Gu Chenchen, Su Jun, Wang Jigui, Xie Qianqian, Wu Jing, Xiao Jun, Liu Weiquan
State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, No. 2, Yuanmingyuan West Road, Beijing, China.
Department of Geriatrics, The Eight Medical Centre, Chinese PLA General Hospital, Beijing, China.
J Cancer Res Clin Oncol. 2023 Sep;149(12):9903-9918. doi: 10.1007/s00432-023-04878-w. Epub 2023 May 30.
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.
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 △60, pCMV-F△107, and pCMV-F△114) or with site-directed mutagenesis in the 3' region (pCMV-FA602-610) 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-F△60 and rCDV-FA602-610) 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.
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.
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.
犬瘟热病毒（CDV）已被证明对原发性犬类肿瘤具有溶瘤活性。本实验室先前的研究证实，CDV可诱导犬乳腺肿瘤（CMT）细胞凋亡，但其分子机制仍不清楚。
在CDV-L中鉴定出CDV的N、P、M、F、H、L、C和V基因，并分别进行克隆。构建了F基因5'区域缺失（pCMV-F△60、pCMV-F△107和pCMV-F△114）或3'区域定点突变（pCMV-FA602-610）的突变体。用Hoechst 33342检测晚期凋亡细胞。用膜联蛋白V-FITC/PI检测早期凋亡细胞。进行定量实时PCR检测凋亡和NF-κB途径靶基因的mRNA水平。进行蛋白质免疫印迹分析检测凋亡或NF-κB途径靶蛋白的表达或磷酸化水平。进行免疫荧光分析检测p65蛋白的核转位。通过基于BHK-T7的系统拯救重组病毒（rCDV-F△60和rCDV-FA602-610）。使用5周龄雌性BALB/c裸鼠检测重组病毒的溶瘤活性。
在本研究中，首先证实疫苗株CDV-L的结构或非结构蛋白均不能单独诱导犬乳腺管状腺癌细胞（CIPp）或导管内乳头状癌细胞（CMT-7364）凋亡。然而，当CIPp或CMT-7364细胞与CDV-L的糖蛋白融合（F）和血凝素（H）蛋白共转染时，观察到核碎裂，检测到高比例的早期凋亡细胞，以及裂解的半胱天冬酶-3、半胱天冬酶-8和聚（ATP核糖）聚合酶（PARP）。凋亡广谱抑制剂Z-VAD-FMK和半胱天冬酶-8途径抑制剂Z-IETD-FMK下调了裂解的半胱天冬酶-3和PARP，证实F和H蛋白通过半胱天冬酶-8和半胱天冬酶-3途径在CMT细胞中共诱导凋亡。F和H蛋白共诱导p65和IκBα的磷酸化以及p65的核转位，证实NF-κB途径的激活，抑制该途径可下调裂解的半胱天冬酶-3和裂解的PARP。具有增强融合活性的重组F蛋白和H蛋白比亲本F蛋白共诱导更多的裂解半胱天冬酶-3和PARP，而相应的重组病毒在CIPp细胞和皮下异种移植小鼠模型中均表现出相同的特性。
CDV-L的F和H蛋白在CMT细胞中共诱导凋亡，而NF-κB途径和F蛋白的融合活性在此过程中起重要作用。
