title: Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53.
abstract: Nutlin-3 is a small-molecule inhibitor that acts to inhibit MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. Whether Nutlin-3 alters cell toxicity following DNA damage under oxic versus hypoxic conditions has not been studied. The potential radiosensitization (0-10 Gy) properties of Nutlin-3 (dose range, 2-10 micromol/L for up to 24 h) were investigated in vitro using three prostate cancer cell lines, 22RV1 [wild-type p53 (WTp53)], DU145 (mutated p53), and PC-3 (p53-null) under oxic (21% O(2)), hypoxic (0.2% O(2)), and anoxic (0% O(2)) conditions. As a single agent, Nutlin-3 (2-10 micromol/L) stabilized p53 and p21(WAF) levels and was toxic to WTp53-22RV1 cells (IC(50), 4.3 micromol/L) but had minimal toxicity toward p53-deficient cells (IC(50), >10 micromol/L). When combined with radiation under oxic conditions, Nutlin-3 decreased clonogenic survival in all three cell lines: 22RV1 [sensitizing enhancement ratio (SER), 1.24], DU145 (SER, 1.27), and PC-3 (SER, 1.12). Anoxia induced p53 protein expression in 22RV1 cells and this was augmented by Nutlin-3 treatment. Furthermore, Nutlin-3 was more effective as a radiosensitizer under hypoxic conditions particularly in WTp53-expressing cells: 22RV1 (SER, 1.78), DU145 (SER, 1.31), and PC-3 (SER, 1.28). The decrease in clonogenic survival with Nutlin-3 was not correlated to altered levels of radiation-induced apoptosis within the three cell lines. Our results indicate that Nutlin-3 can act as a radiosensitizer via p53-independent mechanisms under low O(2) levels. Nutlin-3 may be a useful adjunct to improve the therapeutic ratio using precision radiotherapy targeted to hypoxic cells and warrants further study in vivo.
authors.affiliation: Princess Margaret Hospital (University Health Network), Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Supiot
authors.forename: Richard P
authors.initials: RP
authors.lastname: Hill
authors.forename: Robert G
authors.initials: RG
authors.lastname: Bristow
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology*; D008297:Male; D010879:Piperazines / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000532:radiotherapy*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D013237:Stereoisomerism; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D010879:Piperazines; D011838:Radiation-Sensitizing Agents; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D020169:Caspases