RESUMO
Oncolytic virus is an effective therapeutic strategy for cancer treatment, which exploits natural or manipulated viruses to selectively target and kill cancer cells. However, the innate antiviral system of cancer cells may resistant to the treatment of oncolytic virus. M1 virus is a newly identified oncolytic virus belonging to alphavirus species, but the molecular mechanisms underlying its anticancer activity are largely unknown. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. RNA seq analysis was used to analyze the gene alternation after M1 virus infection. Small interfering RNAs transfection for gene knockdown was used for gene functional tests. Caspase-3/7 activity was detected by Caspase-Glo Assay Systems. A mice model of orthotopic bladder tumor was established to determine the oncolytic effectiveness of the M1 virus. The expression of cleaved-Caspase 3 as well as Ki-67 in tumor cells were detected by immunohistochemical analysis. To further define the molecular factors involved in M1 virus-mediated biological function, we knocked down genes related to alphavirus' activity and found that CCDC6 plays an important role in the oncolytic activity of M1 virus. Moreover, knocked down of CCDC6 augments the reproduction of M1 virus and resulted in endoplasmic reticulum (ER) stress-induced cell apoptosis in vitro as well as in vivo orthotopic bladder cancer model. Our research provides a rational new target for developing new compounds to promote the efficacy of oncolytic virus therapy.
Assuntos
Proteínas do Citoesqueleto/genética , Vetores Genéticos/genética , Neoplasias/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , RNA Interferente Pequeno , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Viruses are obligate parasites that depend on host cells to provide the energy and molecular precursors necessary for successful infection. The main component of virus-induced metabolic reprogramming is the activation of glycolysis, which provides biomolecular resources for viral replication. However, little is known about the crosstalk between oncolytic viruses and host glycolytic processes. METHODS: A MTT assay was used to detect M1 virus-induced cell killing. Flow cytometry was used to monitor infection of M1 virus expressing the GFP reporter gene. qPCR and western blotting were used to detect gene expression. RNA sequencing was performed to evaluate gene expression under different drug treatments. Scanning electron microscopy was performed to visualize the endoplasmic reticulum (ER). Caspase activity was detected. Last, a mouse xenograft model was established to evaluate the antitumor effect in vivo. Most data were analyzed with a two-tailed Student's t test or one-way ANOVA with Dunnett's test for pairwise comparisons. Tumor volumes were analyzed by repeated measures of ANOVA. The Wilcoxon signed-rank test was used to compare nonnormally distributed data. RESULTS: Here, we showed that the glucose analog 2-deoxy-D-glucose (2-DG) inhibited infection by M1 virus, which we identified as a novel type of oncolytic virus, and decreased its oncolytic effect, indicating the dependence of M1 replication on glycolysis. In contrast, lonidamine, a reported hexokinase 2 (HK2) inhibitor, enhanced the infection and oncolytic effect of M1 virus independent of HK2. Further transcriptomic analysis revealed that downregulation of the antiviral immune response contributes to the lonidamine-mediated potentiation of the infection and oncolytic effect of M1 virus, and that MYC is the key factor in the pool of antiviral immune response factors inhibited by lonidamine. Moreover, lonidamine potentiated the irreversible ER stress-mediated apoptosis induced by M1 virus. Enhancement of M1's oncolytic effect by lonidamine was also identified in vivo. CONCLUSIONS: This research demonstrated the dependence of M1 virus on glycolysis and identified a candidate synergist for M1 virotherapy.
RESUMO
Alpha virus M1 is an oncolytic virus that targets zinc-finger antiviral protein (ZAP)-defective cancer cells, and may be useful for treatment of hepatocellular carcinoma (HCC). Most of HCC patients have hepatitis and need long-term antiviral medication. Thus, it is necessary to clarify whether anti-virus medicines influence oncolytic effect of M1. We examined the effect of drugs used to treat hepatitis B/C on M1-mediated oncolysis in vitro and in vivo. Interferon (IFN)-α induces expression of antiviral IFN-stimulated genes (ISGs) in HCC cells with moderate sensitivity to M1 virus. This leads to reduced replication of M1, and blocking of M1-mediated apoptosis. The antagonistic effect of IFN-α is positively related with the expressive level of ISGs. We also examined a population of 147 HCC patients. A total of 107 patients (73%) had low ZAP expression in liver tissues relative to adjacent tissues. Among these 107 patients, 77% were positive for hepatitis B and 2% were positive for hepatitis C. A combination of M1 virus and IFN should be avoided in those patients with HBV or HCV infection, of who ZAP expression is low but ISGs expression is moderate. In conclusion, this study provides a basis for anti-viral regimens for HCC patients with hepatitis B or C who are given oncolytic virus M1.
Assuntos
Antivirais/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Interferon-alfa/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Antivirais/farmacologia , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Interferon-alfa/farmacologia , Neoplasias Hepáticas/patologia , Masculino , Vírus Oncolíticos/genéticaRESUMO
Oncolytic virotherapy is an emerging and promising treatment modality that uses replicating viruses as selective antitumor agents. Here, we report that a classical protein kinase A (PKA) inhibitor, H89, synergizes with oncolytic virus M1 in various cancer cells through activation of Epac1 (exchange protein directly activated by cAMP 1). H89 substantially increases viral replication in refractory cancer cells, leading to unresolvable Endoplasmic Reticulum stress, and cell apoptosis. Microarray analysis indicates that H89 blunts antiviral response in refractory cancer cells through retarding the nuclear translocation of NF-κB. Importantly, in vivo studies show significant antitumor effects during M1/H89 combination treatment. Overall, this study reveals a previously unappreciated role for H89 and demonstrates that activation of the Epac1 activity can improve the responsiveness of biotherapeutic agents for cancer.
Assuntos
Alphavirus/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Isoquinolinas/uso terapêutico , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Sulfonamidas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Terapia Combinada/métodos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Isoquinolinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neoplasias/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Fator de Transcrição RelA/metabolismo , Replicação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Motivated by the latest findings in a recent medical experiment [19] which identify a naturally occurring alphavirus (M1) as a novel selective killer targeting zinc-finger antiviral protein (ZAP)-deficient cancer cells, we propose a mathematical model to illustrate the growth of normal cells, tumor cells and the M1 virus with limited nutrient. In order to better understand biological mechanisms, we discuss two cases of the model: without competition and with competition. In the first part, the explicit threshold conditions for the persistence of normal cells (or tumor cells) is obtained accompanying with the biological explanations. The second part indicates that when competing with tumor cells, the normal cells will extinct if M1 virus is ignored; Whereas, when M1 virus is considered, the growth trend of normal cells is similar to the one without competition. And by using uniformly strong repeller theorem, the minimum effective dosage of medication is explicitly found which is not reported in [19]. Furthermore, numerical simulations and corresponding biological interpretations are given to support our results.