title: Chloroquine, an autophagy inhibitor, potentiates the radiosensitivity of glioma initiating cells by inhibiting autophagy and activating apoptosis.
abstract: BACKGROUND
  Glioblastoma is refractory to conventional treatment, which is combined of surgery, chemotherapy and radiotherapy. Recent studies have shown that glioma initiating cells (GICs) contribute to tumorigenesis and radioresistance. Recently, other studies showed that the GICs use the autophagy as the major pathway to survive. Chloroquine, an anti-malarial chemical, is an autophagic inhibitor which blocks autophagosome fusion with lysosome and slows down lysosomal acidification. The aim of this study was to explore the mechanisms of chloroquine on the radiosensitivity of GICs.
  
  
  METHODS
  Human glioblastoma cell lines U87 were investigated. MTT and clonogenic survival assay were used to evaluate the cell viability and survival from radiation. The formation of autophagosomes were evaluated by immunofluorescence. Annexin V-FITC/PI staining and flow cytometry were used to quantify the apoptotic cells. The expression levels of proteins were analyzed by Western blot. Cell cycle status was analyzed by checking DNA content after staining with PI. A comet assay was used to assess the DNA repair in the cells. Tumorsphere assay was used for evaluating GICs' renewal ability.
  
  
  RESULTS
  Treatment of U87 GICs with chloroquine (10-80 nmol/L) alone inhibited the cell growth in a dose-dependent manner. A dose of chloroquine (20 nmol/L) obviously enhanced the radiation sensitivity of U87 GICs., we found more punctate patterns of microtubule-associated protein LC3 immunoreactivity in radiation-treated U87 GICs, and the level of membrane-bound LC3-II was obviously enhanced. A combination of radiation and chloroquine obviously enhanced the U87 GICs' apoptosis, as demonstrated by the enhanced levels of caspase-3, and reduced level of Bcl-2. In additon, combination of radiation and chloroquine cause G1/G0 cell cycle arrest. what's more, Chloroquine obviously weakened the repair of radiation-induced DNA damage as reflected by the tail length of the comet. Combination treatment of irradiation and chloroquine has synergistic effects on decreasing the GICs' tumorsphere number and diameter.
  
  
  CONCLUSIONS
  Chloroquine enhances the radiosensitivity of GICs in vitro, suggesting the feasibility of joint treatment with chloroquine with radiation for GBM.
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Hongxing
authors.identifier: http://orcid.org/0000-0001-9649-812X
authors.initials: H
authors.lastname: Ye
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Mantao
authors.initials: M
authors.lastname: Chen
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Cao
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Hongguang
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, People's Republic of China. zhanry1960@163.com.
authors.forename: Renya
authors.initials: R
authors.lastname: Zhan
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, People's Republic of China. zxj8065@sina.com.
authors.forename: Xiujue
authors.initials: X
authors.lastname: Zheng
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects* / Q000528:radiation effects; D001932:Brain Neoplasms / Q000473:pathology / Q000532:radiotherapy*; D045744:Cell Line, Tumor / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005910:Glioma / Q000473:pathology / Q000532:radiotherapy*; D006801:Humans; D011836:Radiation Tolerance; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*
chemical_list: D011838:Radiation-Sensitizing Agents; D002738:Chloroquine
keywords: Apoptosis; Autophagy; Chloroquine; Glioma initiating cells; Irradiation; Radiosensitivity