Abstract
Background
Canine distemper virus (CDV) has been shown to have oncolytic activity against primary canine tumors. Previous studies from this laboratory had confirmed that CDV induces apoptosis in canine mammary tumor (CMT) cells, although the molecular mechanism remains unknown.
Methods
The CDV N, P, M, F, H, L, C, and V genes were identified in CDV-L and cloned separately. Mutants with deletions in the 5' region (pCMV-F L△60, pCMV-FL△107, and pCMV-FL△114) or with site-directed mutagenesis in the 3' region (pCMV-FLA602-610) of the F gene were generated. Late-stage apoptotic cells were detected by Hoechst 33342. Early-stage apoptotic cells were detected by AnnexinV-FITC/PI. Quantitative real-time PCR was performed to detect the mRNA levels of target genes of apoptotic and NF-κB pathway. Western blot analysis was performed to detect the expression or phosphorylation levels of target proteins of apoptotic or NF-κB pathway. Immunofluorescence assay was performed to detect the nuclear translocation of p65 protein. Recombinant viruses (rCDV-FL△60 and rCDV-FLA602-610) were rescued by a BHK-T7-based system. 5-week-old female BALB/c nude mice were used to detect the oncolytic activity of recombinant viruses.
Results
In this study, it was first confirmed that none of the structural or non-structural proteins of CDV-L, a vaccine strain, was individually able to induce apoptosis in canine mammary tubular adenocarcinoma cells (CIPp) or intraductal papillary carcinoma cells (CMT-7364). However, when CIPp or CMT-7364 cells were co-transfected with glycoprotein fusion (F) and hemagglutinin (H) proteins of CDV-L, nuclear fragmentation was observed and a high proportion of early apoptotic cells were detected, as well as cleaved caspase-3, caspase-8 and poly (ATP ribose) polymerase (PARP). Cleaved caspase-3 and PARP were down-regulated by apoptosis broad-spectrum inhibitor Z–VAD–FMK and caspase-8 pathway inhibitor Z–IETD–FMK, confirming that the F and H proteins coinduced apoptosis in CMT cells via the caspase-8 and caspase-3 pathways. F and H proteins co-induced phosphorylation of p65 and IκBα and nuclear translocation of p65, confirming activation of the NF-κB pathway, inhibition of which down-regulated cleaved caspase-3 and cleaved PARP. Recombinant F protein with enhanced fusion activity and H protein co-induced more cleaved caspase-3 and PARP than parental F protein, while the corresponding recombinant virus exhibited the same properties both in CIPp cells and in a subcutaneous xenograft mouse model.
Conclusions
F and H proteins of CDV-L co-induce apoptosis in CMT cells, while the NF-κB pathway and fusion activity of F protein paly essential roles in the process.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We deeply appreciate Professor Nobuo Sasaki (University of Tokyo, Japan) for the generous gifts of CIPp cells. We deeply appreciate Professor Degui Lin (China Agricultural University, China) for the generous gifts of CMT-7364 cells.
Funding
This work was supported by the National Key Research and Development Program of China (2016YFD0501001).
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Chenchen Gu, Jun Su and Jigui Wang. The first draft of the manuscript was written by Chenchen Gu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Mouse studies were performed in accordance with the China Agricultural University Institutional Animal Care and Use Committee guidelines (ID: SYXK [Beijing] 2021-0012) and approved by the Animal Ethical and Welfare Committee of China Agricultural University.
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Gu, C., Su, J., Wang, J. et al. Fusion protein and hemagglutinin of canine distemper virus co-induce apoptosis in canine mammary tumor cells. J Cancer Res Clin Oncol 149, 9903–9918 (2023). https://doi.org/10.1007/s00432-023-04878-w
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DOI: https://doi.org/10.1007/s00432-023-04878-w