In Parkinson’s disease (PD) research, human neuroblastoma and immortalized neural cell lines have been widely used as in vitro models. The advancement in the field of reprogramming technology has provided tools for generating patient-specific induced pluripotent stem cells (hiPSCs) as well as human induced neuronal progenitor cells (hiNPCs). These cells have revolutionized the field of disease modeling, especially in neural diseases. Although the direct reprogramming to hiNPCs has several advantages over differentiation after hiPSC reprogramming, such as the time required and the simple procedure, relatively few studies have utilized hiNPCs. Here, we optimized the protocol for hiNPC reprogramming using pluripotency factors and Sendai virus. In addition, we generated hiNPCs of two healthy donors, a sporadic PD patient, and a familial patient with the LRRK2 G2019S mutation (L2GS). The four hiNPC cell lines are highly proliferative, expressed NPC markers, maintained the normal karyotype, and have the differentiation potential of dopaminergic neurons. Importantly, the patient hiNPCs show different apoptotic marker expression. Thus, these hiNPCs, in addition to hiPSCs, are a favorable option to study PD pathology.
Cell Line ; Dopaminergic Neurons ; Fibroblasts ; Humans ; In Vitro Techniques ; Induced Pluripotent Stem Cells ; Karyotype ; Neuroblastoma ; Neurons ; Pathology ; Sendai virus ; Stem Cells ; Tissue Donors

OBJECTIVEThe current study aims to investigate the effect of Hemagglutinating virus of Japan envelope (HVJ-E) on induction of apoptosis and autophagy in human prostate cancer PC3 cells, and the underlying mechanisms.

METHODSPC3 cells were treated with HVJ-E at various multiplicity of infection (MOI), and the generated reactive oxygen species (ROS), cell viability, apoptosis, and autophagy were detected, respectively. Next, the role of ROS played in the regulation of HVJ-E-induced apoptosis and autuphagy in PC3 cells were analysed. In the end, the relationship between HVJ-E-induced apoptosis and autuophagy was investigated by using rapamycin and chloroquine.

RESULTSFlow cytometry assay revealed that HVJ-E treatment induced dose-dependent apoptosis and that the JNK and p38 MAPK signaling pathways were involved in HVJ-E-induced apoptosis in PC3 cells. In addition, HVJ-E was able to induce autophagy in PC3 cells via the class III PI3K/beclin-1 pathway. The data also implyed that HVJ-E-triggered autophagy and apoptosis were ROS dependent. When ROS was blocked with N-acetylcysteine (NAC), HVJ-E-induced LC3-II conversion and apoptosis were reversed. Interestingly, HVJ-E-induced apoptosis was significantly increased by an inducer of autophagy, rapamycin pretreatment, both in vitro and in vivo.

CONCLUSIONHVJ-E exerts anticancer effects via autophagic cell death in prostate cancer cells.

Apoptosis ; physiology ; Autophagy ; physiology ; Cell Line, Tumor ; Cell Survival ; Humans ; Male ; Oncolytic Virotherapy ; Prostatic Neoplasms ; metabolism ; Reactive Oxygen Species ; metabolism ; Sendai virus ; immunology ; physiology ; Virus Inactivation

OBJECTIVEThis paper aims to investigate the apoptotic effect of inactivated Sendai virus (hemagglutinating virus of Japan-enveloped, HVJ-E) on murine melanoma cells (B16F10) and the possible mechanisms involved in the putative apoptotic reactions.

METHODSB16F10 cells were treated with HVJ-E at various multiplicities of infection (MOI), and the reactive oxygen species (ROS), cell viability, and apoptosis were measured. Next, the roles of ROS in the regulation of Bcl-2/Bax and the activation of mitogen-activated protein kinase (MAPK) pathways in HVJ-E-treated B16F10 cells were analyzed. To further evaluate the cytotoxic effect of HVJ-E-generated ROS on B16F10 cells, HVJ-E was intratumorally injected, both with and without N-acetyl-L-cysteine (NAC), into melanoma tumors on BALB/c mice. Tumor volume was then monitored for 3 weeks, and the tumor proteins were separated for immunoblot assay.

RESULTSTreatment of B16F10 cells with HVJ-E resulted in a dose-dependent inhibition of cell-viability and an induction of apoptosis. The latter effect was associated with the generation of ROS. Inhibition of ROS generation by NAC resulted in a significant reduction of HVJ-E-induced Erk1/2, JNK, and p38 MAPK activation. Additionally, ROS inhibition caused a decrease in the Bcl-2/Bax ratio as well as promoting activation of apoptosis both in vitro and in vivo.

CONCLUSIONThese results suggest that HVJ-E possesses potential anticancer activity in B16F10 cells through ROS-mediated mitochondrial dysfunction involving the MAPK pathway.

Animals ; Apoptosis ; Cell Line, Tumor ; Mice ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Respirovirus Infections ; virology ; Sendai virus ; physiology ; Virus Inactivation

OBJECTIVETo explore the anti-tumor effect and its mechanism of Sendai virus Tianjin strain defective interfering particles (DIP) on mouse models of colon carcinoma.

METHODSCT26 cells (5×10(6)/0.1 ml) were subcutaneously injected into the back of Bal B/c mice to establish murine colon carcinoma model. After the tumors reached 5 mm in diameter, the mice were randomly divided into Tianjin strain DIP group and saline control group. The former was intratumorally injected with Tianjin strain DIP (0.1 ml) once a day on day 4, 7, 10 and 13 after CT26 cell inoculation. The latter was intratumorally injected with the same volume of saline. Tumor volume and survival rate of the mice were calculated to confirm the anti-tumor effect of DIP. Flow cytometry and ELISA were used to examine the maturation and release of cytokines IL-6, IFN-α and TNF-α from murine myeloid dendritic cells (DCs) induced by Tianjin strain DIP. Moreover, real-time RT-PCR and immunohistochemistry were performed to identify whether the Tianjin strain DIP could induce infiltration of CD11c(+) DCs, CD4(+) and CD8(+) T cells in the tumors.

RESULTSOn day 22 after CT26 cell inoculation, the average tumor volume of the Tianjin strain DIP group was (33.2 ± 2.0) mm(3), significantly smaller than that of the control group [(2 376.0 ± 130.8)mm(3), P < 0.01]. On day 50 after CT26 cell inoculation, the survival rate of mice was 90.0% in the Tianjin strain DIP group, much higher than that of the control group (30.0%, P < 0.01). Flow cytometry analysis showed that the expression of markers of DCs maturation, including CD40, CD80 and CD86, was dose-dependently increased by DIP or intact virus. No statistically significant difference was found betweent the DIP and intact virus groups. ELISA results showed that DIP could stimulate the secretion of IL-6, IFN-α and TNF-α from mouse DCs. The secretion of all of the cytokines was dose-dependently increased by DIP or intact virus. Real-time RT-PCR revealed that the expression of CD4, CD8 and CD11c mRNAs was increased in tumors treated with DIP compared with that of the saline group at all time points. Moreover, the expression level of all of them remained maximal at 120 h after the last treatment. Immunohistochemical staining revealed that the ratios of CD4(+), CD8(+) T cells or CD11c(+) DCs to total cells were (21.60 ± 1.49)%, (22.12 ± 2.84)% and (23.05 ± 2.91)%, respectively, in the DIP-treated tumors. In the tumors treated by saline, the ratios were (2.62 ± 0.60)%, (4.05 ± 0.12)% and (3.10 ± 0.09)%, respectively. The difference between experimental group and control group had statistical significance.

CONCLUSIONSTianjin strain DIP may exert anti-tumor effect on tumor-bearing mice. The mechanism is related with the antitumor immunity induced by DCs and T cells.

Animals ; Cell Line, Tumor ; Colonic Neoplasms ; metabolism ; pathology ; Cytokines ; metabolism ; Defective Viruses ; immunology ; Dendritic Cells ; metabolism ; Female ; Interferon-alpha ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Sendai virus ; immunology ; T-Lymphocytes ; metabolism ; Tumor Burden ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVEInactivated Sendai virus particle [hemagglutinating virus of Japan envelope (HVJ-E)] has a potential oncolytic effect due to its ability to induce apoptosis in tumor cells. However, the molecular mechanism of apoptosis induction in cancer cells mediated by HVJ-E has not been fully elucidated. This paper aims to investigate the underlying mechanism of apoptosis induction by HVJ-E in prostate cancer cells (PC3).

METHODSPC3 cells were treated with HVJ-E at various MOI, and then interferon-β (IFN-β) production, and the cell viability and apoptosis were detected by ELISA, MTT-based assay and flow cytometry, respectively. Next, the roles of Jak-Stat, MAPK and Akt pathways played in HVJ-E-induced apoptosis in PC3 cells were analyzed by immunoblot assay. To further evaluate the cytotoxic effect of HVJ-E on PC3 cells, HVJ-E was intratumorally injected into prostate cancers on BALB/c-nude mice, and the tumor volume was monitored for 36 days.

RESULTSHVJ-E induced IFN-β production and activated Jak-Stat signaling pathway, which resulted in the activation of caspase-8, caspase-3, and PARP in PC3 prostate cancer cells post HVJ-E treatment. Furthermore, we observed for the first time that p38 and Jnk MAPKs in PC3 cells contributed to HVJ-E-induced apoptosis. In addition, intratumoral HVJ-E treatment displayed a direct inhibitory effect in an in vivo BALB/c nude mouse prostate cancer model.

CONCLUSIONOur findings have provided novel insights into the underlying mechanisms by which HVJ-E induces apoptosis in tumor cells.

Animals ; Apoptosis ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Oncolytic Virotherapy ; Prostatic Neoplasms ; Sendai virus ; immunology ; physiology ; Vaccines, Inactivated ; immunology

PURPOSE: Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. MATERIALS AND METHODS: The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a zeta-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. RESULTS: The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. CONCLUSION: This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.
Cell Line, Tumor ; HN Protein/genetics ; Humans ; Jurkat Cells ; RNA, Small Interfering ; Sendai virus/genetics ; Transfection/*methods ; Viral Fusion Proteins/genetics ; Virosomes

Animals ; Apoptosis ; Cell Line, Tumor ; Down-Regulation ; Melanoma, Experimental ; pathology ; virology ; Mice ; Receptor, IGF Type 1 ; physiology ; Sendai virus ; pathogenicity

In this study, we constructed the plasmid of Sendai virus (SeV) BB1 strain minigenome with Gaussia luciferase (Gluc) as reporter and compared the rescue efficiency of SeV minigenome mediated by T7 promoter with that by CMV promoter. Firstly, the sequence was designed and synthesized which contained hammerhead ribozyme, sequence composed of the trailer, untranslated region of L gene, untranslated region of N gene, and the leader from SeV, and mutant hepatitis delta virus ribozyme sequence. Then, the synthesized sequence was inserted into pVAX1 containing CMV and T7 promoters and the general vector for SeV minigenome pVAX-miniSeV was obtained. Furthermore, pVAX-miniSeV-Gluc (+) and pVAX-miniSeV-Gluc(-) were obtained by inserting Gluc gene into pVAX-miniSeV. From the supernatant of BHK-21 cell transfected with pVAX-miniSeV-Gluc(+), high level of Gluc expression was detection indicating the normal transcription function of CMV promoter. pVAX-SeV-miniGluc(-) and plasmids expressing N,P and L protein of SeV were co-transfected into BST T7/5 cell which derived from BHK-21 and expressed T7 RNA polymerase stably. And high expression of Gluc was found, which indicated that SeV minigenome was efficiently rescued. However, we failed to repeat the result on BHK-21 cell, implying that T7 promoter and CMV promoter may have different effects on the rescue efficiency of SeV minigenome. Therefore, we further constructed SeV minigenome vectors pT7-miniSeV-Gluc (-) and pCMV-miniSeV-Gluc(-) with single promoter of T7 or CMV. Then, these vectors were transfected into BSR T7/ 5 cells respectively accompanied with the N, P, and L protein expression vectors. The result demonstrated that high expression of Gluc was found in the group of pT7-miniSeV-Gluc(-), which failed in the group of pCMV-miniSeV-Gluc(-). It indicated that T7 promoter significantly increased the rescue efficiency of SeV minigenome. We successfully constructed a SeV minigenome vector with secreted luciferase gene as report er and proved T7 promoter can enhance the rescue efficiency of SeV minigenome, which provides basis for construction of infectious clone containing SeV full-length genome.
Animals ; Cell Line ; Cricetinae ; Cytomegalovirus ; genetics ; DNA-Directed RNA Polymerases ; genetics ; Genome, Viral ; Humans ; Promoter Regions, Genetic ; Respirovirus Infections ; virology ; Sendai virus ; genetics ; physiology ; Viral Proteins ; genetics ; metabolism

To explore the infectivity characteristics and susceptibility of Sendai strain Tianjin in 129Sv, DBA/2, Kunming and BALB/c mice and determine the optimal small rodent animal model for strain Tianjin research, the Sendai strain Tianjin was propagated for 72h in 9-11 day-old chicken embryos, the allantoic fluids were then harvested and the virus titer (1:1280) was detected by hemagglutination assay. Four different kinds of mice were intranasally inoculated with 5 microl and the diluted 30 microl virus solution. The weight loss of mice was monitored for 12 consecutive days and the survival rate was observed. Kunming and BALB/c mice were sacrificed on the first day prior to infection and on the fourth and seventh days post infection of the diluted 30 microl Sendai strain Tianjin. Their left lobes of lung were fixed with 4% formalin for histopathologic examination. The maximum percentage of average weight loss of 129Sv, DBA/2 were 13.0%, 4.7% with 100% survival rate when 129Sv, DBA/2, Kunming and BALB/c were inoculated with 5 microl virus solution, while the mice were inoculated with diluted 30 microl virus solution, the maximum percentage of average weight loss reached 21.7%, 30.3%, 16.7% and 9.7% with the survival rate of 20%, 0%, 80% and 100%. Lung infections of mice Kunming on the fourth and seventh day post infection were more severe than that of BALB/c, showing a large number of inflammatory cell exudation and thickening of the submucosa. It suggested that DBA/2 was the most susceptible to the infection of strain Tianjin. The mice susceptibility ranked as DBA/2>129Sv>Kunming>BALB/c. Mice DBA/2 and 129Sv could be used as the optimal small rodent animal models in the research of pathogenicity and vaccine of Sendai strain Tianjin.
Animals ; Disease Susceptibility ; Female ; Humans ; Lung ; pathology ; virology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred DBA ; Respirovirus Infections ; physiopathology ; virology ; Sendai virus ; classification ; physiology

OBJECTIVEThis paper aims to investigate the anti-tumor mechanism of inactivated Sendai virus (Hemagglutinating virus of Japan envelope, HVJ-E) for murine melanoma (B16F10).

METHODSThe murine dendritic cells (DCs) were treated with HVJ-E, and then the cytokines secreted from DCs and costimulation-related molecules on DCs were measured. Meanwhile, the expression of β-catenin in HVJ-E treated murine melanoma cells was detected. In addition, HVJ-E was intratumorally injected into the melanoma on C57BL/6 mice, and the immune cells, CTL response and tumor volume were analyzed.

RESULTSHVJ-E injected into B16F10 melanoma obviously inhibited the growth of the tumor and prolonged the survival time of the tumor-bearing mice. Profiles of cytokines secreted by dendritic cells (DCs) after HVJ-E stimulation showed that the number of cytokines released was significantly higher than that elicited by PBS (1P<0.05). The co-stimulation-related molecules on DCs were comparable to those stimulated by LPS. Immunohistochemical examinations demonstrated the repression of β-catenin in B16F10 melanoma cells after HVJ-E treatment. Meanwhile, real-time reverse transcription PCR revealed that HVJ-E induced a remarkable infiltration of CD11c positive cells, chemokine ligand 10 (CXCL10) molecules, interleukin-2 (IL-2) molecule, CD4(+) and CD8(+) T cells into HVJ-E injected tumors. Furthermore, the mRNA expression level of β-catenin in the HVJ-E injected tumors was also down-regulated. In addition, B16F10-specific CTLs were induced significantly after HVJ-E was injected into the tumor-bearing mice.

CONCLUSIONThis is the first report to show the effective inhibition of melanoma tumors by HVJ-E alone and the mechanism through which it induces antitumor immune responses and regulates important signal pathways for melanoma invasion. Therefore, HVJ-E shows its prospect as a novel therapeutic for melanoma therapy.

Animals ; Cell Line, Tumor ; Cytokines ; genetics ; metabolism ; Dendritic Cells ; immunology ; physiology ; virology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Melanoma ; immunology ; pathology ; virology ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Sendai virus ; physiology ; Virus Inactivation ; Virus Replication ; beta Catenin ; genetics ; metabolism