title: The SMAC mimetic BV6 sensitizes colorectal cancer cells to ionizing radiation by interfering with DNA repair processes and enhancing apoptosis.
abstract: BACKGROUND
  In the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells.
  
  
  METHODS
  Colorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0-4 μM) with or without irradiation (2-8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)).
  
  
  RESULTS
  BV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells.
  
  
  CONCLUSIONS
  The SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells.
authors.affiliation: Department of Radiotherapy and Oncology, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany. stephanie.hehlgans@kgu.de.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hehlgans
authors.affiliation: Department of Radiotherapy and Oncology, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany. julius.oppermann@kgu.de.
authors.forename: Julius
authors.initials: J
authors.lastname: Oppermann
authors.affiliation: Department of Radiotherapy and Oncology, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany. sebastian.reichert@kgu.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Reichert
authors.affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe University Frankfurt, Komturstr. 3a, 60528, Frankfurt am Main, Germany. simone.fulda@kgu.de.|German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. simone.fulda@kgu.de.|German Cancer Consortium (DKTK) partner site: Frankfurt, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. simone.fulda@kgu.de.
authors.forename: Simone
authors.initials: S
authors.lastname: Fulda
authors.affiliation: Department of Radiotherapy and Oncology, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany. claus.roedel@kgu.de.|German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. claus.roedel@kgu.de.|German Cancer Consortium (DKTK) partner site: Frankfurt, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. claus.roedel@kgu.de.
authors.forename: Claus
authors.initials: C
authors.lastname: Rödel
authors.affiliation: Department of Radiotherapy and Oncology, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany. franz.roedel@kgu.de.
authors.forename: Franz
authors.initials: F
authors.lastname: Rödel
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000473:pathology*; D004260:DNA Repair / Q000187:drug effects*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014162:Transfection
chemical_list: C582180:BV6 peptide; D051034:Inhibitor of Apoptosis Proteins; D009842:Oligopeptides; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents