title: Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs.
abstract: BACKGROUND
  Cancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.
  
  
  METHODS
  The human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.
  
  
  RESULTS
  Poorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.
  
  
  CONCLUSIONS
  Our novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.
authors.forename: Noriaki
authors.initials: N
authors.lastname: Hashimoto
authors.affiliation: Department of Digestive Surgery and Surgical Oncology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. tsune-r@yamaguchi-u.ac.jp.
authors.forename: Ryouichi
authors.initials: R
authors.lastname: Tsunedomi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yoshimura
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Shoichi
authors.initials: S
authors.lastname: Hazama
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oka
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000080924:Aldehyde Dehydrogenase 1 Family; D000970:Antineoplastic Agents / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular; D054337:Cell Dedifferentiation; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000502:physiology; D016601:RNA-Binding Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D050697:Retinal Dehydrogenase; D018874:Spheroids, Cellular / Q000187:drug effects
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; C497442:HIF1A protein, human; D018398:Homeodomain Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C516326:LIN28B protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase