Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53

Abstract

Background: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells.

Methods: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3.

Results: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity.

Conclusion: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients.

Figure 1

Nutlin-3 inhibits the proliferation of p53 wild-type laryngeal squamous cell carcinoma cells. Growth curves obtained for laryngeal squamous cell carcinoma cells in the presence and absence of Nutlin-3. A total of 1–2 × 10⁵ cells, depending on the cell line adherence and growth characteristics, were seeded in six-well plates and allowed to attach for 24 h after which they were either left untreated, treated with DMSO (the vehicle for Nutlin-3) or treated with Nutlin-3 at a range of concentrations, as indicated. Cells were harvested 24 and 48 h later and counted in a Beckman Coulter Counter. Error bars represent the s.e.m. from three independent wells. The results shown are from a typical experiment repeated on at least three occasions.

Figure 2

Nutlin-3 treatment induces p53 target gene expression in p53 wild-type laryngeal squamous cell carcinoma cells. Western blot analysis of laryngeal squamous cell carcinoma cells harvested 48 h after either no treatment, treatment with DMSO (vehicle control) or treatment with Nutlin-3 at 0.5 μM or 5.0 μM, as indicated. All lanes were loaded with 50 μg of protein. Blots were probed with antibodies for p53, MDM2, p21 and β-actin (protein loading control) as indicated. Note the absence of any detectable p53 in UM-SCC-12 cells, known to be p53 null from DNA sequence analysis (Q104Stop, LOH). UM-SCC-5 shows two distinct bands of p53 protein, indicative of a well-documented polymorphism in codon 72, which was also confirmed by DNA sequence analysis (codon 72 is Pro/Arg, data not shown). The results shown are from a typical experiment repeated on at least three occasions.

Figure 3

Nutlin-3 induces cell cycle arrest in p53 wild-type laryngeal squamous cell carcinoma cells. Histograms summarising data obtained by flow cytometry after propidium iodide staining (PI) of fixed laryngeal squamous cell carcinoma cells (also see Supplementary Data Figure 2). Cells were seeded and either left untreated (No Tx) or were treated for 48 h with DMSO, 0.5 μM Nutlin-3 or 5 μM Nutlin-3 as indicated. Cells were harvested, fixed in ethanol and stained with propidium iodide. The results shown are from a typical experiment repeated on at least three occasions.

Figure 4

Nutlin-3 radiosensitises p53 wild-type laryngeal squamous cell carcinoma cells. Clonogenic assays were performed on laryngeal squamous cell carcinoma cells exposed to the indicated doses of γ-irradiation. Cells were pre-treated with either DMSO or 5 μM Nutlin-3 for 30 min and then irradiated with 0, 2, 4 or 6 Gy as indicated. Cells were then re-seeded and allowed to form colonies for a period of 2–3 weeks. A colony was defined as containing at least 50 cells, equivalent to greater than five cell doublings. Colonies were fixed, stained and counted. The number of cells seeded varied according to the clonogenic characteristics of each cell line: 6400 for UM-SCC-5, 32 000 for UM-SCC-10A, 1600 for UM-SCC-11B, 400 for UM-SCC-12, 25 000 for UM-SCC-17AS, 6400 for UM-SCC-17A and 1600 for UM-SCC-81B. The quadratic equation S(D)/S(0)=exp(−(αD+βD²)) was used to obtain survival parameters, and all r² values were >0.92. Note that cells expressing full-length mutant p53, in the absence of endogenous wild-type p53, were relatively radiosensitive compared with cells harbouring all other genotypes. The results shown are from a typical experiment repeated on at least three occasions.

Figure 5

Analysis of apoptosis in laryngeal squamous cell carcinoma cells treated with ionising radiation and/or Nutlin-3. Summary of flow cytometric analysis of LSCC cells after either irradiation with 6 Gy or mock treatment. As indicated, cells were pre-treated for 30 min with either DMSO or 5 μM Nutlin-3. After treatment, cells were re-seeded for 48 h before harvesting. Cells were then incubated with Annexin-V-FITC and propidium iodide to measure apoptosis and membrane integrity respectively. The log/log plot were divided into four quadrants as shown in Supplementary Data Figure 3, and the percentage of cells in each quadrant is presented in the histograms. Cells in the lower right quadrant stained with Annexin-V (AV+/PI−) are indicative of cells undergoing apoptosis and cells that have died are expected in the upper right quadrant staining for both Annexin-V and propidium iodide (AV+/PI+). Healthy/intact cells are negative for Annexin-V and propidium iodide (AV−/PI−). The results shown are from a typical experiment repeated on at least three occasions.

Figure 6

Laryngeal squamous cell carcinoma cells display increased complexity and/or size after exposure to ionising radiation and/or Nutlin-3. Flow cytometric analysis of the same samples shown in Figure 5. Forward vs side scatter plots were analysed to identify cells displaying unusual complexity and/or size (increased side scatter and/or forward scatter, respectively). Gates were applied to define the main population of cells and numbers of cells lying outside the gates were recorded. The data are presented in the table as the percentage of gated cells (denoted as ‘in’) and the percentage of more complex and/or larger cells lying outside the gates (denoted as ‘out’) shown as the darker bar. Cells lying outside the gates are larger and more granular, a phenotype typical of senescent cells. The results shown are from a typical experiment repeated on at least three occasions.

Figure 7

Increasing radiosensitivity in response to nutlin-3 treatment is associated with increased senescence. Senescence-associated β-galactosidase (SA-β-gal) assay of laryngeal squamous cell carcinoma cells after irradiation at 6 Gy after ∼30 min of pre-treatment with DMSO or 5 μM Nutlin-3. Cells were treated and seeded for 48 h. A β-galactosidase assay was used at low pH (6.0), which stains senescent cells blue/green. A total of 300 cells were counted from three independent samples per condition, and the percentage of senescent cells as a fraction of the total was calculated. Error is expressed as the s.e.m. There is a clear increase in the number of senescent cells with Nutlin-3 treatment in TP53 wild-type cells (UM-SCC-17AS and UM-SCC-17A), and also a further significant increase in senescence after irradiation, which is statistically significant (Student's t-test). Cells expressing full-length mutant p53 with LOH (UM-SCC-11B and UM-SCC-81B, the most radiosensitive) did not show any detectable senescence under any of the conditions examined. The results shown are from a typical experiment repeated on at least three occasions.