title: HMGN5 knockdown sensitizes prostate cancer cells to ionizing radiation.
abstract: BACKGROUND
  High Mobility Group N (HMGN) proteins are a family of chromatin structural proteins that specifically bind to nucleosome core particles. HMGN5 is a novel and characteristic member of the HMGN protein family. We have previously found that HMGN5 is upregulated in prostate cancer and its downregulation had been demonstrated to induce apoptosis and G2-M cell cycle arrest.
  
  
  METHODS
  The radiosensitization effect of HMGN5 knockdown on PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. The DNA double-strand break (DSB) repair kinetics of HMGN5 knockdown and control cells after radiation exposure was evaluated using immunocytofluorescence. The mitochondrial reactive oxygen species (ROS) levels were estimated using Dihydrorhodamine 123 (DHR 123) probes. Expression of mitochondrial antioxidant MnSOD was measured by real-time PCR and Western blot. The expression of antiapoptotic proteins Bcl-2 and Bcl-xL as well as cleavage of caspase-3, caspase-9, and PARP were also measured using Western blot.
  
  
  RESULTS
  HMGN5 knockdown cells exhibit decreased clonogenic survival and increased apoptosis rate in response to 2-8 Gy ionizing radiation (IR). Loss of HMGN5 does not affect the DSB repair kinetics after radiation exposure. HMGN5 knockdown cells demonstrated increased mitochondrial ROS level and suppressed induction of MnSOD upon radiation compared with control cells upon radiation. Further, MnSOD knockdown resulted in inhibited cell viability as well as increased mitochondrial ROS level and apoptosis upon radiation in PC3 and DU145 cells. Finally, HMGN5 knockdown cells showed significantly decreased levels of antiapoptotic proteins Bcl-2 and Bcl-xL as well as increased cleavage of caspase-3, caspase-9, and PARP compared with control cells after radiation.
  
  
  CONCLUSIONS
  HMGN5 knockdown sensitizes prostate cancer cells to ionizing radiation, and the radiosensitization effect may be partially mediated through suppressed induction of MnSOD and enhanced activation of apoptosis pathway in response to IR.
authors.affiliation: Department of Urology, Peking University First Hospital and the Institute of Urology, Peking University, Beijing, China; National Urological Cancer Center, Beijing, China.
authors.forename: Boxing
authors.initials: B
authors.lastname: Su
authors.forename: Bentao
authors.initials: B
authors.lastname: Shi
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tang
authors.forename: Zhongqiang
authors.initials: Z
authors.lastname: Guo
authors.forename: Xi
authors.initials: X
authors.lastname: Yu
authors.forename: Xinyong
authors.initials: X
authors.lastname: He
authors.forename: Xuesong
authors.initials: X
authors.lastname: Li
authors.forename: Xianshu
authors.initials: X
authors.lastname: Gao
authors.forename: Liqun
authors.initials: L
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D024001:HMGN Proteins / Q000235:genetics*; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology / Q000532:radiotherapy*; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics*; D014410:Tumor Stem Cell Assay
chemical_list: D024001:HMGN Proteins; C429289:HMGN5 protein, human; D011838:Radiation-Sensitizing Agents; D017382:Reactive Oxygen Species; D015534:Trans-Activators; D013482:Superoxide Dismutase
keywords: high mobility group N 5 (HMGN5); ionizing radiation; prostate cancer