title: In vitro study of combined cilengitide and radiation treatment in breast cancer cell lines.
abstract: BACKGROUND
  Brain metastasis from breast cancer poses a major clinical challenge. Integrins play a role in regulating adhesion, growth, motility, and survival, and have been shown to be critical for metastatic growth in the brain in preclinical models. Cilengitide, an αvβ3/αvβ5 integrin inhibitor, has previously been studied as an anti-cancer drug in various tumor types. Previous studies have shown additive effects of cilengitide and radiation in lung cancer and glioblastoma cell lines. The ability of cilengitide to enhance the effects of radiation was examined preclinically in the setting of breast cancer to assess its possible efficacy in the setting of brain metastasis from breast cancer.
  
  
  METHODS
  Our panel of breast cells was composed of four cell lines: T-47D (ER/PR+, Her2-, luminal A), MCF-7 (ER/PR+, Her2-, luminal A), MDA-MB-231 (TNBC, basal B), MDA-MB-468 (TNBC, basal A). The presence of cilengitide targets, β3 and β5 integrin, was first determined. Cell detachment was determined by cell counting, cell proliferation was determined by MTS proliferation assay, and apoptosis was measured by Annexin V staining and flow cytometry. The efficacy of cilengitide treatment alone was analyzed, followed by assessment of combined cilengitide and radiation treatment. Integrin β3 knockdown was performed, followed by cilengitide and radiation treatment to test for incomplete target inhibition by cilengitide, in high β3 expressing cells.
  
  
  RESULTS
  We observed that all cell lines examined expressed both β3 and β5 integrin and that cilengitide was able to induce cell detachment and reduced proliferation in our panel. Annexin V assays revealed that a portion of these effects was due to cilengitide-induced apoptosis. Combined treatment with cilengitide and radiation served to further reduce proliferation compared to either treatment alone. Following β3 integrin knockdown, radiosensitization in combination with cilengitide was observed in a previously non-responsive cell line (MDA-MB-231). Clonogenic assays suggested little radiosensitization effects of cilengitide.
  
  
  CONCLUSIONS
  Cilengitide appears to enhance radiation response in preclinical models of breast cancer. These data suggest that the combination of radiation therapy and cilengitide may prove to be effective where radiation is utilized for the treatment of gross disease in breast cancer, such as in the setting of brain metastasis.
authors.affiliation: Department of Radiation Oncology, The Ohio State University, Columbus, OH 43210, USA. TL@osumc.edu.
authors.forename: Tim
authors.initials: T
authors.lastname: Lautenschlaeger
authors.forename: James
authors.initials: J
authors.lastname: Perry
authors.forename: David
authors.initials: D
authors.lastname: Peereboom
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Ahmed
authors.initials: A
authors.lastname: Ibrahim
authors.forename: Alexander
authors.initials: A
authors.lastname: Huebner
authors.forename: Wei
authors.initials: W
authors.lastname: Meng
authors.forename: Julia
authors.initials: J
authors.lastname: White
authors.forename: Arnab
authors.initials: A
authors.lastname: Chakravarti
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001932:Brain Neoplasms / Q000188:drug therapy / Q000532:radiotherapy / Q000556:secondary; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003131:Combined Modality Therapy; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D039641:Integrin beta Chains / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism; D009362:Neoplasm Metastasis; D012910:Snake Venoms / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D039641:Integrin beta Chains; D039661:Integrin beta3; D012910:Snake Venoms; C084344:integrin beta5; C422910:Cilengitide