Koji Ichiyama

In Vivo and In Vitro Studies Suggest a Possible Involvement of HPV Infection in the Early Stage of Breast Carcinogenesis via APOBEC3B Induction

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application.

Curdlan sulfate (CRDS), a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

Sulfated Alkyl Oligosaccharides Inhibit Human Immunodeficiency virus in vitro and Provide Sustained Drug Levels in Mammals

This study provides an estimate of the relative anti-human immunodeficiency virus (HIV) activities of synthetic sulfated alkyl oligosaccharides in vitro and of their mechanism of action, and an assessment of the levels of alkyl oligosaccharides in small mammals. The antiviral activities of the compounds against several human immunodeficiency virus type-1 and type-2 strains were determined in human CD4+ cells, including primary lymphocytes and macrophages. Laser flow cytometry and a cell-based syncytium assay were used to elucidate the anti-binding/fusion properties of the oligosaccharides. The sulfated alkyl laminarioligosaccharide DL-110 was shown to be the most potent and selective anti-HIV agent in culture with a median inhibitory concentration of 0.2 μM in primary human lymphocytes. This compound did not markedly interact with the CD4+ receptor on lymphocytes at 50 μM, but demonstrated potent anti-syncytium properties in vitro at submicromolar concentrations. DL-110 had no anti-coagulation activity at 38 μM. Mice, rabbits and beagle dogs were given an intravenous injection of test compounds and the drug levels in serum were quantified. When 32 mg kg−1 of DL-110 was administered to mice, significant antiviral concentrations in serum were achieved even 12 h after treatment. Similarly, prolonged antiviral effects were noted in rabbits and dogs 24 h after injection of DL-110. The half-life of DL-110 in mice, rabbits and dogs was estimated to be 5 h. DL-110 and some of its derivatives are promising candidates for further evaluation of the prophylaxis and therapy of HIV infections.

Sulfated pentagalloyl glucose (Y-ART-3) inhibits HIV replication and cytopathic effects in vitro, and reduces HIV infection in hu-PBL-SCID mice.

To evaluate the efficacy of Y-ART-3 as an antiviral drug for HIV infections, its anti-HIV activity was assessed in vitro in cell culture systems and in vivo in hu-PBL-SCID mice. The results indicated that Y-ART-3 invariably inhibited not only HIV-1, but also HIV-2 and SIV strains. Its mechanism of action is ascribed to inhibition of viral adsorption to CD4-positive cells. In an in vivo study, human Ig- and CD4-positive cells were detected at similar levels in Y-ART-3-treated hu-PBL-SCID mice that were infected with HIV, and in PBS-treated control hu-PBL SCID mice that were not infected with HIV. If HIV positivity was calculated using the number of tests in which HIV was detected (i.e. PCR, and p24 from co-cultures of spleen and peritoneal wash cells), a significant effect of Y-ART-3 at a dose of 4 mg/kg was noted. Therefore, Y-ART-3 may be considered to be a potent and effective anti-HIV compound.

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

Macrophage response to oncolytic paramyxoviruses potentiates virus‐mediated tumor cell killing

Tumor‐associated macrophages (TAMs) are known to regulate tumor response to many anti‐cancer therapies, including oncolytic virotherapy. Oncolytic virotherapy employing oncolytic paramyxoviruses, such as attenuated measles (MeV) and mumps (MuV) viruses, has demonstrated therapeutic potential against various malignancies. However, the response of TAMs to oncolytic paramyxoviruses and the consequent effect on virotherapeutic efficacy remains to be characterized. Here, we demonstrate that the presence of human monocyte‐derived macrophages (MDMs), irrespective of initial polarization state, enhances the virotherapeutic effect of MeV and MuV on breast cancer cells. Notably, our finding contrasts those of several studies involving other oncolytic viruses, which suggest that TAMs negatively impact virotherapeutic efficacy by impeding virus replication and dissemination. We found that the enhanced virotherapeutic effect in the presence of MDMs was due to slightly delayed proliferation and significantly elevated cell death that was not a result of increased virus replication. Instead, we found that the enhanced virotherapeutic effect involved several macrophage‐associated anti‐tumor mediators, and was associated with the modulation of MDMs towards an anti‐tumor phenotype. Our findings present an alternative view on the role of TAMs in oncolytic virotherapy, and highlight the immunotherapeutic potential of oncolytic paramyxoviruses; possibly contributing towards the overall efficacy of oncolytic virotherapy.

Chemically sulfated natural galactomannans with specific antiviral and anticoagulant activities

Naturally occurring galactomannans were sulfated to give sulfated galactomannans with degrees of substitution of 0.7-1.4 per sugar unit and molecular weights of M¯n=0.6×10(4)-2.4×10(4). Sulfated galactomannans were found to have specific biological activities in vitro such as anticoagulant, anti-HIV and anti-Dengue virus activities. The biological activities were compared with those of standard dextran and curdlan sulfates, which are polysaccharides with potent antiviral activity and low cytotoxicity. It was found that sulfated galactomannans had moderate to high anticoagulant activity, 13.4-36.6unit/mg, compared to that of dextran and curdlan sulfates, 22.7 and 10.0unit/mg, and high anti-HIV and anti-Dengue virus activities, 0.04-0.8μg/mL and 0.2-1.1μg/mL, compared to those curdlan sulfates, 0.1μg/mL, respectively. The cytotoxicity on MT-4 and LCC-MK2 cells was low. Surface plasmon resonance (SPR) of sulfated galactomannans revealed strong interaction with poly-l-lysine as a model compound of virus proteins, and suggested that the specific biological activities might originate in the electrostatic interaction of negatively charged sulfate groups of sulfated galactomannans and positively charged amino groups of surface proteins of viruses. These results suggest that sulfated galactomannans effectively prevented the infection of cells by viruses and the degree of substitution and molecular weights played important roles in the biological activities.

Combination of vaccine-strain measles and mumps virus synergistically kills a wide range of human hematological cancer cells: Special focus on acute myeloid leukemia

Through combining vaccine-derived measles and mumps viruses (MM), we efficiently targeted a wide range of hematopoietic cancer cell lines. MM synergistically killed many cell lines including acute myeloid leukemia (AML) cell lines. Further investigation suggested that enhanced oncolytic effect of MM was due to increased apoptosis induction. In an U937 xenograft AML mouse model, MM displayed greater tumor suppression and prolonged survival. Furthermore, MM efficiently killed blasts from 16 out of 20 AML patients and elicited more efficient killing effect on 11 patients when co-administered with Ara-C. Our results demonstrate that MM is a promising therapeutic candidate for hematological malignancies.

In vitro study on cytotoxic effect and anti-DENV2 activity of Carica papaya L. leaf

Dengue fever is the most deleterious and rapidly spreading mosquito-borne viral disease, and to date it has resisted attempts to eradicate it. Carica papaya L. leaf extract is traditionally used to cure dengue fever and its associated symptoms. However, no in vitro studies have been reported for the anti-dengue efficacy of this extract. So, the present study attempted to determine the phytochemicals present in Carica papaya L. leaf extracts, as well as their cytotoxic effect and anti-DENV2 activity on the LLC-MK2 cell line. Methanolic extracts, containing triterpenoids and flavonoids, showed cytotoxic effects (CC50=0.6156 mg ml−1), whereas a chloroform extract, rich in alkaloids, tannin and saponin, was non-cytotoxic (CC50= >1 mg ml−1) to LLC-MK2 cells and it showed inhibitory activity (EC50= >1 mg ml−1) against DENV2 with a selectivity index value of±>1. This indicates that the crude chloroform extract has moderate or less inhibitory action against DENV2 growth in in vitro conditions. The current study will help in the future development of new and novel drugs against dengue pathogens with high efficacy.

A novel assay system for anti-human immunodeficiency virus type 1 (HIV-1) activity using a subclone of a monocytic cell line, U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti‐HIV‐1 activity of some antiviral compounds was evaluated in HIV‐1‐infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV‐1 replication in cl.1–14 cells, its 50% effective concentration (EC50) values was 80 times higher than that in HIV‐1 infected MT‐4 cells; the EC50 of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT‐4 cells, respectively. In contrast, the anti‐HIV‐1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT‐4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT‐4 cells. The antiviral activity of several compounds in the HIV‐1‐infected cl.1–14 cells was similar to that in the HIV‐1jr‐fl‐infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.