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Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53
Stéphane Supiot1, Richard P Hill, Robert G Bristow
1Princess Margaret Hospital (University Health Network), Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.
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.
Insights
Nutlin-3 enhances radiation therapy effectiveness, particularly in low-oxygen environments, by acting as a radiosensitizer. This suggests potential for improved prostate cancer treatment, especially when targeting hypoxic cells.
Area of Science:
- Oncology
- Molecular Biology
- Radiotherapy
Background:
- Nutlin-3 inhibits MDM2-p53 interaction, affecting DNA damage signaling.
- The impact of Nutlin-3 on cell toxicity under varying oxygen conditions post-DNA damage is unstudied.
- Prostate cancer cell lines with different p53 statuses were used to investigate radiosensitization.
Purpose of the Study:
- To investigate the radiosensitizing properties of Nutlin-3 in prostate cancer cells under oxic, hypoxic, and anoxic conditions.
- To determine if Nutlin-3 alters cell toxicity and survival following radiation therapy.
- To explore the role of p53 status and oxygen levels in Nutlin-3's radiosensitizing effects.
Main Methods:
- In vitro study using three prostate cancer cell lines (22RV1, DU145, PC-3) with varying p53 status.
- Nutlin-3 (2-10 µmol/L) was administered for up to 24 hours, followed by radiation (0-10 Gy).
- Experiments were conducted under oxic (21% O2), hypoxic (0.2% O2), and anoxic (0% O2) conditions.
Main Results:
- Nutlin-3 alone showed toxicity in wild-type p53 cells but minimal toxicity in p53-deficient cells.
- Nutlin-3 decreased clonogenic survival in all cell lines under oxic conditions when combined with radiation.
- Nutlin-3 demonstrated enhanced radiosensitization under hypoxic conditions, particularly in wild-type p53 cells (SER up to 1.78).
- The radiosensitizing effect was not correlated with altered radiation-induced apoptosis levels.
Conclusions:
- Nutlin-3 acts as a radiosensitizer, potentially through p53-independent mechanisms under low oxygen.
- Nutlin-3 may improve radiotherapy outcomes when targeted to hypoxic cancer cells.
- Further in vivo studies are warranted to validate Nutlin-3 as an adjunct therapy.