Newcastle Disease Virus Induces Apoptosis In Cisplatin-resistant Human Lung Adenocarcinoma A549Cells In Vitro And In Vivo

Cisplatin (DDP) is a powerful anti-cancer drug widely used in lung cancer chemotherapy. However, cisplatin resistance developped in cancer cells weakened the oncolytic efficiency in clinical treatment. This study aims to investigate whether Newcastle disease virus (NDV) displayed an oncolytic effect on cisplatin-resistant A549lung cancer cells. It was demonstrated that NDV induced apoptosis in A549/DDP cells via the caspase pathway, particularly the caspase-8pathway. In addition, the mitogen-activated protein kinase (MAPK) and Akt pathways also contributed to NDV-induced apoptotic induction. Furthermore, NDV exhibited oncolytic effects in a mouse A549/DDP lung cancer model. Collectively, our results indicate that NDV could overcome the cisplatin resistance in lung cancer cells in vitro and in vivo. The main results are summarized as follows:1.TCID50assay was used to quantificate the viral content in A549/DDP cells. Our results indicated that FMW virus replicated effectively in A549/DDP cells.2. Annexin V/PI double staining assay was performed to analyze FMW-infected A549/DDP cells collected from different time points after infection. It was demonstrated that infection with FMW virus resulted in remarkable apoptosis in A549/DDP cells in a time-dependent manner.3. Apoptotic nuclear changes were examined using Hoechst33258staining. Apoptotic chromatin condensation was readily observed in A549/DDP cells infected with FMW virus, whereas no chromatin condensation was observed in control cells.4. Activation of caspase-3, caspase-8, caspase-9, MAPK pathways, Akt pathways and PARP cleavage was detected by Western Blot. The results indicated that the death receptor apoptotic pathway was activated and played a more important role in the apoptotic process induced by FMW infection, while the intrinsic apoptotic pathway was involved in the induction of apoptosis. 5. A549/DDP cells were pretreated with a pan caspase inhibitor Z-VAD-FMK (50μM), and apoptosis was analyzed by flow cytometry. Apoptosis was markedly inhibited in the cells treated with Z-VAD-FMK, therefore indicating that caspase activity was required for FMW-triggered apoptosis in A549/DDP cells.6. Cell viability was determined by MTT assay. These results suggested that FMW virus induced apoptosis in A549/DDP cells via signaling through MAPK and AKT.7. H&E staining and TUNEL assay were used to detect apoptosis in tumor sections. H&E staining of FMW-treated tumor sections showed characteristic apoptotic cells, and the TUNEL assay results revealed pyknotic chromatin in FMW-treated tumor sections. In contrast, fewer tumor necroses or apoptotic cells were observed in PBS-treated tumor sections.8. QRT-PCR and TCID50assay were used to quantify the in vivo replication kinetics of FMW in tumor-bearing mice. The number of M gene copies was markedly increased within tumor tissues from either A549-or A549/DDP-bearing mice. Similarly, NDV titers steadily increased within tumor sections, indicating that FMW replicated effectively in tumor tissues transplanted with either A549or A549/DDP cells.9. The statistical analysis was performed by SPSS V17.0software. The progression of established tumors was abrogated in mice injected with either A549or A549/DDP cells after intratumoral injection of FMW (p<0.05), in contrast to control mice in which tumors progressed rapidly and FMW exhibited nearly the same oncolytic effect on tumor growth in both the A549and A549/DDP groups.In summary, we provide evidence that FMW infection triggered apoptosis in cisplatin-resistant A549cells both in vitro and in vivo.