title: Fractionated radiotherapy is the main stimulus for the induction of cell death and of Hsp70 release of p53 mutated glioblastoma cell lines.
abstract: BACKGROUND
  Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite a multimodal therapy consisting of resection followed by fractionated radiotherapy (RT) combined with the chemotherapeutic agent (CT) temozolomide (TMZ), its recurrence is almost inevitable. Since the immune system is capable of eliminating small tumor masses, a therapy should also aim to stimulate anti-tumor immune responses by induction of immunogenic cell death forms. The histone deacetylase inhibitor valproic acid (VPA) might foster this.
  
  
  METHODS
  Reflecting therapy standards, we applied in our in vitro model fractionated RT with a single dose of 2Gy and clinically relevant concentrations of CT. Not only the impact of RT and/or CT with TMZ and/or VPA on the clonogenic potential and cell cycle of the glioblastoma cell lines T98G, U251MG, and U87MG was analyzed, but also the resulting cell death forms and release of danger signals such as heat-shock protein70 (Hsp70) and high-mobility group protein B1 (HMGB1).
  
  
  RESULTS
  The clonogenic assays revealed that T98G and U251MG, having mutated tumor suppressor protein p53, are more resistant to RT and CT than U87MG with wild type (WT) p53. In all glioblastoma cells lines, fractionated RT induced a G2 cell cycle arrest, but only in the case of U87MG, TMZ and/or VPA alone resulted in this cell cycle block. Further, fractionated RT significantly increased the number of apoptotic and necrotic tumor cells in all three cell lines. However, only in U87MG, the treatment with TMZ and/or VPA alone, or in combination with fractionated RT, induced significantly more cell death compared to untreated or irradiated controls. While necrotic glioblastoma cells were present after VPA, TMZ especially led to significantly increased amounts of U87MG cells in the radiosensitive G2 cell cycle phase. While CT did not impact on the release of Hsp70, fractionated RT resulted in significantly increased extracellular concentrations of Hsp70 in p53 mutated and WT glioblastoma cells.
  
  
  CONCLUSIONS
  Our results indicate that fractionated RT is the main stimulus for induction of glioblastoma cell death forms with immunogenic potential. The generated tumor cell microenvironment might be beneficial to include immune therapies for GBM in the future.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Rubner
authors.forename: Carolin
authors.initials: C
authors.lastname: Muth
authors.forename: Annedore
authors.initials: A
authors.lastname: Strnad
authors.forename: Anja
authors.initials: A
authors.lastname: Derer
authors.forename: Renate
authors.initials: R
authors.lastname: Sieber
authors.forename: Rolf
authors.initials: R
authors.lastname: Buslei
authors.forename: Benjamin
authors.initials: B
authors.lastname: Frey
authors.forename: Rainer
authors.initials: R
authors.lastname: Fietkau
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. udo.gaipl@uk-erlangen.de.
authors.forename: Udo S
authors.initials: US
authors.lastname: Gaipl
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage; D017209:Apoptosis; D001932:Brain Neoplasms / Q000532:radiotherapy*; D002453:Cell Cycle; D016923:Cell Death; D045744:Cell Line, Tumor; D003606:Dacarbazine / Q000008:administration & dosage / Q000031:analogs & derivatives; D019583:Dose Fractionation, Radiation; D016195:G2 Phase; D005909:Glioblastoma / Q000188:drug therapy* / Q000235:genetics* / Q000532:radiotherapy*; D024243:HMGB1 Protein / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009336:Necrosis; D000077204:Temozolomide; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D014635:Valproic Acid / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; D018840:HSP70 Heat-Shock Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D014635:Valproic Acid; D003606:Dacarbazine; D000077204:Temozolomide