Fermented mistletoe extract as a multimodal antitumoral agent in gliomas

Abstract

In Europe, commercially available extracts from the white-berry mistletoe (Viscum album L.) are widely used as a complementary cancer therapy. Mistletoe lectins have been identified as main active components and exhibit cytotoxic effects as well as immunomodulatory activity. Since it is still not elucidated in detail how mistle toe extracts such as ISCADOR communicate their effects, we analyzed the mechanisms that might be responsible for their antitumoral function on a molecular and functional level. ISCADOR-treated glioblastoma (GBM) cells down-regulate central genes involved in glioblastoma progression and malignancy such as the cytokine TGF-β and matrix-metalloproteinases. Using in vitro glioblastoma/immune cell co-cultivation assays as well as measurement of cell migration and invasion, we could demonstrate that in glioblastoma cells, lectin-rich ISCADOR M and ISCADOR Q significantly enforce NK-cell-mediated GBM cell lysis. Beside its immune stimulatory effect, ISCADOR reduces the migratory and invasive potential of glioblastoma cells. In a syngeneic as well as in a xenograft glioblastoma mouse model, both pretreatment of tumor cells and intratumoral therapy of subcutaneously growing glioblastoma cells with ISCADOR Q showed delayed tumor growth. In conclusion, ISCADOR Q, showing multiple positive effects in the treatment of glioblastoma, may be a candidate for concomitant treatment of this cancer.

Figures

Figure 1

In GBM cells, ISCADOR Q changes the expression of cancer associated genes. (a) PCR-based microarray expression analysis. LNT-229 cells were treated with ISCADOR Q (100 μg/mL) or were left untreated. 24 h later, the cells were harvested, mRNA was prepared, reverse transcribed into cDNA and used for microarray-based expression analysis on a panel of 96 migration-/invasion-angiogenesis-involved genes. The best downregulated genes with a threshold >3 are shown. (b) RT-PCR-based validation of differentially regulated genes detected in (a) at 12 or 24 h of ISCADOR Q treatment (n = 4, SEM). (c) TGF-β ELISA of ISCADOR Q treated LNT-229 cells. The cells were treated with ISCADOR Q (100 μg/mL, 24 h) followed by incubation in serum-free medium for 48 h. Supernatants were harvested and TGF-β 1 or TGF-β 2 was quantified using ELISA (n = 4, SEM). (d) The cells were treated with ISCADOR Q (100 μg/mL, 12 or 24 h). Supernatants were harvested 48 h later. Angiopoietin-1 content was analyzed using ELISA (n = 4, SEM). (e) The cells were treated as in (c). Supernatants were harvested and VEGF content was analyzed using ELISA (n = 4, SEM). (f) Contingency table analysis of the PCR-based microarray expression data together with the corresponding genes of the REMBRANDT database. Nominal scaled response variable (genes significantly up- (1), non- (0), or down- (−1) regulated in glioblastomas versus normal brain tissue expression pattern in the REMBRANDT database) and nominal explanatory variable (same gene up- or downregulated in our PCR-based microarray expression analysis after ISCADOR treatment) were analysed and subsequently tested by the likelihood ratio test (P = 0.0046).

Figure 2

ISCADOR reduces glioma cell growth in a dose dependent manner. LNT-229 (a) or SMA560 cells (b) were treated of 24 or 48 h with increasing concentrations of ISCADOR or were left untreated. Cell density was assessed by crystal violet staining (n = 3, SEM).

Figure 3

ISCADOR does not induce cell death at a concentration of 100 μg/mL. (a) LNT-229 cells were treated with ISCADOR (100 μg/mL, 24 h), or were left untreated, and then allowed to grow. Cell density was assessed by crystal violet staining every 24 h (n = 9 for LNT-229 cells, n = 4 for SMA560 cells, SEM). (b) The cells were treated as in (a). Viable cells were determined by trypan blue staining (n = 3, SEM). (c) Fluorescence based Caspase 3/7 activity assay. The cells were treated as indicated and caspase activity was analyzed 16, 24 and 48 h later using the fluorescent substrate DEVM-amc. As a positive control, the cells were treated with CD95L (100 U/mL + 10 μg/mL CHX) for 4 h (n = 3, SEM).

Figure 4

Treatment of GBM cells with ISCADOR enhances NK cell mediated tumor cell lysis. (a) LNT-229-Luc cells were treated with the appropriated ISCADOR variant (100 μg/mL) for 24 h. The cells were washed and incubated with human PBLs at different E/T cell ratio for 4 h. Cell lysis was measured by quantifying luciferase activity. (b) LNT-229-Luc cells were treated as in (a). PBLs were incubated with neutralizing NKG2D antibody (R&D Systems, Wiesbaden, Germany, 10 μg/mL) or left untreated prior to coincubation with GBM cells at an E/T of 40 : 1. (c). LNT-229-Luc cells were treated as in (a) and coincubated with purified polyclonal NK cells ((a) to (c): one representative out of three independent experiments is shown, bars = SD). (d) CFSE labeled purified NK cells were cocultivated with LNT-229 glioma cells. After extensive washing, NK cells (upper panel) or coculture (lower panels) were microscopically documented (n = 3, one representative experiment is shown). (e) Quantification of NK cell attachment on LNT-229 cells (n = 3, SEM,  *P = 0.035).

Figure 5

ISCADOR reduces cell motility and invasiveness in human and mouse GBM cell lines. (a) Boyden chamber migration assays. The cells were treated for 24 h with ISCADOR P, M, or Q (100 μg/mL). 20.000 treated cells were seeded in upper parts of Boyden chambers. Migrated cells were counted 21 h later (n = 3 for ISCADOR P, n = 5 for ISCADOR M, n = 7 for ISCADOR Q, SEM). (b) Scratch assay. The cells were seeded and treated as in (a) 24 h after treatment a scratch was set and migration of cells were documented photographically every 24 h. For better visualization, migration borders were marked with black lines (n = 3, one representative experiment is shown). (c) Boyden chamber matrigel invasion assay. The cells were treated as in (a) (n = 3, SEM).

Figure 6

ISCADOR Q mediated reduction of cell motility is caused by reduced MMP-expression and activity. (a) LNT-229 cells were treated with ISCADOR Q or were left untreated. 24 h later, cellular supernatants (SN) or lysates were prepared. MMP or TIMP expression was analyzed by immunoblot (Ab; specific antibody, one representative experiment is shown). (b) Quantification of protein expression (n = 3 for MMP-1, n = 9 for MMP-2, n = 3 for MMP-3, n = 8 for MMP-9, n = 2 for MMP-10, n = 4 for MMP-14, n = 5 for TIMP-2). (c) Gelatinase zymogram analyzing MMP activity (n = 10, one representative experiment is shown). (d) Quantification of MMP gelatinase activity (n = 8 for MMP-9, n = 10 for MMP-2, n = 4 for 36 kD active MMP-2; SEM). Note that the activated form of MMP-9 could not be separated strictly from MMP-2. Therefore quantification of MMP-9 activity was only done for proMMP-9.

Figure 7

ISCADOR Q reduces the growth of subcutaneously growing GBM in mice. (a) and (b) The cells were pretreated with ISCADOR Q (100 μg/mL) or were left untreated. One million viable LNT-229 cells were implanted into the right flank of nude mice (a), or one million SMA560 cells into VMDk-mice (b) Tumor size was measured 3 times per week. Inlet: As a control and to avoid the measurement of artifacts induced by ISCADOR Q mediated inhibition of cell growth, proliferation of implanted cells was assessed by crystal violet staining in parallel. (c) One million LNT-229 cells were implanted into the right flank of nude mice and tumors were allowed to grow for six days. At day 7 subcutaneous injections twice a week with PBS or a weekly increasing dose of ISCADOR Q (1 up to 100 μg in total, arrows indicate ISCADOR injections) were given on the contralateral body site. After two weeks of treatment with the highest dosage, injections were stopped and tumor size was measured as in (a). (d) One million SMA560 cells were implanted into the right flank of VMDk mice and tumors were allowed to grow for six day. On day 7, a single intratumoral injection of ISCADOR Q (20 μL, 100 μg/mL, indicated by an arrow) was given into the palpable tumors. Tumor size was measured as in (a).