title: Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer.
abstract: BACKGROUND
  Pro-survival Bcl-2 family members can promote cancer development and contribute to treatment resistance. Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. Inhibition of anti-apoptotic Bcl-2 family members therefore represents an appealing strategy to overcome resistance to anti-cancer therapies. The aim of this study was to evaluate combined effects of radiation and the pan-Bcl-2 inhibitor AT-101 in HNSCC in vitro. In addition, we determined human plasma levels of AT-101 obtained from a phase I/II trial, and compared these with the effective in vitro concentrations to substantiate therapeutic opportunities.
  
  
  METHODS
  We examined the effect of AT-101, radiation and the combination on apoptosis induction and clonogenic survival in two HNSCC cell lines that express the target proteins. Apoptosis was assessed by bis-benzimide staining to detect morphological nuclear changes and/or by propidium iodide staining and flow-cytometry analysis to quantify sub-diploid apoptotic nuclei. The type of interaction between AT-101 and radiation was evaluated by calculating the Combination Index (CI) and by performing isobolographic analysis. For the pharmacokinetic analysis, plasma AT-101 levels were measured by HPLC in blood samples collected from patients enrolled in our clinical phase I/II study. These patients with locally advanced HNSCC were treated with standard cisplatin-based chemoradiotherapy and received dose-escalating oral AT-101 in a 2-weeks daily schedule every 3 weeks.
  
  
  RESULTS
  In vitro results showed that AT-101 enhances radiation-induced apoptosis with CI's below 1.0, indicating synergy. This effect was sequence-dependent. Clonogenic survival assays demonstrated a radiosensitizing effect with a DEF37 of 1.3 at sub-apoptotic concentrations of AT-101. Pharmacokinetic analysis of patient blood samples taken between 30 min and 24 h after intake of AT-101 showed a dose-dependent increase in plasma concentration with peak levels up to 300-700 ng/ml between 1.5 and 2.5 h after intake.
  
  
  CONCLUSIONS
  AT-101 is a competent enhancer of radiation-induced apoptosis in HNSCC in vitro. In addition, in vitro radiosensitization was observed at clinically attainable plasma levels. These finding support further evaluation of the combination of AT-101 with radiation in Bcl-2-overexpressing tumors.
authors.affiliation: Department of Biological Stress Response, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Shuraila F
authors.initials: SF
authors.lastname: Zerp
authors.affiliation: Department of Radiation Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: T Rianne
authors.initials: TR
authors.lastname: Stoter
authors.affiliation: Department of Radiation Oncology (MAASTRO), GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: Frank J P
authors.initials: FJ
authors.lastname: Hoebers
authors.affiliation: Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Michiel W M
authors.initials: MW
authors.lastname: van den Brekel
authors.affiliation: Department of Radiotherapy, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Ria
authors.initials: R
authors.lastname: Dubbelman
authors.affiliation: Department of Radiation Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Gitta K
authors.initials: GK
authors.lastname: Kuipers
authors.affiliation: Department of Radiation Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: M Vincent M
authors.initials: MV
authors.lastname: Lafleur
authors.affiliation: Department of Radiation Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Slotman
authors.affiliation: Department of Biological Stress Response, The Netherlands Cancer Institute, Amsterdam, The Netherlands. m.verheij@nki.nl.|Department of Radiotherapy, The Netherlands Cancer Institute, Amsterdam, The Netherlands. m.verheij@nki.nl.|The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands. m.verheij@nki.nl.
authors.forename: Marcel
authors.initials: M
authors.lastname: Verheij
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000493:pharmacokinetics / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000532:radiotherapy; D045744:Cell Line, Tumor; D059248:Chemoradiotherapy / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006072:Gossypol / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D006258:Head and Neck Neoplasms / Q000188:drug therapy* / Q000532:radiotherapy; D006801:Humans; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D011838:Radiation-Sensitizing Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D000077195:Squamous Cell Carcinoma of Head and Neck
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D019253:Proto-Oncogene Proteins c-bcl-2; D011838:Radiation-Sensitizing Agents; D006072:Gossypol; C028178:gossypol acetic acid