Apoptin enhances the oncolytic activity of vaccinia virus in vitro

The chicken anemia virus gene that encodes apoptin, a selective killer of cancer cells, was synthe-sized and inserted into the vaccinia virus (strain L-IVP) genome. The insertion replaces a major part of the viral C11R gene that encodes viral growth factor, which is important for virulence. The recombinant virus VVdGF-ApoS24/2 was obtained by transient dominant selection using the gene of puromycin resistance as a selective marker. The expression of the apoptin gene from a synthetic early-late promoter of vaccinia virus ensured the efficient accumulation of the target protein in VVdGF-ApoS24/2-infected cells. Although recombinant apoptin carried the signal peptide of the virus growth factor at the N-end, the protein was not secreted into the culture medium. The recombinant virus VVdGF-ApoS24/2 exhibited significantly higher selective lytic activity in human cancer cell lines (A549, A431, U87MG, RD, and MCF7) than the parent strain L-IVP and its VVdGF2/6 variant with C11R deletion. These results suggest that the use of apoptin can be an efficient means of enhancing the natural anticancer activity of vaccinia virus.     

Engineering and combining oncolytic measles virus for cancer therapy

Cancer immunotherapy using tumor-selective, oncolytic viruses is an emerging therapeutic option for solid and hematologic malignancies. A considerable variety of viruses ranging from small picornaviruses to large poxviruses are currently being investigated as potential candidates. In the early days of virotherapy, non-engineered wild-type or vaccine-strain viruses were employed. However, these viruses often did not fully satisfy the major criteria of safety and efficacy. Since the advent of reverse genetics systems for manipulating various classes of viruses, the field has shifted to developing genetically engineered viruses with an improved therapeutic index. In this review, we will summarize the concepts and strategies of multi-level genetic engineering of oncolytic measles virus, a prime candidate for cancer immunovirotherapy. Furthermore, we will provide a brief overview of measles virus-based multimodal combination therapies for improved tumor control and clinical efficacy.     

Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions

Newcastle disease virus (NDV) is an avian paramyxovirus, which has been demonstrated to possess significant oncolytic activity against mammalian cancers. This review summarizes the research leading to the elucidation of the mechanisms of NDV-mediated oncolysis, as well as the development of novel oncolytic agents through the use of genetic engineering. Clinical trials utilizing NDV strains and NDV-based autologous tumor cell vaccines will expand our knowledge of these novel anticancer strategies and will ultimately result in the successful use of the virus in the clinical setting.     

Hizikia fusiforme extract enhances dendritic cell maturation in vitro and in vivo

ABSTRACT

Dendritic cells (DCs) are play critical roles in the priming and regulation of immune responses. DCs rapidly process and convey these antigens to prime antigen-specific T cells. Therefore, regulation of DCs functions is important for immunity and immunotherapies. Immune adjuvants for DCs activation are needed to improve the efficacy of vaccines against tumors and many infectious diseases. Therefore, we demonstrate that H. fusiformis extract can regulate DCs maturation and activation. H. fusiformis extract induced costimulatory molecules (CD 80 and CD86), antigen-presenting molecules (major histocompatibility complex (MHC) I and II), CCR7 expression, and interleukin (IL)-12 production in DCs. These effects are associated with upregulation of mitogen-activated protein kinase (MAPK) signaling pathway. In addition, H. fusiformis extract induces costimulatory molecules on splenic DCs and activated CD8⁺ T cells in vivo. Taken together, these findings suggest that H. fusiformis extract may be a potential efficient immune therapeutic compound in DCs-mediated immunotherapies.     

Therapeutic potency of oncolytic virotherapy in breast cancer targeting,current status and perspective

Breast cancer is the most common cause of cancer death in women and presents a serious therapeutic challenge worldwide. Traditional treatments are less successful at targeting cancer tumors, leading to recurrent treatment-resistant secondary malignancies. Oncolytic virotherapy (OV) is a novel anticancer strategy with therapeutic implications at targeting cancer cells by using mechanisms that differ from conventional therapies. Administration of OVs either alone or in combination with standard therapies provide new insights regarding the effectiveness and improvement of treatment responses for breast cancer patients. This review summarizes cellular, animal and clinical studies investigating therapeutic potency of oncolytic virotherapy in breast cancer treatment for a better understanding and hence a better management of this disease.     

新城疫病毒FMW株体外溶瘤作用及其机制分析

[目的]筛选出能高效抑制多种人肿瘤细胞生长增殖的新城疫(New Castle disease virus,NDV)毒株,为进一步构建重组高效靶向溶瘤毒株奠定基础.[方法]以体外噻唑蓝法测定NDV对A549、SMMC7721等肿瘤细胞及人胚干细胞L-02、人胚肾细胞HEK293等的生长抑制率,空斑试验确定病毒滴度及感染复数.利用形态学观察、Hoechst荧光染色、流式细胞术及免疫印迹等分析了NDV-FMW诱导肿瘤细胞凋亡的细胞生物学变化及其机制.[结果]从近50株NDV中筛选出NDV-FMW,以20 MOI病毒作用A549、SMMC7721等肿瘤细胞48 h,细胞生长抑制率达60%,NDV-FMW诱导肿瘤细胞发生凋亡,效应呈时间和剂量的依赖性,凋亡细胞出现核染色质断裂、浓缩及二倍体亚峰,细胞周期阻滞于GO/G1期,此外,病毒感染A549细胞16 h后开始检测到活化的Caspase-3裂解片段及PARP裂解大片段.[结论]NDV-FMW株体外能高效抑制肿瘤细胞的增殖,并经Caspase-3途径诱导肿瘤细胞凋亡.FMW株具有自主知识产权,其良好的体外溶瘤能力为进一步探讨体内抗肿瘤及临床试验的进行奠定了基础,并有可能为恶性肿瘤的治疗提供新的生物制剂.     

新城疫病毒FMW株体外溶瘤作用及其机制

摘要：【目的】筛选出能高效抑制多种人肿瘤细胞生长增殖的新城疫（New Castle disease virus, NDV）毒株,为进一步构建重组高效靶向溶瘤毒株奠定基础。【方法】以体外噻唑蓝法测定NDV对A549、 SMMC7721等肿瘤细胞及人胚干细胞L-O2、人胚肾细胞HEK293等的生长抑制率,空斑试验确定病毒滴度及感染复数。利用形态学观察、 Hoechst荧光染色、流式细胞术及免疫印迹等分析了NDV-FMW诱导肿瘤细胞凋亡的细胞生物学变化及其机制。【结果】从近50株NDV中筛选出NDV-FMW,以     

Brazilian red propolis extract enhances expression of antioxidant enzyme genes in vitro and in vivo

ABSTRACT

Brazilian red propolis reportedly has reactive oxygen species (ROS) scavenging effects in vitro, but the cellular mechanisms remain unclear. In the present study, the effects of an ethanol extract of Brazilian red propolis (EERP) on the Nrf2-ARE intracellular antioxidant pathway were examined in vitro and in vivo. EERP and its constituents transactivated the reporter gene through the ARE sequence and enhanced the expression of Nrf2-regulated genes in HEK293 cells. It also increased Nrf2 protein in the nucleus, which was partially inhibited by kinase inhibitors. Furthermore, EERP suppressed ROS generation and cytotoxicity induced by tert-butyl hydroperoxide. In vivo, orally administered EERP increased the expression of Nrf2-regulated genes in mice liver. These results suggest that EERP is a potential resource for preventing oxidative stress-related diseases as an Nrf2 inducer.     

Replication and cytopathic effect of oncolytic vesicular stomatitis virus in hypoxic tumor cells in vitro and in vivo

Tumor hypoxia presents an obstacle to the effectiveness of most antitumor therapies, including treatment with oncolytic viruses. In particular, an oncolytic virus must be resistant to the inhibition of DNA, RNA, and protein synthesis that occurs during hypoxic stress. Here we show that vesicular stomatitis virus (VSV), an oncolytic RNA virus, is capable of replication under hypoxic conditions. In cells undergoing hypoxic stress, VSV infection produced larger amounts of mRNA than under normoxic conditions. However, translation of these mRNAs was reduced at earlier times postinfection in hypoxia-adapted cells than in normoxic cells. At later times postinfection, VSV overcame a hypoxia-associated increase in alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation and initial suppression of viral protein synthesis in hypoxic cells to produce large amounts of viral protein. VSV infection caused the dephosphorylation of the translation initiation factor eIF-4E and inhibited host translation similarly under both normoxic and hypoxic conditions. VSV produced progeny virus to similar levels in hypoxic and normoxic cells and showed the ability to expand from an initial infection of 1% of hypoxic cells to spread through an entire population. In all cases, virus infection induced classical cytopathic effects and apoptotic cell death. When VSV was used to treat tumors established in nude mice, we found VSV replication in hypoxic areas of these tumors. This occurred whether the virus was administered intratumorally or intravenously. These results show for the first time that VSV has an inherent capacity for infecting and killing hypoxic cancer cells. This ability could represent a critical advantage over existing therapies in treating established tumors.     

Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.     

Development of a second-generation oncolytic Herpes simplex virus expressing TNFalpha for cancer therapy

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) therapy is a promising anti-cancer treatment when combined with radiotherapy due to its potent radio sensitising effects, but systemic toxicity has limited its clinical use. Previously, non-replicative adenovirus vectors have been used to deliver TNFalpha directly to the tumour, including under the control of a radiation sensitive promoter. Here, we have used an ICP34.5 deleted, oncolytic herpes simplex virus (HSV) for delivery to increase expression levels and spread through the tumour, and the use of the US11 true late HSV promoter to limit expression to where the virus replicates, i.e. selectively in tumour tissue. METHODS: TNFalpha expression under the CMV or US11 promoter was compared on cell lines CT26, BHK and Fadu. To further compare the activities of the promoters, expression of human TNFalpha was analysed in the presence and absence of acyclovir--an inhibitor of viral DNA replication and on HSV/ICP34.5- non-permissive cell line 3T6. The in vivo efficacy and toxicity of TNFalpha viruses were compared using A20 double flank tumour model in Balb/C mice and Fadu tumour model in nude mice. RESULTS: The results demonstrated that the US11 promoter significantly reduced and delayed TNFalpha expression as compared to use of the CMV promoter, especially in non-permissive cells or in the presence of acyclovir. Despite the reduced and more selective expression levels, US11 driven TNFalpha showed improved anti-tumour effects compared to CMV driven TNFalpha, and without the toxic side effects. CONCLUSIONS: This approach is therefore beneficial in increasing localised TNFalpha expression as compared to the use of non-replicative approaches, and combines the effects of TNFalpha with oncolytic virus replication which is expected to further enhance the efficacy of radiotherapy in a combined treatment approach.     

应用新城疫病毒治疗肿瘤的研究进展

新城疫病毒可以特异地杀伤肿瘤细胞,而对正常细胞没有伤害,目前在临床实验中认为是安全、有效的溶瘤试剂。随着近年来反向遗传操作技术的日趋成熟,该技术开始应用到新城疫病毒溶瘤效果的优化方面,通过改造新城疫病毒的F基因,及表达重组粒细胞巨噬细胞集落刺激因子,干扰素-γ,白细胞介素-2和肿瘤坏死因子-α等肿瘤杀伤因子,使该病毒具备更加优越的肿瘤杀伤能力,成为肿瘤治疗领域一个新兴的亮点,为癌症的临床治疗提供了崭新的前景。以下将简要介绍应用反向遗传操作技术重组新城疫病毒优化肿瘤治疗效果的研究进展,以及本实验室在相关领域的研究情况。     

Purging metastases in lymphoid organs using a combination of antigen-nonspecific adoptive T cell therapy, oncolytic virotherapy and immunotherapy

In many common cancers, dissemination of secondary tumors via the lymph nodes poses the most significant threat to the affected individual. Metastatic cells often reach the lymph nodes by mimicking the molecular mechanisms used by hematopoietic cells to traffic to peripheral lymphoid organs. Therefore, we exploited naive T cell trafficking in order to chaperone an oncolytic virus to lymphoid organs harboring metastatic cells. Metastatic burden was initially reduced by viral oncolysis and was then eradicated, as tumor cell killing in the lymph node and spleen generated protective antitumor immunity. Lymph node purging of tumor cells was possible even in virus-immune mice. Adoptive transfer of normal T cells loaded with oncolytic virus into individuals with cancer would be technically easy to implement both to reduce the distribution of metastases and to vaccinate the affected individual in situ against micrometastatic disease. As such, this adoptive transfer could have a great therapeutic impact, in the adjuvant setting, on many different cancer types.     

Activation of the alternative complement pathway by Newcastle disease virus. I) Effect of the virus on the complement system in vivo and in vitro

The authors have studied the effect of the paramyxovirus of Newcastle disease on the complement system in the mouse. The results obtained show that NDV activate both in vivo and in vitro the alternative complement pathway, suggesting that the intervention of complement system during viral infections represents a general biological phenomenon.     

In vivo and in vitro activity of Streptococcus faecium extract on Herpes simplex virus

The effects of substance or substance extracted from Str. faecium SF 68 on HSV-1 are evaluated. The "in vivo" assay show that bacterial extract introduced i.p. in mice simultaneously with HSV-1 brought about 100% of survival, but bacterial extract after virus challenge brought about complete mortality of mice. "In vitro" assays show that bacterial extract reduce significantly PFU number. It seemed that Str. faecium extract affected the virus at the stage of adsorption on the host cells.     

Anti-interferon globulin inhibits macrophage phagocytic enhancement in vivo by tilorone or Newcastle disease virus

Administration of the interferon inducers tilorone or Newcastle disease virus to mice enhances the in vitro uptake of opsonized erythrocytes (EA) by peritoneal macrophages. To evaluate the role of induced interferon (IF) in the macrophage stimulation, sheep anti-IF, or control globulins were given to mice prior to and after the administration of the inducers. Both IF titers and uptake of EA by macrophages were reduced by anti-IF but not by control globulin. In contrast, phagocytic stimulation by a lipopolysaccharide, a weak IF inducer, was unaffected by the anti-IF globulin. The results indicate that endogenously generated type I IF may participate in the control of macrophage function in vivo.