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.

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

AIM: To investigate the anti-cancer mechanisms of Korean mistletoe lectin (Viscum album coloratum agglutinin, VCA) using a human colon cancer cell line (COLO). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Critical role of reactive oxygen species and mitochondrial membrane potential in Korean mistletoe lectin-induced apoptosis in human hepatocarcinoma cells.

Viscum album L. coloratum agglutinin (VCA), isolated from Korean mistletoe, is a strong inducer of apoptosis in a variety of tumor cells; however, the underlying molecular mechanisms responsible are not clear. Here, we show that VCA induces apoptotic killing, as demonstrated by DNA fragmentation, Hoechst 33258 staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and flow cytometry analysis in hepatocarcinoma Hep3B cells. VCA treatment results in a significant increase in reactive oxygen species (ROS) and loss of mitochondrial membrane potential (DeltaPsim). Furthermore, treatment with the antioxidant N-acetyl-L-cysteine reduces ROS induction by VCA, preventing apoptosis in Hep3B cells, indicating that oxidative stress is involved in VCA-mediated cell death. Our results also show rapid changes in mitochondrial transition permeability, Bax translocation, cytochrome c release, caspase-3 activity, and poly(ADP-ribose) polymerase degradation in Hep3B cells occurring in VCA-induced apoptosis. There is much evidence that implicates c-Jun NH2-terminal kinase (JNK) activation with apoptosis in a variety of cellular and animal models. In this study, we show that VCA induces JNK phosphorylation, which is abolished with pretreatment with a JNK inhibitor. Moreover, Hep3B cells overexpressing JNK1 or stress-activated protein kinase kinase (SEK1) seem to be more susceptible to cell death from ROS and loss of DeltaPsim induced by VCA, whereas expression of dominant-negative JNK1 or SEK1 in Hep3B cells do not. These data suggest that JNK phosphorylation may be a major regulator involved in VCA-induced apoptosis. Together, these results suggest that VCA induces apoptosis by inducing ROS production and a loss of DeltaPsim, in which JNK phosphorylation plays a critical role in these events.

Preparation of alginate/chitosan microcapsules and enteric coated granules of mistletoe lectin.

The aqueous extract of European mistletoe (Viscum album, L.) has been used in cancer therapy. The purified mistletoe lectins, main components of mistletoe, have demonstrated cytotoxic and immune-system-stimulating activities. Korean mistletoe (Viscum album L. coloratum), a subspecies of European mistletoe, has also been reported to possess anticancer and immunological activities. A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. coloratum agglutinin, VCA) with Mr 60 kDa was isolated from Korean mistletoe. Mistletoe preparations have been given subcutaneously due to the low stability of lectin in the gastrointestinal (GI) tract. In the present study, we investigated the possibility of alginate/chitosan microcapsules as a tool for oral delivery of mistletoe lectin. In addition, our strategy has been to develop a system composed of stabilizing cores (granules), which contain mistletoe lectin, extract or powder, coated by a biodegradable polymer wall. Our results indicated that successful incorporation of VCA into alginate/chitosan microcapsules has been achieved and that the alginate/chitosan microcapsule protected the VCA from degradation at acidic pH values. And coating the VCA with polyacrylic polymers, Eudragit, produced outstanding results with ideal release profiles and only minimal losses of cytotoxicity after manufacturing step. The granules prepared with extract or whole plant produced the best results due to the stability in the extract or whole plant during manufacturing process.

Mistletoe lectin induces apoptosis and telomerase inhibition in human A253 cancer cells through dephosphorylation of Akt.

Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.

Korean mistletoe lectin-induced apoptosis in hepatocarcinoma cells is associated with inhibition of telomerase via mitochondrial controlled pathway independent of p53.

The extract of European mistletoe (Viscum album, L) has been used in adjuvant chemotherapy of cancer and mistletoe lectins are considered to be major active components. The present work was performed to investigate the effects of Korean mistletoe lectin (Viscum album L. coloratum agglutinin, VCA) on proliferation and apoptosis of human hepatoma cells as well as the underlying mechamisms for these effects. We showed that VCA induced apoptosis in both SK-Hep-1 (p53-positive) and Hep 3B (p53-negative) cells through p53- and p21-independent pathways. VCA induced apoptosis by down-regulation of Bcl-2 and by up-regulation of Bax functioning upstream of caspase-3 in both cell lines. In addition, we observed down-regulation of telomerase activity in both VCA-treated cells. Our results provide direct evidence of the anti-tumor potential of this biological response which comes from inhibition of telomerase and consequent inducing apoptosis. VCA-induced apoptosis is regulated by mitochondrial controlled pathway independently of p53. These findings are important for the therapy with preparation of mistletoe because they show that telomerase-dependent mechanism can be targeted by VCA in human hepatocarcinoma. Taken together, our results suggest that the VCA, considered as a telomerase-inhibitor, can be envisaged as a candidate for enhancing sensitivity of conventional anticancer drugs.