Songshu Meng

Caspase- and p38-MAPK-dependent induction of apoptosis in A549 lung cancer cells by Newcastle disease virus

Newcastle disease virus (NDV) has a potential oncolytic effect due to its ability to induce apoptosis in tumor cells. However, previous studies have indicated discrepancies regarding the apoptosis signaling pathways induced by NDV in tumor cells. Here, we show that NDV infection induces simultaneous activation of intrinsic and extrinsic death pathways in A549 human lung cancer cells. In contrast, endoplasmic reticulum (ER) stress is not activated in NDV-induced apoptosis. We demonstrate for the first time that mitogen-activated protein kinase (MAPK) pathways are activated in NDV-infected A549 cells, and p38 MAPK is involved in NDV-induced cell death. Together, our findings provide novel insights into the underlying mechanisms by which NDV induces apoptosis in tumor cells.

Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication

Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

ABSTRACT Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.

Avian reovirus triggers autophagy in primary chicken fibroblast cells and Vero cells to promote virus production

Avian reovirus (ARV) is an important cause of disease in poultry. Although ARV is known to induce apoptosis in infected cells, the interaction between ARV and its target cells requires further elucidation. In this report, we show that the ARV isolate strain GX/2010/1 induces autophagy in both Vero and primary chicken embryonic fibroblast (CEF) cells based on the appearance of an increased number of double-membrane vesicles, the presence of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) dot formation, and the elevated production of LC3II. We further demonstrate that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway contributes to autophagic induction by ARV infection. Moreover, treatment of ARV-infected cells with the autophagy inducer rapamycin increased viral yields, while inhibition of the autophagosomal pathway using chloroquine led to a decrease in virus production. Altogether, our studies strongly suggest that autophagy may play a critical role in determining viral yield during ARV infection.

Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells

Encorafenib (LGX818) is a new-generation BRAF inhibitor that is under evaluation in clinical trials. However, the underlying mechanism remains to be elucidated. Here we show that LGX818 potently decreased ERK phosphorylation and inhibited proliferation in BRAFV600E melanoma cell lines. Moreover, LGX818 downregulated CyclinD1 in a glycogen synthase kinase 3β-independent manner and induced cell cycle arrest in the G1 phase, Surprisingly, LGX818 triggered cellular senescence in BRAFV600E melanoma cells, as evidenced by increased β-galactosidase staining, while no appreciable induction of apoptosis was detected, as determined by Annexin V and propidium iodide staining and immunoblot analysis of caspase-3 processing and poly (ADP-ribose) polymerase cleavage. Increased p27KIP1 expression and retinoblastoma protein activation were detected during LGX818-induced senescence. Additionally, inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 1B by AZ191 reversed LGX818-induced CyclinD1 turnover and senescence. Interestingly, autophagy is triggered through inhibition of the mTOR/70S6K pathway during LGX818-induced senescence. Moreover, autophagy inhibition by pharmacological and genetic regulation attenuates LGX818-induced senescence. Notably, combining LGX818 with autophagy modulators has anti-proliferative effect in LGX818-resistant BRAF mutant melanoma cells. Altogether, we uncovered a mechanism by which LGX818 exerts its anti-tumor activity in BRAFV600E melanoma cells.

Newcastle disease virus induces apoptosis in cisplatin-resistant human lung adenocarcinoma A549 cells in vitro and in vivo

Cisplatin (DDP) is widely used in lung cancer chemotherapy. However, cisplatin resistance represents a major obstacle in effective clinical treatment. This study aims to investigate whether Newcastle disease virus (NDV) exhibits an oncolytic effect on cisplatin-resistant A549 lung cancer cells. We found that NDV induced A549/DDP cell apoptosis via the caspase pathway, particularly involving caspase-9, while the mitogen-activated protein kinase (MAPK) and Akt pathways also contributed to apoptotic induction. Furthermore, NDV displayed oncolytic effects in a mouse A549/DDP lung cancer model. Collectively, our data indicate that NDV could overcome the cisplatin resistance in lung cancer cells in vitro and in vivo.

Apoptin Enhances the Oncolytic Properties of Newcastle Disease Virus

Objective: Naturally occurring strains of Newcastle disease virus (NDV) have demonstrated the potential to kill cancer cells in both preclinical and clinical studies. Previous studies showed that apoptin, the VP3 protein of chicken infectious anemia virus, is a p53-independent, Bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in transformed cells. In this study, we tested the hypothesis that apoptin enhances NDV-mediated tumor cell death. Methods: Reverse genetics was used to engineer an oncolytic NDV strain, FMW, to express apoptin. The antitumor effects of the recombinant virus (rFMW/AP) were also evaluated in the tumor cell lines and tumor-bearing mice. Results: Compared to the parental strain FMW, rFMW/AP was more potent in killing A459 and SMMC7721 tumor cells. Recombinant NDV also exhibited higher efficacy in suppressing tumor growth in mice bearing A549-induced tumors. Furthermore, rFMW/AP did not display apparent toxic effects in either normal cells or control mice. Conclusion: Our results suggest that the recombinant NDV expressing apoptin is a promising novel antitumor agent.

Gamabufotalin, a bufadienolide compound from toad venom, suppresses COX-2 expression through targeting IKKβ/NF-κB signaling pathway in lung cancer cells

BackgroundGamabufotalin (CS-6), a major bufadienolide of Chansu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, the underlying mechanism of CS-6 involved in anti-tumor activity remains poorly understood.MethodsThe biological functions of gamabufotalin (CS-6) were investigated by migration, colony formation and apoptosis assays in NSCLC cells. The nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed after treatment with CS-6. Molecular docking study was used to simulate the interaction of CS-6 with IKKβ. The in vivo anti-tumor efficacy of CS-6 was also analyzed in xenografts nude mice. Western blot was used to detect the protein expression level.ResultsGamabufotalin (CS-6) strongly suppressed COX-2 expression by inhibiting the phosphorylation of IKKβ via targeting the ATP-binding site, thereby abrogating NF-κB binding and p300 recruitment to COX-2 promoter. In addition, CS-6 induced apoptosis by activating the cytochrome c and caspase-dependent apoptotic pathway. Moreover, CS-6 markedly down-regulated the protein levels of COX-2 and phosphorylated p65 NF-κB in tumor tissues of the xenograft mice, and inhibited tumor weight and size.ConclusionsOur study provides pharmacological evidence that CS-6 exhibits potential use in the treatment of COX-2-mediated diseases such as lung cancer.

Neratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells

Receptor tyrosine kinase ErbB2/HER2 is frequently observed to be overexpressed in human cancers, leading to over activation of downstream signaling modules. HER2 positive is a major type of breast cancer for which ErbB2 targeting is already proving to be an effective therapeutic strategy. Apart from antibodies against ErbB2, the small molecule tyrosine kinase inhibitor lapatinib has had successful clinical outcomes, and other inhibitors such as neratinib are currently undergoing clinical investigations. In this study we report the effects of lapatinib and neratinib on the mRNA and protein levels of the ErbB2 receptor. We provide evidence that neratinib-induced down regulation of ErbB2 occurs through ubiquitin-mediated endocytic sorting and lysosomal degradation. At the mechanistic level, neratinib treatment leads to HSP90 release from ErbB2 and its subsequent ubiquitylation and endocytic degradation. Our findings provide novel insights into the mechanism of ErbB2 inhibition by neratinib.

Pharmacological modulation of autophagy enhances Newcastle disease virus-mediated oncolysis in drug-resistant lung cancer cells

BackgroundOncolytic viruses represent a promising therapy against cancers with acquired drug resistance. However, low efficacy limits its clinical application. The objective of this study is to investigate whether pharmacologically modulating autophagy could enhance oncolytic Newcastle disease virus (NDV) strain NDV/FMW virotherapy of drug-resistant lung cancer cells.MethodsThe effect of NDV/FMW infection on autophagy machinery in A549 lung cancer cell lines resistant to cisplatin (A549/DDP) or paclitaxel (A549/PTX) was investigated by detection of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) puncta, formation of double-membrane vesicles and conversion of the nonlipidated form of LC3 (LC3-I) to the phosphatidylethanolamine-conjugated form (LC3-II). The effects of autophagy inhibitor chloroquine (CQ) and autophagy inducer rapamycin on NDV/FMW-mediated antitumor activity were evaluated both in culture cells and in mice bearing drug-resistant lung cancer cells.ResultsWe show that NDV/FMW triggers autophagy in A549/PTX cells via dampening the class I PI3K/Akt/mTOR/p70S6K pathway, which inhibits autophagy. On the contrary, NDV/FMW infection attenuates the autophagic process in A549/DDP cells through the activation of the negative regulatory pathway. Furthermore, combination with CQ or knockdown of ATG5 significantly enhances NDV/FMW-mediated antitumor effects on A549/DDP cells, while the oncolytic efficacy of NDV/FMW in A549/PTX cells is significantly improved by rapamycin. Interestingly, autophagy modulation does not increase virus progeny in these drug resistant cells. Importantly, CQ or rapamycin significantly potentiates NDV/FMW oncolytic activity in mice bearing A549/DDP or A549/PTX cells respectively.ConclusionsThese results demonstrate that combination treatment with autophagy modulators is an effective strategy to augment the therapeutic activity of NDV/FMW against drug-resistant lung cancers.