title: Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth.
abstract: Although gemcitabine (2',2'-difluorocytidine monohydrochloride) is a common anticancer agent of cholangiocellular carcinoma (CCC), its growth inhibitory effects and gemcitabine resistance in CCC cells are poorly understood. Our aims were to uncover the mechanism underlying the antitumor effect of gemcitabine and to analyze the mechanism regulating in vitro CCC cell gemcitabine resistance. In addition, we sought to identify miRNAs associated with the antitumor effects of gemcitabine in CCCs. Using a cell proliferation assay and flow cytometry, we examined the ability of gemcitabine to inhibit cell proliferation in three types of human CCC cell lines (HuCCT-1, Huh28, TKKK). We also employed western blotting to investigate the effects of gemcitabine on cell cycle-related molecules in CCC cells. In addition, we used array chips to assess gemcitabine-mediated changes in angiogenic molecules and activated tyrosine kinase receptors in CCC cells. We used miRNA array chips to comprehensively analyze gemcitabine-induced miRNAs and examined clusters of differentially expressed miRNAs in cells with and without gemcitabine treatment. Gemcitabine inhibited cell proliferation in a dose- and time-dependent manner in HuCCT-1 cells, whereas cell proliferation was unchanged in Huh28 and TKKK cells. Gemcitabine inhibited cell cycle progression in HuCCT-1 cells from G0/G1 to S phase, resulting in G1 cell cycle arrest due to the reduction of cyclin D1 expression. In addition, gemcitabine upregulated the angiogenic molecules IL-6, IL-8, ENA-78 and MCP-1. In TKKK cells, by contrast, gemcitabine did not arrest the cell cycle or modify angiogenic molecules. Furthermore, in gemcitabine-sensitive HuCCT-1 cells, gemcitabine markedly altered miRNA expression. The miRNAs and angiogenic molecules altered by gemcitabine contribute to the inhibition of tumor growth in vitro.
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Toyota
authors.affiliation: Life Science Research Center, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Iwama
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Kato
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Joji
authors.initials: J
authors.lastname: Tani
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Katsura
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Miwa
authors.initials: M
authors.lastname: Miyata
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Fujiwara
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Fujita
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Fujimori
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Okura
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Kiyoyuki
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tadokoro
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Shima
authors.initials: S
authors.lastname: Mimura
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Nomura
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Miyoshi
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Asahiro
authors.initials: A
authors.lastname: Morishita
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kamada
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Yoneyama
authors.affiliation: Department of Gastroenterological Surgery, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Okano
authors.affiliation: Department of Gastroenterological Surgery, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Gastroenterology and Neurology, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Masaki
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001650:Bile Duct Neoplasms / Q000473:pathology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D018281:Cholangiocarcinoma / Q000473:pathology*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D035683:MicroRNAs; D020411:Oligonucleotide Array Sequence Analysis; D059467:Transcriptome; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D035683:MicroRNAs; D003841:Deoxycytidine; D000093542:Gemcitabine