N-acetylcysteine amide augments the therapeutic effect of neural stem cell-based antiglioma oncolytic virotherapy

Abstract

Current research has evaluated the intrinsic tumor-tropic properties of stem cell carriers for targeted anticancer therapy. Our laboratory has been extensively studying in the preclinical setting, the role of neural stem cells (NSCs) as delivery vehicles of CRAd-S-pk7, a gliomatropic oncolytic adenovirus (OV). However, the mediated toxicity of therapeutic payloads, such as oncolytic adenoviruses, toward cell carriers has significantly limited this targeted delivery approach. Following this rationale, in this study, we assessed the role of a novel antioxidant thiol, N-acetylcysteine amide (NACA), to prevent OV-mediated toxicity toward NSC carriers in an orthotropic glioma xenograft mouse model. Our results show that the combination of NACA and CRAd-S-pk7 not only increases the viability of these cell carriers by preventing reactive oxygen species (ROS)-induced apoptosis of NSCs, but also improves the production of viral progeny in HB1.F3.CD NSCs. In an intracranial xenograft mouse model, the combination treatment of NACA and NSCs loaded with CRAd-S-pk7 showed enhanced CRAd-S-pk7 production and distribution in malignant tissues, which improves the therapeutic efficacy of NSC-based targeted antiglioma oncolytic virotherapy. These data demonstrate that the combination of NACA and NSCs loaded with CRAd-S-pk7 may be a desirable strategy to improve the therapeutic efficacy of antiglioma oncolytic virotherapy.

Figures

Figure 1

N-acetylcysteine amide (NACA) prevents the apoptosis of NSC carriers loaded with CRAd-S-pk7. (a) HB1.F3.CD NSCs were treated with various concentrations of NACA (1, 2.5, 5, and 10 mmol/l), CRAd-S-pk7 (1, 5, 10, and 50 IU) and the combination of NACA with CRAd-S-pk7. Cell viability was evaluated by MTT assay 72 hours after treatment. Representative digital microscopic image of viable cells are seen in the bottom of Figure 1a. Scale bar, 200 μm. (b) HB1.F3.CD cells were infected with CRAd-S-pk7 at 10 and 50 IU/cell. Media was changed 1 hour after virus infection and then exposed to 1 mmol/l of NACA or were left untreated. Cell viability was measured by MTT assay at the indicated times. (c) The viability of U87 glioma cells after treatment with NACA and CRAd-S-pk7 was determined by MTT assay at 72 hours. The values shown are mean ± SEM. *P < 0.05, **P < 0.01 versus control.

Figure 2

N-acetylcysteine amide (NACA) treatment on CRAd-S-pk7 loaded HB1.F3.CD cells increases viral replication. HB1.F3.CD cells (5 × 104 cell/well) were infected with CRAd-S-pk7 at a concentration of 50-infection units/cell. Infected cells were then treated with/without NACA. Total DNA was isolated and the replication capacity of CRAd-S-pk7 combined with NACA was measured by quantitative real-time PCR. Viral replication was determined by the number of viral E1A copies per ng of DNA from infected NSCs. All samples were analyzed in triplicates. They are presented as mean ± SEM. (a) HB1.F3.CD cells were infected with CRAd-S-pk7 in a dose-dependent manner (1, 5, 10, and 50 IU/cell) and (b) then combined with 1 mmol/l of NACA at 72 hours after treatment. (c) The replication of CRAd-S-pk7 treated with 1 mmol/l of NACA was measured by qRT-PCR in a time-dependent manner.

Figure 3

N-acetylcysteine amide (NACA) enhances CRAd-S-pk7 viral production in HB1.F3.CD NSCs. (a) Media of HB1.F3.CD cells infected with various concentrations of CRAd-S-pk7 (1, 10, 50, and 100 IU/cell) were collected at 72 hours after treatment and assessed by viral titer assay. (b) HB1.F3.CD cells were infected with 50 IU/cell of CRAd-S-pk7 and treated with different concentrations of NACA (1, 2.5, and 5 mmol/l). Viral titer was then evaluated. (c) HB1.F3.CD cells were infected with several doses of CRAd-S-pk7 and subsequently treated with or without 1 mmol/l of NACA. Three days after treatment, supernatant was collected and viral titer assay was performed. (d) Representative microscopic pictures of viral titer assay. Scale bar, 200 μm. (e) HB1.F3.CD cells were infected with CRAd-S-pk7 at 50 IU/cell with or without 1 mmol/l of NACA. Supernatant (cell free) and the infected cells (cell associated) were separately collected at indicated time points. Viral titer was then analyzed.

Figure 4

N-acetylcysteine amide (NACA) treatment of NSCs loaded with CRAd-S-pk7 leads to induction of glioma cell oncolysis. (a) HB1.F3.CD cells were infected with CRAd-S-pk7 (50 IU/cell) and then treated with or without 1 mmol/l of NACA. Supernatant was collected 4 days after initial treatment and used to infect U87 glioma cells. Cell viability was analyzed 4 days later using crystal violet staining (top). Scale bar, 200 μm. Differences in U87 cell viability between CON (control), NACA only, CRAd-S-pk7 only and OV and NACA combination groups were quantified (bottom). (b) HB1.F3.CD cells were infected with CRAd-S-pk7 (50 IU/cell) and then treated with or without 1 mmol/l of NACA. U87-Luc cells were plated in precultured wells containing NSCs loaded with OVs at the following ratio (NSCs: U87-Luc, 1:0.5 and 1:1). NSCs and U87 cells were lysed at 96 hours after coculture and Luciferase activity was measured. Bar graph shows luciferase activity (arithmetic mean ± SEM) in quadruplicates for each coculture. *P < 0.05.

Figure 5

N-acetylcysteine amide (NACA) modulates endogenous ROS and decreases the expression of markers of cellular apoptosis in NSCs infected with CRAd-S-pk7. (a) HB1.F3.CD cells were infected with CRAd-S-pk7 (10 and 50 IU/cell) and then treated with or without 1 mmol/l of NACA for 24 hours. Afterwards, cells were incubated with an oxidant-sensitive fluorogenic reagent, CM-H2DCFDA. Cell image was obtained using a fluorescent microscope. Scale bar, 100 μm. The mean fluorescence of four randomly selected fields was calculated. ROS values were compared with the control group and expressed as the fold of the control level. (b) HB1.F3.CD cells were treated with NACA (1, 2.5, and 5 mmol/l) and CRAd-S-pk7 (1, 10, and 50 IU/cell). After 24 hours, cell lysates were analyzed by immunoblotting. (c) HB1.F3.CD cells were treated with combination of NACA and CRAd-S-pk7 as indicated and cells were collected. Cell lysates (30 μg) were analyzed by immunoblotting with p-Akt, p53 and caspase-3 antibodies. (d) HB1.F3.CD cells were treated with combination of NACA (1 mmol/l) and CRAd-S-pk7 (50 IU/cell) and subjected to western blotting. Equal protein loading was verified by anti-β-actin antibody. The quantitative analysis of p-p38 and p-Akt protein levels was determined by densitometry analysis (normalized to actin), represented as a bar graph.

Figure 6

N-acetylcysteine amide (NACA) combined with CRAd-S-pk7 loaded NSCs prolongs animal survival and increases intratumoral apoptosis. U87 cells (2 × 105 cells per mice) were intracranially injected into nude mice. Four days post tumor implantation animals were intraperitoneally treated with NACA, 250 mg/kg/day for 5 consecutive days. HB1.F3.CD cells incubated with CRAd-S-pk7 oncolytic virus (50 IU/cells) were resuspended in 1X PBS (4 × 105 cells in 2.5 μl/mouse) before intratumoral injection. NSCs loaded with CRAd-S-pk7 were intratumorally injected five days after U87 tumor implantations. (a) Kaplan-Meier survival curve of mice implanted with U87 cells that were randomly divided into four groups: PBS (n = 6), NACA only (250 mg/kg, n = 6), HB1.F3.CD cells loaded with CRAd-S-pk7 (50 IU/cell, n = 7) and combination of NACA and HB1.F3.CD cells loaded with CRAd-S-pk7 (n = 7). Differences between survival curves were compared using a log-rank test and are shown as P values. *P < 0.05 (b) Histological sections of U87 brain tumors were stained with anticaspase-3 antibody. Dotted lines indicate the border between tumor (T) and nontumor (NT) areas.

Figure 7

N-acetylcysteine amide (NACA) treatment of CRAd-S-pk7 loaded NSCs enhances viral replication and increases viral progeny in vivo. Animals treated with the combination of NACA and NSCs loaded with CRAd-S-pk7 oncolytic viruses were sacrificed 21 days post-tumor implantation. Mice brains were collected, sliced into serial coronal sections, and the presence of CRAd-S-pk7 at both normal brain and tumor areas was analyzed by immunohistochemistry staining. (a) Immunohistochemistry staining showing E1A expression in U87 tumor sections (center and border) of mice treated with PBS, NACA only, HB1.F3.CD cells loaded with NACA and combination of NACA and HB1.F3.CD cells loaded with NACA. Scale bar, 35 μm. (b) The right brain hemisphere (the one with implanted tumor cells and intratumoral injection of NSCs) of mice from all four groups (n = 4 per group) was collected and in vivo CRAd-S-pk7 replication was quantified by quantitative real-time PCR. Viral progeny was assessed by viral titer assay.