title: Potent effect of adenoviral vector expressing short hairpin RNA targeting ribonucleotide reductase large subunit M1 on cell viability and chemotherapeutic sensitivity to gemcitabine in non-small cell lung cancer cells.
abstract: BACKGROUND
  Ribonucleotide reductase large subunit (RRM1) is the main enzyme responsible for synthesis of the deoxyribonucleotides used during DNA synthesis. It is also a cellular target for gemcitabine (GEM). Overexpression of RRM1 is reportedly associated with resistance to GEM and the poor prognosis for many types of malignant tumours. Aim of the present study is to establish gene therapy against RRM1-overexpressing tumours.
  
  
  METHODS
  An adenoviral vector that encoded a short hairpin siRNA targeting the RRM1 gene (Ad-shRRM1) was constructed. Two RRM1-overexpressing non-small cell lung cancer (NSCLC) lines, MAC10 and RERF-LC-MA, were used. Finally, a human tumour xenograft model in nude mice was prepared by subcutaneously implanting tumours derived from RERF-LC-MA cells.
  
  
  RESULTS
  Ad-shRRM1 effectively downregulated RRM1 mRNA and protein in both types of NSCLC cells and significantly reduced the percentage of viable cells as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (p<0.005). Caspase 3/7 analysis revealed that transfection with Ad-RRM1 increased the percentage of apoptotic cells in culture containing either type of RRM1-overexpressing cell (p<0.001). Treatment with Ad-shRRM1 exerted a potent antitumour effect against the RRM1-overexpressing RERF-LC-MA xenografts (p<0.05). Furthermore, Ad-shRRM1-mediated inhibition of RRM1 specifically increased sensitivity to gemcitabine of each type of RRM1-overexpressing tumour cell. Combination treatment with Ad-shRRM1 and GEM exerted significantly greater inhibition on cell proliferation than Ad-shRRM1 or GEM treatment alone.
  
  
  CONCLUSIONS
  RRM1 appeared to be a promising target for gene therapy, and Ad-shRRM1 had strong antitumour effects, specifically anti-proliferative and pro-apoptotic effects, against NSCLC cells that overexpressed RRM1. Combination therapy with Ad-shRRM1 and GEM may become a new treatment option for patients with NSCLC.
authors.affiliation: Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Tokunaga
authors.affiliation: Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Electronic address: dgliu@kms.ac.jp.
authors.forename: Dage
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Nakano
authors.affiliation: Department of Urology, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Nii
authors.affiliation: Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Tetsuhiko
authors.initials: T
authors.lastname: Go
authors.affiliation: Kitano Hospital, Medical Research Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480, Japan.
authors.forename: Cheng-Long
authors.initials: CL
authors.lastname: Huang
authors.affiliation: Department of General Thoracic Surgery, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Yokomise
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D008297:Male; D008819:Mice, Nude; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D000067492:RNAi Therapeutics; D012262:Ribonucleoside Diphosphate Reductase; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D000093542:Gemcitabine
keywords: Adenovirus; Chemotherapeutic sensitivity; Gemcitabine; Gene therapy; RRM1; shRNA