Dual blockade of the Hedgehog and ERK1/2 pathways coordinately decreases proliferation and survival of cholangiocarcinoma cells.
OBJECTIVE
The Hedgehog (Hh) and pERK1/2 pathways participate in the tumorigenesis of various tissues, but there has been no report on the involvement of these two pathways in cholangiocarcinoma (CCA). The aim of this study was to evaluate the effects of the Hh pathway inhibitor, cyclopamine, and MEK inhibitor, U0126, as a single agent or in combination on CCA cell proliferation and survival.


METHODS
Seven CCA cell lines were treated with cyclopamine and/or U0126, and cell proliferation was determined by WST-1 assay. The cell cycle was investigated by fluorescence-activated cell sorter analysis. The expression levels of several cell cycle-related genes were determined by western blot analyses.


RESULTS
Cyclopamine decreased cell proliferation and arrested the cell cycle at the G1 phase, while U0126 decreased the proliferation of CCA cells with KRAS mutation stronger than with wild-type KRAS. The combination of both inhibitors had an additive antiproliferative effect, particularly in cells with KRAS mutation, and induced caspase-dependent apoptosis in the CCA cells. The expression levels of cell cycle-related proteins that are targets of the two pathways, such as cyclin D1 and cyclin B1, were strongly decreased in some CCA cell lines after combined inhibitor treatment.


CONCLUSIONS
Our results suggest that the Hedgehog and ERK1/2 pathways are important for CCA cell proliferation, and simultaneous inhibition of the two pathways may lead to stronger decreases in cell growth and viability in a subset of CCA cases.
Jinawath
Artit
A
Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45, Yushima, Tokyo 113-8519, Japan.
Akiyama
Yoshimitsu
Y
Sripa
Banchob
B
Yuasa
Yasuhito
Y
D017209:Apoptosis / Q000187:drug effects; D001650:Bile Duct Neoplasms / Q000188:drug therapy / Q000235:genetics*; D001653:Bile Ducts, Intrahepatic; D002070:Butadienes / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D018281:Cholangiocarcinoma / Q000188:drug therapy / Q000235:genetics*; D019938:Cyclin D1 / Q000378:metabolism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D014704:Veratrum Alkaloids / Q000494:pharmacology
D002070:Butadienes; D004791:Enzyme Inhibitors; D053823:Hedgehog Proteins; D009570:Nitriles; C113580:U 0126; D014704:Veratrum Alkaloids; D019938:Cyclin D1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C000541:cyclopamine