Viscum Album Coloratum Research Articles

Dietary Korean mistletoe enhances cellular non-specific immune responses and survival of Japanese eel ( Anguilla japonica)

The present study was performed to investigate the immunostimulatory effects of Korean mistletoe extract (KM-110; Viscum album Coloratum) on the non-specific immune response and protection against Aeromonas hydrophila infection in Japanese eel ( Anguilla japonica). Eels were fed under 4 regimes, 0%, 0.1%, 0.5% and 1.0% KM-110 mixed diet. On day 14 after feeding, 15 fish from each group were injected i.p. with live A. hydrophila (3 × 10 6 CFU) and the remaining unchallenged fish from each group were used to study the innate immune response. On 14 days post-infection, the total survival rates were 26.6% in control, and 33.3%, 66.6% and 80% in 0.1%, 0.5% and 1% KM-110-treated groups, respectively. The maximum lysozyme activity was observed in the 1% KM-110-treated group. There was no significant difference of lysozyme activity between 0.1% and 0.5% KM-110 group. Superoxide anion (O 2 −) production was significantly ( p < 0.05) augmented in the 0.5% and 1% KM-110 groups compared to the control and 0.1% KM-110 group. No significant difference of (O 2 − production was found between 0.5% and 1% KM-110 group. Likewise, there was a significant increase in phagocytic activity in the 0.5% KM-110 group compared with the 0.1% group ( p < 0.05), but no significant difference between the 0.5% and the 1% KM-110 group indicating that 0.5% KM-110 concentration is suitable for stimulating maximum phagocytic activity resulting in a high amount of ROI production. Considering the present results, KM-110 could be utilized as a promising immunostimulating substance for a diet in aquaculture.

Effects of Korean Mistletoe Lectin(Viscum album coloratum) on Proliferation and Cytokine Expression in Human Peripheral Blood Mononuclear Cells and T-Lymphocytes

The anti-cancer activity of mistletoe has been ascribed to a combination of cytotoxic and immunological effects. We previously showed that Korean mistletoe lectin (Viscum album L. var. coloratum agglutinin, VCA) can stimulate IFN-gamma production and modulate proliferation in murine splenocytes. In this study, we investigated the effects of VCA on human peripheral blood mononuclear cells (hPBMC) and T-lymphocytes. The addition of VCA resulted in a significant inhibition of proliferation at higher concentrations (at 2-8 ng/mL, 1-8 ng/mL in hPBMC and T-lymphocytes, respectively) but an induction at lower concentrations (at 4-16 pg/mL, 4-32 pg/mL in hPBMC and T-lymphocytes, respectively). Further studies were carried out to determine if the pro-proliferative or anti-proliferative activity exhibited by VCA was correlated with apoptosis and cytokine secretion. As a result, the apoptotic cell number increased to 26% after 48 h of VCA treatment (10 ng/mL) in the presence of anti-CD3/CD28 antibodies. On the other hand, without anti-CD3/CD28 antibody stimulants, VCA did not arrest cell cycle. In addition, it was shown that VCA could induce IL-2 secretion was dose-dependently increased by VCA in stimulated (anti-CD3/CD28 antibodies) (at 0.25-2 ng/mL) and non-stimulated (at 3-25 pg/mL) human T-lymphocytes. Also, at low and non-toxic concentrations of VCA, the RT-PCR result confirmed the release of pro-inflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, IL-8, and IFN-gamma. These data may suggest new perspective to modulate the balance between cell growth, cytokine production and programmed cell death therapeutically.

Protective potential of mistletoe (Viscum album coloratum) against oxidative stress and cellular premature senescence

The protective activity from oxidative stress and antiaging potential of mistletoe (Viscum album coloratum) were investigated under in vitro and cellular system using LLC-PK1 renal epithelial cell and WI-38 human lung fibroblast cells. The water extract from the mistletoe (Korean product) showed nitric oxide (NO) and hydroxyl radical scavenging activity as concentration dependent-manner. Moreover, under LLC-PK1 cellular model, the mistletoe extract ameliorated oxidative stress induced by 2,2′-azobis (2-amidinopropane) dihydrochloride, the generator of peroxyl radical. In addition, the protective effect of mistletoe from NO under cellular oxidative stress was also studied. The LLC-PK1 cells showed the decreases in cell viability by the treatment with generators of peroxynitrite, 3-morpholinosydnonimine, and its precurors NO and superoxide anion produced by sodium nitroprusside and pyrogallol, respectively. However, the mistletoe led to the significant increases in the cell viability with dose-dependence. Furthermore, the protective potential from oxidative stress-induced aging was studied using WI-38 cells. The WI-38 cells exerted the characteristics of cellular premature senescence by the treatment of hydrogen peroxide, but mistletoe protected against stress-induced cellular senescence. The present study suggests that mistletoe extract would have the protective activity from oxidative stress caused by free radicals and the promising anti-aging potential against oxidative stress-induced aging process.

Mechanisms involved in Korean mistletoe lectin-induced apoptosis of cancer cells

To investigate the anti-cancer mechanisms of Korean mistletoe lectin (Viscum album coloratum agglutinin, VCA) using a human colon cancer cell line (COLO). Cytotoxic effects of VCA on COLO cells were determined by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay in vitro and tumor-killing effects in vivo. To study the mechanisms involved, the expression of various pro-caspases, anti-apoptotic proteins, and death receptors was determined by western blot. To determine which death receptor is involved in VCA-induced apoptosis of COLO cells, cytotoxicity was examined by MTT assay after treatment with agonists or antagonists of death receptors. VCA killed COLO cells in a time- and dose-dependent manner and induced complete regression of tumors in nude mice transplanted with COLO cells. Treatment of COLO cells with VCA activated caspase-2, -3, -8, and -9 and decreased expression of anti-apoptotic molecules including receptor interacting protein, nuclear factor-kappaB, X-linked inhibitor of apoptosis protein, and Akt/protein kinase B. We then examined the involvement of death receptors in VCA-induced apoptosis. Only tumor necrosis factor receptor 1, among the death receptors examined, was involved in apoptosis of COLO cells, evidenced by inhibition of VCA-induced apoptosis and decreased activation of caspases, particularly caspase-8, by tumor necrosis factor receptor 1 antagonizing antibody. VCA-induced apoptotic COLO cell death is due to the activation of caspases and inhibition of anti-apoptotic proteins, in part through the tumor necrosis factor receptor 1 signaling pathway.

Mistletoe Lectin (Viscum album coloratum) Modulates Proliferation and Cytokine Expressions in Murine Splenocytes

It is well documented that an extract of European mistletoe has a variety of biological effects, such as the stimulation of cytokine production from immune cells, and additional immunoadjuvant activities. While the European mistletoe has been studied intensively, we know less about Korean mistletoe as a therapeutic plant, especially as a possible immunomodulating drug. This study will investigated the effects of Korean mistletoe lectin (Viscum album L. var. coloratum agglutinin, VCA) on murine splenocytes to investigate whether VCA acts as an immunomodulator, which could lead to improved immune responses in these cells. The results showed that VCA inhibited cell proliferation at higher concentrations (at 1-8 ng/ml) and enhanced cell proliferation at lower concentrations (at 4-32 pg/ml). Further studies were carried out to determine if the proproliferative or anti-proliferative activity exhibited by VCA was correlated with cytokine secretion. Consequently, interferon (IFN)-gamma secretion was decreased in concanavalin A (ConA)-stimulated murine splenocytes by VCA (4-64 ng/ml), but there was no change in IL-4 levels. This suggests that VCA has the ability to modulate murine splenocyte proliferation and can possibly act on the balance of Th1/Th2 cellular immune responses.

Molecular Characterization of the Recombinant A-chain of a Type II Ribosome-Inactivating Protein (RIP) from Viscum album coloratum and Structural Basis on its Ribosome-Inactivating Activity and the Sugar-binding Properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.

Antitumor activity of the Korean mistletoe lectin is attributed to activation of macrophages and NK cells.

Inhibitory effect of the lectins (KML-C) isolated from Korean mistletoe (KM; Viscum album coloratum) on tumor metastases produced by murine tumor cells (B16-BL6 melanoma, colon 26-M3.1 carcinoma and L5178Y-ML25 lymphoma cells) was investigated in syngeneic mice. An intravenous (i.v.) administration of KML-C (20-50 ng/mouse) 2 days before tumor inoculation significantly inhibited lung metastases of both B16-BL6 and colon 26-M3.1 cells. The prophylactic effect of 50 ng/mouse of KML-C on lung metastasis was almost the same with that of 100 microg/mouse of KM. Treatment with KML-C 1 day after tumor inoculation induced a significant inhibition of not only the experimental lung metastasis induced by B16-BL6 and colon 26-M3.1 cells but also the liver and spleen metastasis of L5178Y-ML25 cells. Furthermore, multiple administration of KML-C given at 3 day-intervals after tumor inoculation led to a significant reduction of lung metastasis and suppression of the growth of B16-BL6 melanoma cells in a spontaneous metastasis model. In an assay for natural killer (NK) cell activity, i.v. administration of KML-C (50 ng/mouse) significantly augmented NK cytotoxicity against Yac-1 tumor cells 2 days after KML-C treatment. In addition, treatment with KML-C (50 ng/mouse) induced tumoricidal activity of peritoneal macrophages against B16-BL6 and 3LL cells. These results suggest that KML-C has an immunomodulating activity to enhance the host defense system against tumors, and that its prophylactic and therapeutic effect on tumor metastasis is associated with the activation of NK cells and macrophages.

CDNA Cloning and Sequence Analysis of the Lectin Genes of the Korean Mistletoe (Viscum album coloratum)

cDNA Cloning and Sequence Analysis of the Lectin Genes of the Korean Mistletoe (Viscum album coloratum)

Cellular and humoral adjuvant activity of lectins isolated from Korean mistletoe ( Viscum album colaratum)

The adjuvant effect of lectins (KML-C) isolated from Korean mistletoe ( Viscum album coloratum) on induction of humoral and cellular immune responses against keyhole limpet hemocyanine (KLH) was examined. When mice were immunized subcutaneously (s.c.) with KLH (20 μg/mouse) admixed with or without 50 ng/mouse of KML-C (KLH+KML-C), mice immunized with KLH+KML-C showed significantly higher antibody titers against KLH than those immunized with KLH alone, showing the highest titer 5 weeks after immunization. Furthermore, boost immunization with KLH+KML-C at 2-week interval elicited much higher activity than single immunization to enhance antibody responses against KLH. The assay for determining isotypes of antibodies revealed that KML-C augmented KLH-specific antibody titers of IgG1, IgG2a and IgG2b. The culture supernatants obtained from the splenocytes of mice treated with KLH+KML-C also showed a higher level of both KLH-specific Th-1 (IL-2 and IFN-γ) and Th-2 type cytokine (IL-4). In an in vitro analysis of T lymphocyte proliferation to KLH on week 4, the splenocytes of mice treated with KLH+KML-C showed a significantly higher proliferating activity than those treated with KLH alone. In addition, mice immunized twice with KLH+KML-C and followed by intrafootpad (i.f.) injection of KLH (50 μg/site) 14 weeks after the primary immunization induced a higher delayed-type hypersensitivity (DTH) reaction than mice treated with KLH alone. These results suggest that KML-C is a potent immunoadjuvant to enhance cellular and humoral immune responses.

GAMMA-INTERFERON (IFN-γ) AUGMENTS APOPTOTIC RESPONSE TO MISTLETOE LECTIN-II VIA UPREGULATION OF FAS/FAS L EXPRESSION AND CASPASE ACTIVATION IN HUMAN MYELOID U937 CELLS

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.

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 μM/ml, respectively. Further biochemical analyses indicated that KML-C consists of four chains ( M r = 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 (p I), 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 Zn 2+, an inhibitor of Ca 2+/Mg 2+-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 Ca 2+/Mg 2+-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.) adminstration of KM-110 (100 μg/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 μg) 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 μg) 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.

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 μg/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 (1–10μg/ml) for 30 min followed by 24 h incubation in fresh medium showed a strong tumour necrosis factor-α (TNF-α) 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-α.