Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation

Abstract

Background: Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored.

Methods: Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit.

Results: We show that despite similar reovirus replication in p53(+/+) and p53(-/-) cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53(-/-) or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors.

Conclusion: Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.

Figure 1

p53 stabilisation by Nutlin-3a enhances virus-associated cytotoxicity and release of progeny virions. (A) Upper panel: Human colon cancer HCT116 cells (p53^+/+ and p53^−/−) pretreated with 5 μM Nutlin-3a for 6 h were infected at an MOI of 1. Total virus samples were collected at 18 hpi and quantified by plaque titration (±s.e.m., n=3). Lower panel: western blot analysis to determine levels of p53 upon reovirus or Nutlin-3 treatment at 24 hpi. (B) Percentage of reovirus released into the media was determined by dividing reovirus titres from infected cell media by the total reovirus titre (±s.e.m., n=3). (C) p53-dependent cytotoxicity enhancement caused by the combination of reovirus and Nutlin-3a. HCT116 p53^+/+ or p53^−/− cells were mock infected or infected at an MOI of 1 in the presence or absence of 5 μM Nutlin-3a. Images were taken at 24 hpi. Unless otherwise indicated, no treatment indicates that they were treated with dimethyl sulfoxide (DMSO, vehicle). Student's t-test was used to compare two groups of data; NS: not significant; ^**P<0.001.

Figure 2

Nutlin-3a treatment induces significantly higher level of cell death in reovirus-infected p53^+/+ tumour cells. (A) Percentage of cell death in HCT116 p53^+/+ cells caused by reovirus, Nutlin-3a or the combination of the two quantified by Annexin V and 7-AAD staining. The apoptotic fraction was calculated as the sum of Annexin V-positive live cells and dead cells (upper and lower right quadrants). Samples were collected at 12, 15, 18, 24, 36 and 48 hpi. (B and C) Cell death induced by reovirus in the presence or absence of Nutlin-3a treatment in U2OS (n=3, ±s.e.m.) or in A549 (n=3, ±s.e.m.) cells. A549 and U2OS cells were infected at an MOI of 100 and 500, respectively. Samples were collected at 24 and 48 h, respectively, for Annexin V and 7-AAD staining. (D) Caspase inhibitor ZVAD blocked apoptosis induced by reovirus or the combination of Nutlin-3a and reovirus. Cells were treated with ZVAD (50 μM) at the same time of Nutlin-3a treatment and throughout infection. Student's t-test was used to compare two groups of data; ^*P<0.05; ^**P<0.001.

Figure 3

Enhanced cell death induced by the combination of Nutlin-3a and reovirus is dependent on p53. (A) The combination of reovirus and Nutlin-3a caused significantly higher levels of cell death in HCT116 p53^+/+, but not in p53^−/− cells. HCT116 p53^+/+ and p53^−/− were either mock infected or infected with reovirus at an MOI of 1. Cells were collected at 24 hpi, subjected to Annexin V and 7-AAD staining and quantified by FACS analysis. (n=4, ±s.e.m.). (B) Extent of cell death of A549 p53 knockdown cells (A549 p53-shRNA) and non-silencing control cells (A549 control) induced by Nutlin-3a, reovirus (MOI of 100) or the combination of the two reagents (upper panel). p53 levels after Nutlin-3a treatment (lower panel) were determined by western blotting. (C) Extent of cell death in reovirus-infected HCT116 p53^−/− cells transfected with either p53 expression (pcDNA3-p53) or control pcDNA3 plasmid. HCT116 p53^−/− were transfected by pcDNA3-p53 or pcDNA3 plasmids and mock infected or infected with reovirus at an MOI of 1. (D) Induction of p53 protein accumulation at different concentrations of RITA (left panel) and percentage of apoptosis in reovirus-infected HCT116 p53^+/+ and p53^−/− cells with or without 0.63 μM RITA treatment (right panel). Student's t-test was used to compare two groups of data; NS: not significant; ^**P<0.001; ^***P<0.0001.

Figure 4

Differential expression levels of p53 target genes (A) Noxa, (B) PUMA, (C) Bax or (D) p21, induced by reovirus, Nutlin-3a or the combination of the two. Total RNA extracted from HCT116 p53^+/+ or p53^−/−, with or without Nutlin-3a treatment or reovirus infection for 24 h (as indicated) was assessed for expression levels of GAPDH, Noxa, PUMA, Bax and p21 by real-time qPCR. Levels of genes were normalised to housekeeping gene GAPDH. (E) Fold difference of HCT116 knockout cells’ apoptosis induced by the combination of Nutlin-3a and reovirus compared with HCT116 p53^+/+cells. Percentage of apoptosis induced by Nutlin-3a and reovirus in the knockout cells were normalised to p53^+/+ cells (n=4, ±s.e.m.). Student's t-test was used to compare two groups of data; NS: not significant; ^*P<0.05; ^**P<0.001.

Figure 5

Cell death induced by Nutlin-3a and reovirus requires NF-κB activation. Levels of NF-κB transcription activity after reovirus infection in HCT116 p53^+/+ cells (A) and U2OS cells (C) (n=4, ±s.e.m.). HCT116 cells were infected at an MOI of 1 and lysates were collected at 12 hpi. U2OS cells were infected at an MOI of 500 and lysates were collected at 48 hpi. (B) Nutlin-3a treatment enhanced levels of NF-κB p65 nuclear translocation. Arrows point to infected cells. (D) NF-κB activation inhibitor N (InSolution NF-κB activation inhibitor at 1 : 100 000 dilution) treatment reduced the level of cell death induced by the combination of reovirus and Nutlin-3a in p53^+/+ HCT116 cells. Cell death was determined by quantifying the sub-G1 population of PI-stained cells. Student's t-test was used to compare two groups of data; NS: not significant; ^*P<0.05; ^**P<0.001.

Figure 6

Upregulation of p53 target genes (A) Noxa, (B) PUMA, (C) Bax and (D) p21 induced by the combination of Nutlin-3a and reovirus was inhibited by NF-κB inhibitor N. HCT116 p53^+/+ cells were pretreated with NF-κB inhibitor N for 1 h before Nutlin-3a treatment. At 6 hpi, cells were infected by reovirus at an MOI of 1. RNA samples were collected at 24 hpi and subjected to RNA purification, cDNA synthesis and real-time PCR (representative of two respective experiments, ±s.e.m.).

Figure 7

Nutlin-3a significantly enhanced both reovirus-induced cytotoxicity and reovirus dissemination in HCT116 p53^+/+ cells. (A) IC₅₀ values of reovirus in HCT116 p53^+/+ or p53^−/− cells in the presence or absence of 5 μM Nutlin-3a at 48 hpi. (B and C) Immunohistochemical staining of reovirus-infected HCT116 cells (B) and U2OS cells (C) in the presence or absence of Nutlin-3a treatment. (B, lower panel) Quantification of the plaque size on reovirus-infected HCT116 cells (plaque number n=14, ±s.e.m.). Cells were fixed at 7-day post-infection and proceed to staining using anti-reovirus antibody, shown as the representative of three respective experiments. Student's t-test was used to compare two groups of data; NS: not significant; ^**P<0.001; ^***P<0.0001.