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. 2013;8(1):e54006.
doi: 10.1371/journal.pone.0054006. Epub 2013 Jan 16.

Activation of p53 by chemotherapeutic agents enhances reovirus oncolysis

Da Pan  1 , Paola Marcato, Dae-Gyun Ahn, Shashi Gujar, Lu-Zhe Pan, Maya Shmulevitz, Patrick W K Lee
Affiliations

Activation of p53 by chemotherapeutic agents enhances reovirus oncolysis

Da Pan et al. PLoS One. 2013.

Abstract

Mammalian reovirus is a benign virus that possesses the natural ability to preferentially infect and kill cancer cells (reovirus oncolysis). Reovirus exploits aberrant Ras signalling in many human cancers to promote its own replication and spread. In vitro and in vivo studies using reovirus either singly or in combination with anti-cancer drugs have shown very encouraging results. Presently, a number of reovirus combination therapies are undergoing clinical trials for a variety of cancers. Previously we showed that accumulation of the tumor suppressor protein p53 by Nutlin-3a (a specific p53 stabilizer) enhanced reovirus-induced apoptosis, and resulted in significantly higher levels of reovirus dissemination. In this study, we examined the role of p53 in combination therapies involving reovirus and chemotherapeutic drugs. We showed that sub-lethal concentrations of traditional chemotherapy drugs actinomycin D or etoposide, but not doxorubicin, enhanced reovirus-induced apoptosis in a p53-dependent manner. Furthermore, NF-κB activation and expression of p53-target genes (p21 and bax) were important for the p53-dependent enhancement of cell death. Our results show that p53 status affects the efficacy of combination therapy involving reovirus. Choosing the right combination partner for reovirus and a low dosage of the drug may help to both enhance reovirus-induced cancer elimination and reduce drug toxicity.

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Conflict of interest statement

Competing Interests: The authors have the following interests. Nutlin-3a for this study was provided by Hoffmann-LaRoche Inc. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Sub-lethal concentrations of specific chemotherapy drugs enhance reovirus-induced apoptosis.
Cell viability of HCT116 p53+/+ cells after exposure to various concentrations of (A) ActD, (B) Dox or (C) Etp. MTS reagents were added at 48 h post treatment. Accumulation of p53 was determined by western blot analysis using p53-specific antibody. Representative data from two independent experiments (triplicate wells, ±s.e.m.) is shown. D. Apoptosis induced by reovirus alone, sub-lethal concentrations of ActD/Etp/Dox alone (Nutlin-3 as control), or the combination of reovirus and the drug. HCT116 p53+/+ and p53−/− cells were infected by reovirus at an MOI of 1 for 1 h and then supplemented with media with or without low concentrations of Dox, Etp or ActD. 1/10,000 dilution of DMSO in cell culture media was used as vehicle control. Percentage of apoptotic cells was determined by double-staining with Annexin V and 7-AAD at 24 hpi. Student’s t-test was used to compare percentage of cell death between reovirus infected cells and cells treated by the combination of reovirus and drugs; *p<0.05; **p<0.001; ***p<0.0001.
Figure 2
Figure 2. Enhanced apoptosis induced by reovirus and ActD/Etp depends on virus replication and caspase activities.
A. Percentage of cells that were infected by reovirus with or without the treatment of Dox, Etp, or ActD at 24 hpi, determined by FACS analysis using anti-reovirus antibody. B. Caspase inhibitor ZVAD blocks the enhancement of cell death induced by the combination of reovirus and a low concentration of ActD or Etp. HCT116 p53+/+ and p53−/− cells were treated with ZVAD for 1 h before reovirus infection. Following reovirus infection, ZVAD and a low concentration of ActD or Etp were added to culture media of reovirus-infected cells. Cell death was determined by Annexin V and 7-AAD staining at 24 hpi. Student’s t-test was used to compare two groups of data; *p<0.05 and ***p<0.0001. C. Immunohistochemical staining of the plaques formed on reovirus-infected HCT116 cells in the presence or absence of ActD (0.63 nM) or Etp (1.6 µM).
Figure 3
Figure 3. Reovirus combined with ActD/Etp significantly enhances p53-target genes expression.
(A) noxa, (B) puma, (C) bax and (D) p21 expression levels in HCT116 p53+/+ and p53−/− cells treated with reovirus (reo), Etp/ActD or the combination of Etp/ActD and reovirus. RNA samples were collected at 24 hpi and processed for real-time q-PCR analysis using gene-specific primers (±s.e.m., n = 3). Student’s t-test was used to compare expression levels between reovirus-infected cells and cells treated with the combination of reovirus and a drug; *p<0.05, **p<0.001 and ***p<0.0001.
Figure 4
Figure 4. Enhanced apoptosis induced by the combination of reovirus and ActD/Etp requires bax and p21.
HCT116 p53+/+ and its isogenic knockout (p53−/−, PUMA−/−, Bax−/−, p21−/−, PUMA−/−p21−/− and Bax−/−p21−/−) cells and HCT116 p53+/+ cells infected with lentivirus containing shRNAs control (shRNA control) or sequences against noxa (Noxa-kd1, Noxa-kd2), were infected with reovirus at an MOI of 1 in the presence or absence of (A) ActD or (B) Etp treatment. Cells were collected at 24 hpi and subjected to Annexin V and 7-AAD staining to determine the percentage of cell death (±s.e.m., n = 3). Student’s t-test was used to compare cell death between knockout (or knockdown) cells and p53+/+ cells; *p<0.05, **p<0.001 and ***p<0.0001.
Figure 5
Figure 5. NF-κB activation is important forenhanced apoptosis induced by reovirus and ActD/Etp.
A. NF-κB activation was determined by NF-κB p65 subunit nuclear translocation. HCT116 p53+/+ and p53−/− cells were infected with reovirus at an MOI of 1 in the presence or absence of ActD (0.63 nM) or Etp (1.6 µM) treatment. Cells were fixed at 12-h post ActD/Etp treatment and cells were stained with anti-p65, anti-reo antibody and To-pro-3 for nucleus staining. B. NF-κB inhibitor N significantly reduces cell death induced by the combination of reovirus and ActD/Etp. HC116 p53+/+ cells were treated with NF-κB inhibitor N for 1 h before reovirus infection in the absence or presence of either ActD (0.63 nM) or Etp (1.6 µM) treatment. Cells were collected at 24 hpi for PI staining and the percentage of sub-G1 for each sample was normalized to reovirus infection alone (±s.e.m., n = 3). Student’s t-test was used to compare two groups of data; *p<0.05.
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