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Research Articles: Therapeutics, Targets, and Development

Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53

Princess Margaret Hospital (University Health Network), Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Robert G. Bristow, Princess Margaret Hospital (University Health Network), Departments of Medical Biophysics and Radiation Oncology, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2936; Fax: 416-946-4586. E-mail: Rob.Bristow{at}rmp.uhn.on.ca

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 µmol/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% O₂), hypoxic (0.2% O₂), and anoxic (0% O₂) conditions. As a single agent, Nutlin-3 (2-10 µmol/L) stabilized p53 and p21^WAF levels and was toxic to WTp53-22RV1 cells (IC₅₀, 4.3 µmol/L) but had minimal toxicity toward p53-deficient cells (IC₅₀, >10 µmol/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 O₂ 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. [Mol Cancer Ther 2008;7(4):993–9]

Grant support: Terry Fox PMH Hypoxia Program Project Grant (R.G. Bristow and R.P. Hill), National Cancer Institute of Canada Operating Grant (R.G. Bristow), Fondation de France Research Grant (S. Supiot), and PMH-DRO Fellows Program Grant (S. Supiot). R.G. Bristow is a Canadian Cancer Society Research Scientist.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/28/07; revised 1/22/08; accepted 2/28/08.