Yung Choon Yoo

Bovine Lactoferrin and Lactoferricin, a Peptide Derived from Bovine Lactoferrin, Inhibit Tumor Metastasis in Mice

We investigated the effect of a bovine milk protein, lactoferrin (LF–B), and a pepsin–generated peptide of LF–B, lactoferricin (Lfcin–B), on inhibition of tumor metastasis produced by highly metastatic murine tumor cells, B16–BL6 melanoma and L5178Y–ML25 lymphoma cells, using experimental and spontaneous metastasis models in syngeneic mice. The subcutaneous (s.c.) administration of bovine apo–lactoferrin (apo–LF–B, 1 mg/mouse) and Lfcin–B (0.5 mg/monse) 1 day after tumor inoculation significantly inhibited liver and lung metastasis of L5178Y–ML25 cells. However, human apo–lactoferrin (apo–LF–H) and bovine holo–lactoferrin (holo–LF–B) at the dose of 1 mg/mouse failed to inhibit tumor metastasis of L5178Y–ML25 cells. Similarly, the s.c. administration of apo–LF–B as well as Lfcin–B, but not apo–LF–H and holo–LF–B, 1 day after tumor inoculation resulted in significant inhibition of lung metastasis of B16–BL6 cells in an experimental metastasis model. Furthermore, in in vivo analysis for tumor–induced angiogenesis, both apo–LF–B and Lfcin–B inhibited the number of tumor–induced blood vessels and suppressed tumor growth on day 8 after tumor inoculation. However, in a long–term analysis of tumor growth for up to 21 days after tumor inoculation, single administration of apo–LF–B significantly suppressed the growth of B16–BL6 cells throughout the examination period, whereas Lfcin–B showed inhibitory activity only during the early period (8 days). In spontaneous metastasis of B16–BL6 melanoma cells, multiple administration of both apo–LF–B and Lfcin–B into tumor–bearing mice significantly inhibited lung metastasis produced by B16–BL6 cells, though only apo–LF–B exhibited an inhibitory effect on tumor growth at the time of primary tumor amputation (on day 21) after tumor inoculation. These results suggest that apo–LF–B and Lfcin–B inhibit tumor metastasis through different mechanisms, and that the inhibitory activity of LF–B on tumor metastasis may he related to iron (Fe3+)–saturation.

Lectins isolated from Korean mistletoe (Viscum album coloratum) induce apoptosis in tumor cells

Cytotoxic lectins (KML-C) were isolated from an extract of Korean mistletoe [Viscum album C. (coloratum)] by affinity chromatography on a hydrolysed Sepharose 4B column, and the chemical and biological properties of KML-C were examined, partly by comparing them with a lectin (EML-1) from European mistletoe[Viscum album L. (loranthaceae)]. The hemagglutinating activity of KML-C was inhibited by N-acetyl-D-galactosamine and D-galactose at the minimum concentrations of 6.3 and 12.5 microM/ml, respectively. Further biochemical analyses indicated that KML-C consists of four chains (Mr = 27.5, 30, 31 and 32.5 kDa) which, in some of the molecules, are disulfide-linked, and that the chains of KML-C are distributed over a broad range of isoelectric points (pI), 8.0 to 9.0, whereas the range for EML-1 is 6.6-7.0. A difference was also observed between the N-terminal sequences of KML-C and EML-1. The isolated lectins showed strong cytotoxicity against various human and murine tumor cells, and the cytotoxic activity of KML-C was higher than that of EML-1. Tumor cells treated with KML-C exhibited typical patterns of apoptotic cell death, such as apparent morphological changes and DNA fragmentation, and its apoptosis-inducing activity was blocked by addition of Zn2+, an inhibitor of Ca2+/Mg2+ -dependent endonucleases, in a dose-dependent manner. These results suggest that KML-C is a novel lectin related to the cytotoxicity of Korean mistletoe, and that its cytotoxic activity against tumor cells is due to apoptosis mediated by Ca2+/Mg2+ -dependent endonucleases.

Prophylactic effect of Korean mistletoe (Viscum album coloratum) extract on tumor metastasis is mediated by enhancement of NK cell activity

We here demonstrated the prophylactic effect of an extract (KM-110) from Viscum album coloratum, a Korean mistletoe, on tumor metastasis produced by highly metastatic tumor cells, colon 26-M3.1 carcinoma, B16-BL6 melanoma and L5178Y-ML25 lymphoma cells, using experimental models in mice. Intravenous (i.v.) administration of KM-110 (100 microg/mouse) 2 days before tumor inoculation significantly inhibited lung metastasis of B16-BL6 and colon 26-M3.1 cells, and liver and spleen metastasis of L5178Y-ML25 cells. The prophylactic effect of KM-110 on tumor metastasis was evident with various administration routes, i.e. subcutaneous, oral, intranasal as well as i.v., and was dependent upon the dose of KM-110 administered. Furthermore, mice given KM-110 (100 microg) 2 days before tumor inoculation showed significantly prolonged survival rates compared with the untreated mice. In a time course analysis of NK activity, i.v. administration of KM-110 (100 microg) significantly augmented NK cytotoxicity to Yac-a tumor cells from 1 to 3 days after KM-110 treatment. Furthermore, depletion NK cells by injection of rabbit anti-asialo GM1 serum completely abolished the inhibitory effect of KM-110 on lung metastasis of colon 26-M3.1 cells. These results suggest that KM-110 possesses immunopotentiating activity which enhances the host defense system against tumors, and that its prophylactic effect on tumor metastasis is mediated by NK cell activation.

Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins

SummaryRecombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD50 (survival rate: 43%) or 4 LD50 (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.

Inhibitory effect of Korean mistletoe (Viscum album coloratum) extract on tumour angiogenesis and metastasis of haematogenous and non-haematogenous tumour cells in mice

We examined the inhibitory effect of an aqueous extract (referred to as KM-110) from Viscum album coloratum, a Korean mistletoe, on tumour metastasis produced by highly metastatic murine tumour cells, B16-BL6 melanoma, colon 26-M3.1 carcinoma and L5178Y-ML25 lymphoma cells, using experimental and spontaneous metastasis models in syngeneic mice. In experimental metastasis of B16-BL6 and colon 26-M3.1 cells, intravenous (i.v.) administration of KM-110 (100 micrograms/mouse) 1 day after tumour inoculation significantly inhibited lung metastasis of both tumour cells. The administration of KM-110 also exhibited a therapeutic effect on liver and spleen metastasis of L5178Y-ML25 lymphoma cells. Furthermore, in spontaneous metastasis of B16-BL6 melanoma cells, multiple administration of KM-110 into tumour-bearing mice resulted in significant inhibition of lung metastasis by tumour cells, as well as the suppressive activity to the growth of primary tumour. In in vivo analysis for tumour-induced angiogenesis, the i.v. administration of KM-110 suppressed tumour growth and inhibited the number of blood vessels oriented towards the tumour mass. In a bioassay, the culture supernatant (KM-110-treated medium) of murine peritoneal macrophages that had been stimulated with KM-110 (1-10 micrograms/ml) for 30 min followed by 24 h incubation in fresh medium showed a strong tumour necrosis factor-alpha (TNF-alpha) activity. In addition, KM-110-treated medium significantly inhibited the growth of in vitro cultures of rat lung endothelial (RLE) cells. These results suggested that the extract of Korean mistletoe inhibits tumour metastasis caused by haematogenous as well as non-haematogenous tumour cells, and that its antimetastatic effect results from the suppression of tumour growth and the inhibition of tumour-induced angiogenesis by inducing TNF-alpha.

Adjuvant activity of muramyl dipeptide derivatives to enhance immunogenicity of a hantavirus-inactivated vaccine.

The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-1 virus (B-1 vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined. When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-1 vaccine admixed with or without 100 micrograms mouse-1 of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-1 vaccine alone. Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-1 vaccine alone during 3-9 weeks after the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-1 alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid protein (rNP) of Hantaan 76-118 strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a significantly higher proliferating activity than those treated with B-1 vaccine alone. In addition, when mice were immunized once with B-1 vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-1 vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(L18) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-1 vaccine alone. These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-1 vaccine to induce a humoral and cellular response to HFRS virus.

Effects of muramyl dipeptide derivatives as adjuvants on the induction of antibody response to recombinant hepatitis B surface antigen

The ability of two muramyl dipeptide (MDP) derivatives, B30-MDP and MDP-Lys(L18), to enhance the immunogenicity of recombinant hepatitis B surface antigen (rHBsAg) was examined. When mice were immunized intraperitoneally with rHBsAg together with each MDP derivative, the antibody titres were higher than those in mice immunized with alum-adsorbed rHBsAg, which is a commercially available hepatitis B vaccine. When mice were given a subcutaneous or intramuscular injection of rHBsAg and either MDP derivative, the antibody titres were the same as those in mice given alum-adsorbed rHBsAg. These results indicate the usefulness of MDP derivatives as immunoadjuvants for a new-generation vaccine.

Comparison of Virulence between Seoul Virus Strain SR-11 and Hantaan Virus Strain 76-118 of Hantaviruses in Newborn Mice

Virulence of hantavirus strain of SR‐11 Seoul virus and Hantaan 76–118 (HTN) of Hantaan virus were compared. Infections of both strains were lethal in newborn mice. However, inoculum required to cause lethal infection was about 4,000 times higher for strain HTN (1.65 × 103 PFU/mouse/LD50) than for strain SR‐11 (0.36 PFU). Thus, both strains were considered pathogenic to newborn mice but they possessed different levels of virulence. The assay system used for these strains in newborn mice proved to be useful in the study of hantavirus vilurence. Growth curves of the two strains in CV‐7 cell cultures were compared. Strain SR‐11 was shown to have higher activity of virus replication and virus release into the culture fluids than strain HTN. The possibility of a relationship between replication activity and high levels of virulence in mice was suggested.

Effect of MDP-Lys(L18) as a mucosal immunoadjuvant on protection of mucosal infections by Sendai virus and rotavirus

To examine the effect of MDP-Lys(L18), a derivative of muramyl dipeptide (MDP), as a mucosal immunoadjuvant, we investigated its activity to augment host resistance against mucosal infections by Sendai virus and rotavirus in mice. In an experimental infection model using suckling mice (10-day-old) inoculated perorally (p.o.) with 1.5 x 10(6) p.f.u. mouse-1 of rotavirus strain SA11, intrarectal (i.r.) as well as p.o. administration of MDP-Lys(L18) (50 micrograms mouse-1) prior to virus infection markedly reduced rotavirus-induced diarrhea. Furthermore, when MDP-Lys(L18) was administered p.o. (1 mg mouse-1), i.r. (300 micrograms mouse-1) or intranasally (i.n., 100 micrograms mouse-1) various days before Sendai virus infection (2.6 x 10(4) HAD mouse-1), all the mucosal administration of MDP-Lys(L18) significantly protected a lethal infection of Sendai virus, showing a dose-dependent manner. However, the efficacy of MDP-Lys(L18) to induce the prophylactic activity against the viruses somewhat varied according to the administration route and timing. In time course analysis of virus isolation in vivo, the mice administered with MDP-Lys(L18) exhibited a significant reduction of both viruses in the lungs for Sendai virus and in the bowels for rotavirus. These results suggest that MDP-Lys(L18) is a potent mucosal immunoadjuvant to enhance nonspecific host resistance against two mucosal infectious viruses, Sendai virus and rotavirus.

Inhibition of tumor metastasis by Arg-Gly-Asp-Ser (RGDS) peptide conjugated with sulfated chitin derivative, SCM-chitin-RGDS

We have synthesized a new compound in which Arg-Gly-Asp-Ser (RGDS) was conjugated with 6-0-sulfated and 6-O-carboxymethyl-chitin (SCM-chitin), i.e. SCM-chitin-RGDS, and tested the inhibitory effect on lung and liver metastases of three different types of tumors in mice. SCM-chitin-RGDS was more effective for the inhibition of liver metastasis of L5178Y-ML25 lymphoma and lung metastases of colon 26 M3.1 cells than SCM-chitin, RGDS or their mixture. GRGDS peptide, however, required a higher dose (3000 µg) to obtain a sufficiently antimetastatic effect. Intermittent i.v. administration of SCM-chitin-RGDS before or after the i.v. inoculation of L5178Y-ML25 cells caused significant inhibition of liver metastasis as compared with the multiple administration of RGDS, SCM-chitin or untreated control. Co-injection of lymphoma cells with SCM-chitin-RGDS or multiple treatment of SCM-chitin-RGDS after tumor inoculation showed significantly enhanced survival rate. SCM-chitin-RGDS also showed the spontaneous lung metastasis produced by intrafootpad injection of B16-BL6 melanoma cells by the multiple i.v. administrations. These results demonstrate that the conjugation of RGDS peptide with SCM-chitin led to augmentation of therapeutic potential to cancer metastasis, thus implying an importance of the conjugation of cell-adhesive RGDS peptide with structurally heparin-like SCM-chitin, which possesses binding ability to the heparin-binding domain of fibronectin or laminin and extremely low anticoagulant properties.