Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death

Abstract

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic. Here, we present evidence that genetically modified, recombinant NDV strains are cytotoxic to human tumor cell lines of ecto-, endo-, and mesodermal origin. We show that cytotoxicity against tumor cells is due to multiple caspase-dependent pathways of apoptosis independent of interferon signaling competence. The signaling pathways of NDV-induced, cancer cell-selective apoptosis are not well understood. We demonstrate that NDV triggers apoptosis by activating the mitochondrial/intrinsic pathway and that it acts independently of the death receptor/extrinsic pathway. Caspase-8-methylated SH-SY5Y neuroblastoma cells are as sensitive to NDV as other caspase-8-competent cells. This demonstrates that NDV is likely to act primarily through the mitochondrial death pathway. NDV infection results in the loss of mitochondrial membrane potential and the subsequent release of the mitochondrial protein cytochrome c, but the second mitochondrion-derived activator of caspase (Smac/DIABLO) is not released. In addition, we describe early activation of caspase-9 and caspase-3. In contrast, cleavage of caspase-8, which is predominantly activated by the death receptor pathway, is a TNF-related, apoptosis-inducing ligand (TRAIL)-induced late event in NDV-mediated apoptosis of tumor cells. Our data, therefore, indicate that the death signal(s) generated by NDV in tumor cells ultimately converges at the mitochondria and that it acts independently of the death receptor pathway. Our cytotoxicity studies demonstrate that recombinant NDV could be developed as a cancer virotherapy agent, either alone or in combination with therapeutic transgenes. We have also shown that trackable oncolytic NDV could be developed without any reduction in oncolytic efficacy.

FIG. 1.

NDV is cytolytic to human tumor cells and noncytolytic in normal human cells. Shown are CPE induced by rNDV in chicken embryo fibroblast and human tumor cells. DF1 chicken embryo fibroblast cells and human tumor cell lines were either mock infected or infected with rLaSota V.F., rBC, or rBC-Edit strains of NDV at an MOI of 0.01. CPE in the form of cell fusion, syncytium formation, rounding, and destruction of the monolayer in different cells are shown. (A and B) Mock-infected and rBC-Edit-infected HEpG2 cells. (C and D) Mock-infected and rBC-Edit-infected HT1080 cells. (E and F) Mock-infected and rBC-Edit-infected PC3 prostate cancer cells. (G and H) Mock-infected and rBC-Edit-infected CaCo2 colon cancer cells. (I and J) Mock-infected and rBC-Edit-infected HuTu80 intestinal epithelial cells. (K and L) Mock-infected and rBC-Edit-infected DF1 chicken embryo fibroblast cells. (M and N) Mock-infected and rBC-Edit-infected 2fTGH human fibrosarcoma cells. (O and P) Mock-infected and rBC-Edit-infected U3A human fibrosarcoma cells. Magnification, ×40. (Q) SV-HUC1 uroepithelial cells were either mock infected or infected with rLaSota V.F., rBC, or rBC-Edit strains of NDV at MOIs of 0.01, 1.0, or 10. Culture supernatants were assayed for virus content by a plaque assay in DF1 cells at 48 h postinfection, and results of virus titer determination at an MOI of 0.01 were compared with those for viruses assayed under similar conditions in HuTu80 and CaCo2 cells. Results represent mean values + SEM from two independent experiments.

FIG. 2.

Morphological features of apoptosis in rNDV-infected human tumor cells. Cells were either mock infected or infected with rLaSota V.F., rBC, rBC-Edit, or rBC-EGFP strains of NDV at an MOI of 0.01. At 6 and 14 h postinfection, apoptotic cell death was visualized by staining the infected cells with DAPI (1 μg/ml). (A) Condensation of chromatin and nuclear fragmentation of rNDV-infected HuTu80 cells. (B) Fluorescein isothiocyanate-annexin V (10 mg/ml) staining of NDV-infected HuTu80 cells. (B) Phosphatidyl serine externalization to the outer leaflet of the infected cell membrane is evident by green fluorescence of the cell membrane. (C) DNA laddering of infected cells was examined by using an apoptotic DNA laddering kit (Roche) per the manufacturer's instructions. Intranucleosomal DNA fragmentation is evident as a laddering pattern of the cellular DNA in rNDV-infected HuTu80 cells at 8, 10, and 12 h postinfection. (D) DNA laddering of SH-SY5Y neuroblastoma cells at 8, 10, and 12 h postinfection. (E) DNA laddering of rNDV-infected PC3, 2fTGH, U3A, and HT29 cells at 12 h postinfection.

FIG. 3.

Apoptotic signaling in rNDV-infected cells. DF1, SV-HUC1, and human tumor cells were either mock infected or infected with rLaSota V.F., rBC, or rBC-Edit strains of NDV at an MOI of 0.01. Culture supernatants were assayed by ELISA for TNF-α production at 48 h postinfection in HuTu80 cells (A) and SV-HUC1 cells (B). Soluble TRAIL expression was assayed by ELISA in various tumor cells at 48 h postinfection (C), and surface expression of TRAIL was examined in HuTu80 cells by immunoblotting with anti-TRAIL antibody at the indicated times postinfection (D). C, mock infected; L, rLaSota V.F. infected; B, rBC infected; E, rBC-Edit infected; M, molecular weight marker. ELISA results represent mean values + SEM from two independent experiments.

FIG. 4.

Caspase-8 expression in NDV-induced apoptosis of tumor cells. DF1 and various human tumor cells were either mock infected or infected with rLaSota V.F., rBC, or rBC-Edit strains of NDV at an MOI of 0.01. Culture supernatants were assayed by ELISA for caspase-8 production at 48 h postinfection. The relative fluorescence units over mock-infected controls are shown for DF1 and a few representative human tumor cells for caspase-8 (A). (B) Kinetics of caspase-8 induction in HuTu80 cells. (C) Caspase-8 production in caspase-8-methylated SH-SY5Y neuroblastoma cells. Results represent mean values + SEM from two independent experiments.

FIG. 5.

Disruption of mitochondrial membrane potential in rBC-EGFP-infected tumor cells was examined by staining with DAPI and MitoTracker Red CMX-Ros. NDV-infected cells which had a disruption of the Δψ_m and were undergoing apoptosis were shown by the diffuse cytoplasmic pattern of CMX-Ros with condensed chromatin. Tumor cells were infected with rBC-EGFP virus, treated 24 h postinfection with MitoTracker Red CMX-Ros for 2 h, and fixed later. Syncytium formation, EGFP expression, and mitochondrial membrane disruption in rBC-EGFP-infected cells are shown. (A) CaCo2 colon carcinoma cells, bright field; magnification, ×40. (B) Epifluorescence microscopy, ×40. (C) Diffuse staining of cytoplasm with MitoTracker Red merged with fluorescent image, ×40. (D) HEpG2 hepatocarcinoma cells, bright field, ×40. (E) Epifluorescence, ×40. (F) Diffuse cytoplasmic staining of MitoTracker Red with EGFP expression, ×40. (G) PC3 prostate cancer cells, bright field, ×40. (H) Epifluorescence, ×40. (I) Diffuse cytoplasmic staining of MitoTracker Red with EGFP expression, ×40. (J) HuTu80 cells, uninfected control cells, bright field, ×40. (K) Epifluorescence, ×40. (L) Punctuate cytoplasmic staining with MitoTracker Red, ×40.

FIG. 6.

Caspase-9 expression in NDV-induced apoptosis of tumor cells. DF1 and various human tumor cells were either mock infected or infected with rLaSota V.F., rBC, or rBC-Edit strains of NDV at an MOI of 0.01. Culture supernatants were assayed by ELISA for caspase-9 production at 48 h postinfection. (A) The relative fluorescence units over mock-infected controls are shown for DF1 cells and a few representative human tumor cells for caspase-9. (B) Kinetics of caspase-9 production in HuTu80 cells. (C) Caspase-9 production in SH-SY5Y neuroblastoma cells. Results represent mean values + SEM from two independent experiments.

FIG. 7.

Effector caspase-3 expression in NDV-induced apoptosis of tumor cells. DF1 and various human tumor cells were either mock infected or infected with rLaSota V.F., rBC, or rBC-Edit strains of NDV at an MOI of 0.01. Culture supernatants were assayed by ELISA for caspase-3 production at 48 h postinfection. (A) The relative fluorescence units over mock-infected controls are shown for DF1 and a few representative human tumor cells for caspase-3. (B) Kinetics of caspase-3 production in HuTu80 cells. (C) Caspase-3 production in SH-SY5Y neuroblastoma cells. Results represent mean values + SEM from two independent experiments. Broad-spectrum caspase inhibitor does not prevent replication of NDV in human tumor cells. HuTu80 cells were pretreated for 1 h with 100 μM Z-VAD-FMK prior to infection with rLaSota V.F., rBC, or rBC-Edit (MOI = 1) virus or mock infection. Virus content in the supernatant of infected cells was assayed by a plaque assay in DF1 cells at the indicated time points postinfection (D). Virus titers in control cells with dimethyl sulfoxide treatment were not significantly different (P > 0.05). Results represent mean values + SEM from two independent experiments.