Boron-incorporating hemagglutinating virus of Japan envelope (HVJ-E) nanomaterial in boron neutron capture therapy.

Combining immunotherapeutic and radiotherapeutic technique has recently attracted much attention for advancing cancer treatment. If boron-incorporated hemagglutinating virus of Japan-envelope (HVJ-E) having high membrane fusion ability can be used as a boron delivery agent in boron neutron capture therapy (BNCT), a radical synergistic improvement of boron accumulation efficiency into tumor cells and antitumor immunity may be induced. In this study, we aimed to develop novel boron-containing biocompatible polymers modified onto HVJ-E surfaces. The copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC) and methacrylamide benzoxaborole (MAAmBO), poly[MPC-co-MAAmBO], was successfully synthesized by using a simple free radical polymerization. The molecular structures and molecular weight of the poly[MPC-co-MAAmBO] copolymer were characterized by nuclear magnetic resonance and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. The poly[MPC-co-MAAmBO] was coated onto the HVJ-E surface via the chemical bonding between the MAAmBO moiety and the sugar moiety of HVJ-E. DLS, AFM, UV-Vis, and fluorescence measurements clarified that the size of the poly[MPC-co-MAAmBO]-coated HVJ-E, HVJ-E/p[MPC-MAAmBO], to be about 130 ~ 150 nm in diameter, and that the polymer having 9.82 × 106 ~ 7 boron atoms was steadily coated on a single HVJ-E particle. Moreover, cellular uptake of poly[MPC-co-MAAmBO] could be demonstrated without cytotoxicity, and the hemolysis could be successfully suppressed by 20%. These results indicate that the HVJ-E/p[MPC-MAAmBO] may be used as boron nanocarriers in a combination of immunotherapy with BNCT.

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner.

BACKGROUND: Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells. METHODS: Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells. RESULTS: We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown. CONCLUSION: In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

Inactivated Sendai Virus Induces ROS-dependent Apoptosis and Autophagy in Human Prostate Cancer Cells.

OBJECTIVE: The 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. METHODS: PC3 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. RESULTS: Flow 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. CONCLUSION: HVJ-E exerts anticancer effects via autophagic cell death in prostate cancer cells.

Inactivated Sendai virus particle upregulates cancer cell expression of intercellular adhesion molecule-1 and enhances natural killer cell sensitivity on cancer cells.

We have already reported that the inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) has multiple anticancer effects, including induction of cancer-selective cell death and activation of anticancer immunity. The HVJ-E stimulates dendritic cells to produce cytokines and chemokines such as ß-interferon, interleukin-6, chemokine (C-C motif) ligand 5, and chemokine (C-X-C motif) ligand 10, which activate both CD8+ T cells and natural killer (NK) cells and recruit them to the tumor microenvironment. However, the effect of HVJ-E on modulating the sensitivity of cancer cells to immune cell attack has yet to be investigated. In this study, we found that HVJ-E induced the production of intercellular adhesion molecule-1 (ICAM-1, CD54), a ligand of lymphocyte function-associated antigen 1, in several cancer cell lines through the activation of nuclear factor-κB downstream of retinoic acid-inducible gene I and the mitochondrial antiviral signaling pathway. The upregulation of ICAM-1 on the surface of cancer cells increased the sensitivity of cancer cells to NK cells. Knocking out expression of ICAM-1 in MDA-MB-231 cells using the CRISPR/Cas9 method significantly reduced the killing effect of NK cells on ICAM-1-depleted MDA-MB-231 cells. In addition, HVJ-E suppressed tumor growth in MDA-MB-231 tumor-bearing SCID mice, and the HVJ-E antitumor effect was impaired when NK cells were depleted by treatment with the anti-asialo GM1 antibody. Our findings suggest that HVJ-E enhances NK cell sensitivity against cancer cells by increasing ICAM-1 expression on the cancer cell surface.

Inactivated Sendai virus induces apoptosis mediated by reactive oxygen species in murine melanoma cells.

OBJECTIVE: This 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. METHODS: B16F10 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. RESULTS: Treatment 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. CONCLUSION: These results suggest that HVJ-E possesses potential anticancer activity in B16F10 cells through ROS-mediated mitochondrial dysfunction involving the MAPK pathway.

Inactivated Sendai virus induces apoptosis and autophagy via the PI3K/Akt/mTOR/p70S6K pathway in human non-small cell lung cancer cells.

Inactivated Sendai virus (HVJ-E) has shown potential anticancer efficacy in various cancer cells. However, the ability of HVJ-E to regulate cancer cell survival and death remains largely unknown. In the present study we first found that HVJ-E exhibited cytotoxic effects in the non-small cell lung cancer cell (NSCLC) line A549 and cisplatin-resistant A549 cells (A549/DDP). The suppression of cell viability was due to both the activation of caspases and the JNK and p38 MAPK signaling pathways in A549 and A549/DDP human lung cancer cells. In addition, we demonstrated that HVJ-E could induce autophagy in NSCLC cells via the PI3K/Akt/mTOR/p70S6K signaling pathway for the first time. Inhibiting autophagy in A549/DDP cells and inducing autophagy in A549 cells enhanced HVJ-E-induced apoptosis. These findings provide a molecular basis of HVJ-E-mediated cell death and support the notion that combination treatment with autophagy modulators is an effective strategy to augment the cytotoxic effects of HVJ-E in NSCLC cells.

IPS-1 plays a dual function to directly induce apoptosis in murine melanoma cells by inactivated Sendai virus.

Inactivated Sendai virus (HVJ-E) directly kills cancer cells by inducing apoptosis through a mechanism mediated by Janus kinases/signal transducers and activators of transcription (JAK/STAT) signaling pathways. However, whether other signaling pathways are involved remain largely unknown. This study aimed to investigate the mechanism underlying HVJ-E-induced apoptosis in murine B16F10 melanoma cells. We found that HVJ-E induced B16F10 cell apoptosis via the caspase pathway, particularly caspase-9, which mediates the intrinsic apoptotic pathway. Mitogen-activated protein kinase (MAPK) pathway activation also contributed to HVJ-E-induced apoptosis. Whereas caspase pathway involvement depended on both IFN-ß promoter stimulator-1 (IPS-1) and type I interferon (IFN), MAPK pathway activation was independent of type I IFN but involved IPS-1. In addition, intratumoral HVJ-E treatment displayed a direct oncolytic effect in an in vivo BALB/c nude mouse melanoma model. Collectively, our data provides new insights into the mechanism underlying HVJ-E-induced apoptosis in tumor cells.

Induction of apoptosis in hormone-resistant human prostate cancer PC3 cells by inactivated Sendai virus.

OBJECTIVE: Inactivated 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). METHODS: PC3 cells were treated with HVJ-E at various MOI, and then interferon-ß (IFN-ß) 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. RESULTS: HVJ-E induced IFN-ß 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. CONCLUSION: Our findings have provided novel insights into the underlying mechanisms by which HVJ-E induces apoptosis in tumor cells.

Anti-tumor effects of inactivated Sendai virus particles with an IL-2 gene on angiosarcoma.

Cutaneous angiosarcoma is a life-threatening tumor that is resistant to conventional therapies. The therapeutic effects of Sendai virus particles (hemagglutinating virus of Japan envelope: HVJ-E) carrying IL-2 gene (HVJ-E/IL-2) were examined in a mouse model of angiosarcoma. Intra-tumoral injection of HVJ-E/IL-2 effectively inhibited the growth of angiosarcoma cells (ISOS-1) inoculated in mice and improved tumor-free rates. HVJ-E/IL-2 stimulated local accumulation of CD8 (+) T cells and NK cells and reduced regulatory T cells in regional lymph nodes. Notably, the prevalence of myeloid-derived suppressor cells was lower in HVJ-E/IL-2-treated mice than in HVJ-E-treated mice. HVJ-E/IL-2 treatment promoted IFN-γ production from CD8 (+) T cells in response to tumor cells, more significantly than HVJ-E treatment. Greatly improved tumor-free rates were obtained when sunitinib, a tyrosine kinase inhibitor, was administered in combination with HVJ-E/IL-2. Immunogene therapy with HVJ-E/IL-2 with or without sunitinib could be a promising therapeutic option for cutaneous angiosarcoma.

An In Vivo Screening Model for Investigation of Pathophysiology of Human Implantation Failure.

To improve current infertility treatments, it is important to understand the pathophysiology of implantation failure. However, many molecules are involved in the normal biological process of implantation and the roles of each molecule and the molecular mechanism are not fully understood. This review highlights the hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) vector, which uses inactivated viral particles as a local and transient gene transfer system to the murine uterus during the implantation period in order to investigate the molecular mechanism of implantation. In vivo screening in mice using the HVJ-E vector system suggests that signal transducer and activator of transcription-3 (Stat-3) could be a diagnostic and therapeutic target for women with a history of implantation failure. The HVJ-E vector system hardly induces complete defects in genes; however, it not only suppresses but also transiently overexpresses some genes in the murine uterus. These features may be useful in investigating the pathophysiology of implantation failure in women.

CXCL2 combined with HVJ-E suppresses tumor growth and lung metastasis in breast cancer and enhances anti-PD-1 antibody therapy.

Breast cancer has a high risk of metastasis; however, no effective treatment has been established. We developed a novel immunotherapy for breast cancer to enhance cytotoxic T lymphocytes against cancer cells using N1-type neutrophils with anti-tumor properties. For this purpose, we combined CXCL2 (CXC chemokine ligand 2) plasmid DNA with inactivated Sendai virus (hemagglutinating virus of Japan)-envelope (HVJ-E). The combination of CXCL2 DNA and HVJ-E (C/H) suppressed the growth of murine breast cancers in orthotopic syngeneic models by enhancing cytotoxic T lymphocytes and inhibited lung metastasis of breast cancer from primary lesions. N1-type neutrophils (CD11b+ Ly6G+ FAS+) increased in the tumor microenvironment with C/H treatment, and tumor suppression and cytotoxic T lymphocyte activation from C/H was blocked after administrating anti-neutrophil antibodies, which indicates the role of N1-type neutrophils in cancer immunotherapy. We also demonstrated that the anti-tumor activities of C/H treatment were enhanced by the administration of anti-PD-1 antibodies through neutrophil-mediated cytotoxic T lymphocyte activation. Thus, the triple combination of C/H and anti-PD-1 antibody C/H treatment may provide an improvement in cancer immunotherapy.

Hemagglutinating Virus of Japan Envelope (HVJ-E)-Guided Gene Transfer to the Intestinal Epithelium.

The rapidly self-renewing epithelium of the small intestine represents an exquisite model for the stem cell-driven tissue renewal and tumorigenesis. Intestinal stem cells (ISCs) are located in the crypt base, where they produce rapidly dividing progenitors that undergo cell-cycle arrest and terminal differentiation upon several rounds of cell division. So far, genetic studies in mice have played a central role in analyzing function of genes during the stem cell-driven renewal of the intestinal epithelium. However, generation and maintenance of genetically engineered mice are a time-consuming endeavor, which limits the progress in intestinal biology. Recently, we have established a novel method that serves as an alternative to mouse genetics in intestinal biology. The method, termed intestine-specific gene transfer (iGT), enables rapid and efficient delivery of small molecules, such as siRNAs and plasmids, into the intestinal epithelium of living mice by utilizing the hemagglutinating virus of Japan envelope (HVJ-E). Here, we describe a detailed protocol for iGT and discuss how this method can accelerate progress in intestinal biology and elucidate the mechanisms of intestinal epithelium self-renewal.

Cytoplasmic calcium increase via fusion with inactivated Sendai virus induces apoptosis in human multiple myeloma cells by downregulation of c-Myc oncogene.

Because the emergence of drug resistance is a major limitation of current treatments for multiple myeloma (MM), it is necessary to continuously develop novel anticancer strategies. Here, using an inactivated Sendai virus (Hemagglutinating Virus of Japan; HVJ) envelope (HVJ-E), we discovered that increase of cytoplasmic Ca2+ by virus-cell fusion significantly induced apoptosis against human MM cells but not peripheral blood mononuclear cells from healthy donors. Interaction of F protein of HVJ-E with MM cells increased intracellular Ca2+ level of MMs by the induction of Ca2+ efflux from endoplasmic reticulum but not influx from extracellular region. The elevation of the Ca2+ cytoplasmic level induced SMAD1/5/8 phosphorylation and translocation into the nucleus, and SMAD1/5/8 and SMAD4 complex suppressed c-Myc transcription. Meanwhile, HVJ-E decreases S62 phosphorylation of c-Myc and promotes c-Myc protein degradation. Thus, HVJ-E-induced cell death of MM resulted from suppression of c-Myc by both destabilization of c-Myc protein and downregulation of c-Myc transcription. This study indicates that HVJ-E will be a promising tool for MM therapy.

Virus-stimulated neutrophils in the tumor microenvironment enhance T cell-mediated anti-tumor immunity.

The tumor microenvironment (TME) fosters tumors by attenuating anti-tumor immunity, reinforcing tumor cell survival and increasing angiogenesis. Among the constituents of the TME, here, we focused on tumor-associated neutrophils (TANs). First, we found that the combination of poly I:C and inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) synergistically suppressed tumor growth in the B16-F10 melanoma mouse model. In this model, poly I:C contributed to the recruitment of CD11b+Ly6G+ neutrophils to the TME, and co-injection of poly I:C and HVJ-E increased CD11b+Ly6G+FAS+ TAN in the TME. Depletion of neutrophils abolished the synergistic anti-tumor effect of HVJ-E and poly I:C in B16-F10 tumors. We revealed that C-X-C motif chemokine ligand 2 (CXCL2) is produced in the TME by poly I:C, but HVJ-E enhanced neutrophil infiltration of the TME does not occur. An anti-CXCL2 antibody inhibited the tumor suppression by HVJ-E+poly I:C. HVJ-E in combination with recombinant CXCL2 protein or CXCL2 pDNA suppressed mouse melanoma by increasing cytotoxic T lymphocyte activity against B16-F10 melanoma, which was abolished by an anti-Ly6G antibody. HVJ-E directly and indirectly increased FAS and ICAM-1 expression in cultured bone marrow-derived naïve neutrophils. Thus, HVJ-E activates anti-tumor immunity via anti-tumorigenic neutrophils in the TME. An HVJ-E vector containing the CXCL2 gene may be applicable as a novel cancer gene therapy strategy.

Oncolytic Sendai virus-based virotherapy for cancer: recent advances.

Many drugs have been developed and optimized for the treatment of cancer; however, it is difficult to completely cure cancer with anticancer drugs alone. Therefore, the development of new therapeutic technologies, in addition to new anticancer drugs, is necessary for more effective oncotherapy. Oncolytic viruses are one potential new anticancer strategy. Various oncolytic viruses have been developed for safe and effective oncotherapy. Recently, Sendai virus-based oncotherapy has been reported by several groups, and attention has been drawn to its unique anticancer mechanisms, which are different from those of the conventional oncolytic viruses that kill cancer cells by cancer cell-selective replication. Here, we introduce Sendai virus-based virotherapy and its anticancer mechanisms.

Inactivated Sendai Virus (HVJ-E) Immobilized Electrospun Nanofiber for Cancer Therapy.

Inactivated Hemagglutinating Virus of Japan Envelope (HVJ-E) was immobilized on electrospun nanofibers of poly(ε-caprolactone) by layer-by-layer (LbL) assembly technique. The precursor LbL film was first constructed with poly-L-lysine and alginic acid via electrostatic interaction. Then the HVJ-E particles were immobilized on the cationic PLL outermost surface. The HVJ-E adsorption was confirmed by surface wettability test, scanning laser microscopy, scanning electron microscopy, and confocal laser microscopy. The immobilized HVJ-E particles were released from the nanofibers under physiological condition. In vitro cytotoxic assay demonstrated that the released HVJ-E from nanofibers induced cancer cell deaths. This surface immobilization technique is possible to perform on anti-cancer drug incorporated nanofibers that enables the fibers to show chemotherapy and immunotherapy simultaneously for an effective eradication of tumor cells in vivo.