related activation of caspase cascade. Objective:To investigate the role of Smad4in epithelial-mesenchymal transition (EMT), migration and invasion of cholangiocellular carcinoma cells. Methods:Wild type RBE cells were treated with TGF-β1for0,6,12,24and48h, followed by analysis of the expression of N-cadherin and E-cadherin by western blotting. The morphology of wild type RBE cells was observed after stimulation of TGF-β1for24h. Vector control and shSmad4RBE cells were treated with TGF-β1for24h, followed by observation of cell morphology and evaluation of the expression of N-cadherin and E-cadherin by western blotting. A straight wound line was made in wild type HuCCTl cells, which were photographed after treatment with TGF-β1and/or TGBR I inhibitor SB431542for24h. Wild type HuCCTl cells were treated with TGF-β1and/or SB431542for48h and72h, followed by analysis of the expression of MMP-9and Vimentin by western blotting. After wounding, vector control and shSmad4HuCCT1cells were treated with TGF-β1and/or SB431542for24h and then photographed. Vector control and shSmad4HuCCTl cells were seeded into the upper chamber of transwells and were treated with TGF-β1and/or SB431542for24h, after fixing and staining with crystal violet, the cells which passed through the membrane were counted and photographed. Vector control and shSmad4HuCCTl cells were seeded into the upper chamber of transwells coated with matrigel and were treated with TGF-β1and/or SB431542for48h, after fixing and staining with crystal violet, the cells which passed through the membrane were counted and photographed. Vector control and shSmad4HuCCT1cells were treated with TGF-β1and/or SB431542for48h and72h, followed by analysis of the expression of N-cadherin, MMP-9and Vimentin by western blotting. Wild type, vector control and shSmad4HuCCT1cells were injected into the abdominal cavity of nude mice respectively, and the capacity of different group cells to disseminate to the diaphragm and form metastatic colonization was evaluated8weeks later. Results:TGF-β1induced the switch from epithelioid morphology to mesenchymal morphology in wild type RBE cells, and led to increased expression of N-cadherin and decreased expression of E-cadherin. In the absence of TGF-β1, shSmad4RBE cells showed more like epithelioids and higher expression level of E-cadherin than vector control cells. When treated with TGF-β1, shSmad4RBE cells were less prone to mesenchymal morphology accompanied with a lower expression level of N-cadherin, compared with vector control cells. Whereas vector control cells lost the expression of E-cadherin, Smad4knockdown cells still showed a considerable expression level of E-cadherin after TGF-β1treatment. In the wound healing assay, TGF-β1promoted the migration of wild type HuCCTl cells, and shSmad4HuCCTl cells showed lower capacity of migration than vector control cells. In cell migration assay by transwell, after treatment with TGF-β1, the number of shSmad4HuCCTl cells that passed through the membrane was smaller than that of vector control cells. In cell invasion assay by transwell, after treatment with or without TGF-β1, the number of shSmad4HuCCTl cells that passed through the membrane was smaller than that of vector control cells. TGF-β1induced the expression of MMP-9and increased the expression of Vimentin of wild type HuCCTl cells. The expression level of N-cadherin, MMP-9and Vimentin of shSmad4HuCCTl cells was lower than that of vector control cells in the presence or absence of TGF-β1. Compared with wild type and vector control cells, shSmad4HuCCTl cells showed a lower capacity to disseminate to the diaphragm and form metastatic colonization. Conclusion:Smad4mediates the epithelial-mesenchymal transition of RBE cells. Smad4mediates the migration and invasion of HuCCT1cells that involves up-regulation of N-cadherin, MMP-9and Vimentin expression.