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. 2013 Feb;20(2):133-40.
doi: 10.1038/cgt.2012.97. Epub 2013 Jan 25.

Concurrent chemotherapy inhibits herpes simplex virus-1 replication and oncolysis

Y Kulu  1 , H Kawasaki, J M Donahue, H Kasuya, J C Cusack, E W Choi, D K Kuruppu, B C Fuchs, K K Tanabe
Affiliations

Concurrent chemotherapy inhibits herpes simplex virus-1 replication and oncolysis

Y Kulu et al. Cancer Gene Ther. 2013 Feb.

Abstract

Herpes simplex virus-1 (HSV-1) replication in cancer cells leads to their destruction (viral oncolysis) and has been under investigation as an experimental cancer therapy in clinical trials as single agents, and as combinations with chemotherapy. Cellular responses to chemotherapy modulate viral replication, but these interactions are poorly understood. To investigate the effect of chemotherapy on HSV-1 oncolysis, viral replication in cells exposed to 5-fluorouracil (5-FU), irinotecan (CPT-11), methotrexate (MTX) or a cytokine (tumor necrosis factor-α (TNF-α)) was examined. Exposure of colon and pancreatic cancer cells to 5-FU, CPT-11 or MTX in vitro significantly antagonizes both HSV-1 replication and lytic oncolysis. Nuclear factor-κB (NF-κB) activation is required for efficient viral replication, and experimental inhibition of this response with an IκBα dominant-negative repressor significantly antagonizes HSV-1 replication. Nonetheless, cells exposed to 5-FU, CPT-11, TNF-α or HSV-1 activate NF-κB. Cells exposed to MTX do not activate NF-κB, suggesting a possible role for NF-κB inhibition in the decreased viral replication observed following exposure to MTX. The role of eukaryotic initiation factor 2α (eIF-2α) dephosphorylation was examined; HSV-1-mediated eIF-2α dephosphorylation proceeds normally in HT29 cells exposed to 5-FU, CPT-11 or MTX. This report demonstrates that cellular responses to chemotherapeutic agents provide an unfavorable environment for HSV-1-mediated oncolysis, and these observations are relevant to the design of both preclinical and clinical studies of HSV-1 oncolysis.

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Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

We declare that we have no conflict of interest.

Figures

Fig. 1
Fig. 1. Chemotherapy-induced cytotoxicity inhibits viral replication
(A) Capan-2, SW480 and HT29 cells were treated with different concentration of TNF-α, 5-FU, CPT-11, or MTX for 3 days. (B) The cell survival rates of HT29 cells were measured at 3 days following KOS infection, (MOI values ranging from 0.001 to 10) in the presence or absence of TNF-α (10 or 100 ng/ml), 5-FU (10 or 100 µM), CPT-11 (1 or 10 µM), or MTX (2.5 or 25 µM). The number of viable cells was determined using a MTT assay. (C) Viral titers were determined at 0, 24, 40 h following infection of HT29 cells with KOS at MOI = 0.1 in the presence or absence of TNF-α (10 or 100 ng/ml), 5-FU (10 or 100 µM), CPT-11 (1 or 10 µM), or MTX (2.5 or 25 µM). Results are shown as mean ± SD.
Fig. 2
Fig. 2. NF-κB activation is required for efficient HSV-1 replication
(A) HT29 cells were either mock infected or infected with HSV-1 (KOS, 7134, gal4, d27, hrR3, and R3616) at MOI = 1 for up to 36 h, and equal amounts (10 µg) of nuclear extracts were prepared and analyzed by EMSA. (B) HT29 cells were infected with the IκBα-SR (Ad.CMV.IκBα) or control (empty vector) adenovirus (Ad.CMV3) at MOI = 20 for 12 h. These cells were infected with KOS, 7134, and hrR3 at MOI = 0.1 for up to 40 h, and the equal amounts (10 µg) of nuclear extracts were prepared and analyzed by EMSA. Positive control: nuclear extracts from HT29 that were treated with TNF-α for 24 h. Mock: Heat-inactivated KOS. After 0 to 40 h following HSV-1 infection, the cells that were initially infected with the Ad.CMV.IκBα, Ad.CMV3 or saline were harvested and supernatants from these cells were titered on Vero cells or 0-28 cells. (C) Same experiment in Capan-2 cells. (D) Same experiment in SW480 cells. Results are shown as mean ± S.D.
Fig. 3
Fig. 3. Differential effects of chemotherapeutic agents on NF-κB activation
(A) HT29 cells were treated with different concentration of TNF-α, 5-FU, CPT-11, or MTX for 24 h, after which time equal amounts (10 µg) of nuclear extracts from the cells were prepared and analyzed by EMSA. (B) HT29 cells were treated with 10 ng/ml TNF-α, 100 µM 5-FU, 1 µM CPT-11, and 25 µM MTX for up to 36 h, and nuclear extracts were prepared and analyzed by EMSA. Negative control: medium (DMEM with 10 % FBS and antibiotics) alone. (C) HT29 cells were treated with 10 ng/ml TNF-α, 100 µM 5-FU, 1 µM CPT-11, and 25 µM MTX in the presence or absence of HSV-1 KOS infection for 24 h, and nuclear extracts were prepared and analyzed by EMSA. Negative control: medium (DMEM with 10 % FBS and antibiotics) alone. Positive control: nuclear extracts from HT29 that were treated with 10 ng/ml TNF-α for 24 h. The density of each band was determined using UVP image software, and results are shown as mean ± S.D. in graph.
Fig. 4
Fig. 4. Chemotherapy induces in vitro PKR/eIF-2α phosphorylation activity
Protein lysates from HT29 cell that were treated with TNF-α, 5-FU, CPT-11 or MTX for 24 h were examined for their ability to (A) phosphorylate PKR, (B) phosphorylate eIF-2α and (C) dephosphorylate eIF-2α, in the presence or absence of HSV-1 KOS infection. Cells were treated with TNF-α (10 ng/ml), 5-FU (100 µM), CPT-11 (1 µM), and MTX (25 µM) for 24 h and then harvested to assess kinase or phosphatase activity. Control: medium alone (without drug). Mock-infected HT29: HT29 cells incubated with heat-inactivated KOS. Negative control for (A): precipitated cell lysate with protein A-agarose alone (without PKR-antibody). Control for (C): Purified phosphorylated eIF-2α by GST-PKR (without cell lysates). The eIF-2α dephosphorylation assay (C) examines protein lysates from drug-treated (or mock-treated) cells for dephosphorylation of radiolabeled His-tagged eIF-2α bacterial fusion protein over a time interval of 5 minutes (by comparison of His-eIF-2α phosphorylatation after 0 versus 5 minutes of incubation with cell lysate). The optical density of all autoradiography bands was determined using UVP image software, and results are shown as the mean ratio of optical density ± S.D. in graphs beneath each autoradiograph.
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