The fermented wheat germ extract (code name: MSC, trade name: FWGE), with standardized benzoquinone content has been shown to inhibit tumor propagation and metastases formation in vivo. The aim of this study was to understand the molecular and cellular mechanisms of the anti-tumor effect of MSC. Therefore, we have designed in vitro model experiments using T and B tumor lymphocytic cell lines. Tyrosine phosphorylation of intracellular proteins and elevation of the intracellular Ca2+ concentration were examined using immunoblotting with anti-phosphotyrosine antibody and cytofluorimetry by means of Ca2+ sensitive fluorescence dyes, Fluo-3AM and FuraRed-AM, respectively. Apoptosis was measured with cytofluorimetry by staining the DNA with propidium iodide and detecting the ‘sub-G1’ cell population. The level of the cell surface MHC class I molecules was analysed with indirect immunofluorescence on cytofluorimeter using a monoclonal antibody to the non-polymorphic region of the human MHC class I. MSC stimulated tyrosine phosphorylation of intracellular proteins and the influx of extracellular Ca2+ resulted in elevation of intracellular Ca2+ concentration. Prominent apoptosis of 20-40% was detected upon 24 h of MSC treatment of the cell lines. As a result of the MSC treatment, the amount of the cell surface MHC class I proteins was downregulated by 70-85% compared to the non-stimulated control. MSC did not induce a similar degree of apoptosis in healthy peripheral blood mononuclear cells. 

 Tumor cells are capable of avoiding the adaptive immune response of cytotoxic T lymphocytes by expressing only self-antigens instead of tumor specific antigens in the context of MHC class I molecules (4) or by downregulation the MHC class I from the cell surface. Although the downregulation of MHC class I prevents the killing by cytotoxic T cells, the tumor cells become susceptible to natural killer (NK) cell activity (5). The extract of wheat germ fermented with yeast, MSC, has been recently developed with standardized benzoquinone content. It has been previously shown that MSC exerts a potent anti-metastatic activity in animal tumor models (7). Moreover, interim results of a still running, phase II clinical trial with MSC indicate that it is an encouraging candidate of a supportive therapy after surgery and/or adjuvant chemotherapy in colorectal human malignancies (8). MSC treatment of thymectomized mice resulted in shortened rejection time of skin graft indicating that the graft, became sensitized to the immune response (9). MSC also decreased nucleic acid ribose synthesis through the non-oxidative steps of the pentose cycle but increased a direct glucose oxidation through the oxidative steps thus limiting cell proliferation and protecting human cells from oxidative stress (10). MSC is a mixture of ingredients with potential biological activity. One family of the active compounds may be benzoquinones, 2,6-dimethoxip-benzoquinone (DMBQ) and 2-methoxi-p-benzoquinone that are released by glycosidases present in the fermenting yeast. Although benzoquinones exert a strong effect on tumor cell proliferation (11), we have previously shown that benzoquinone may not be the only active compound in MSC (9).

 To gain insight into the cellular and molecular mechanisms by which MSC exerts its anti-tumor effect, the early and late cell response have been studied upon in vitro treatment of lymphoid tumor cells with MSC. Early biochemical events such as induction of tyrosine phosphorylation, modulation of tyrosine phosphatase activity and elevation of intracellular Ca2+ concentration have been investigated. Furthermore, the biological response of the lymphoid tumor cells including expression of the cell surface MHC class I proteins and induction of programmed cell death upon MSC treatment have been analysed.Cells and reagents. The wild-type and two mutant phenotypes of Jurkat leukemic T cells, the p56lck and CD45 deficient variants, JCam and J45.01, respectively, the Burkitt lymphoma B cell lines, Bl41 and Raji and the myelo-monocytic cell line, U937 were cultured in RPMI-1640 (Gibco, Rockville, MD, USA) medium containing 5% fetal calf serum (FCS) (Protein GMK) at 37˚C under 5% CO2. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors by density gradient centrifugation over Ficoll-Hypaque. 2,6-dimethoxi-p-benzoquinone (DMBQ) was chemically synthetized in our laboratory. Reagents were purchased from Sigma (St. Louis, MO, USA) if not stated otherwise. Fermented wheat germ extract, MSC, was produced as described elsewhere (7,9). Water soluble fraction was prepared by dissolving 100 mg MSC in 1 ml water and the insoluble material was removed by centrifugation. The MSC concentration indicated in the experiments was based on the quantity of the dry material before extraction. 

Tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation experiments were carried out as previously described (12). Briefly, Jurkat or BL-41 cells were harvested from growth medium by centrifugation, washed once in RPMI without FCS and were resuspended at 4x107 cells/ml in RPMI without FCS. Stimulation was initiated by adding 10 µg/ml of anti-T cell receptor (TCR) monoclonal antibody (mAb), OKT3 and 15 µg/ml anti-B cell receptor (BCR) mAb (10A/2) (a kind gift from G. Sármay) to the Jurkat and BL-41 cells, respectively for 1 min or 5 mg/ml of MSC for 10 min, or cells were left unstimulated. Activation was stopped with addition of equal volume of 2X concentrated, ice cold lysis buffer (1X lysis buffer: 50 mM HEPES pH 7.4, 1% Triton X-100, 150 mM NaCl, 20 mM NaF, 5 mM Na3VO4, 2 mM EDTA, 1 mM phenylmethyl-sulfonylfluoride (PMSF) and 10 µg/ml leupeptin). Cells were lysed on ice for 30 min and cleared from nuclear/cytoskeletal components by centrifugation at 12,000 x g for 15 min. Postnuclear supernatants were mixed with equal volumes of 2X SDS sample buffer (13) and loaded onto a 10% SDS polyacrylamide gel. Proteins were then transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) in a transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Membranes were blocked using Tris-buffered saline (TBS) containing 0.05% Tween 20 and 3% cold fish gelatin for 1 h at 37˚C and subsequently probed with anti-phosphotyrosine mAb, 4G10 (Upstate Biotechnology Inc., Lake Placid, NY, USA) and rabbit anti-mouse IgG conjugated to horseradish peroxidase (Dako, Carpinteria, CA, USA). 

Immunoreactive proteins were visualized by an enhanced chemiluminescence (ECL plus) detection system (Amersham, Little Chalfout, UK). Prestained molecular weight marker was purchased from Gibco-BRL (Rockville, MD, USA). Phosphatase assay rationale. Phosphatase assay was carried out as previously described (14). Briefly, Jurkat cells (2x106 cells/sample) were lysed for 30 min in ice-cold lysis buffer (1% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 1.0 mM EDTA) supplemented with 10 µg/ml leupeptin and 1 mM PMSF. Postnuclear supernatants were mixed with 10 µl Sepharose beads (Pharmacia, Peapack, NJ, USA) covalently coupled with anti CD45 mAb, GB3 (15), and incubated for 1 h at 4˚C. The immunoprecipitates were washed twice in lysis buffer and once in phosphatase assay buffer (50 mM HEPES pH 7.0, 100 mM KCl, 0.1% Triton X-100, 1 mM EDTA). The beads were incubated in 45 µl phosphatase assay buffer supplemented with 1 mM dithiothreitol and 1.3 mM para-nitrophenyl-phosphate (pNPP) for 30 min at 37˚C. Fifteen µl of supernatants of the reaction mixtures was transferred into the wells of a 96-well microtiter plate (Falcon, Bedford, MA, USA) and the amount of the released inorganic phosphate from pNPP was determined according to Ng et al (16) using 80 µl of malachite green reagent and a spectrophotometer operating at 650 nm wavelength. Whole cellular phosphatase activity was inhibited with an addition of 10 µM sodium vanadate in the indicated experiments.

 Measurement of intracellular Ca2+ concentration [Ca2+]i. Jurkat cells were suspended in 107 cells/ml concentration in culture medium (RPMI-1640 supplemented with 5% FCS) and incubated with 7.5 µM of Fluo-3AM and 7.5 µM of FuraRed-AM (Molecular Probes, Eugene, OR, USA) for 30 min at 37˚C. Cells were then adjusted to a final concentration of 5x105 cells/ml with the addition of cell culture medium and incubated for 30 min at 37˚C. Cells were washed twice with RPMI and resuspended in fresh cell culture medium at 106 cell/ml. Intracellular [Ca2+]i was measured using FACSCalibur (Becton Dickinson, Bedford, MA, USA) and data were presented as the ratio of Fluo-3 and FuraRed fluorescence intensity at 530 and 650 nm, respectively, versus time. In some experiments the influx of Ca2+ was inhibited with addition of EGTA in the indicated concentration. Detection of apoptotic cells. Different cells were treated with either 1 mg/ml MSC, 0,3 µg/ml DMBQ or 6 µg/ml WGA or left unstimulated for 24 h then subjected to DNA content analysis. The cells were then harvested and washed twice with phosphate buffered salt (PBS) containing 0.1% glucose then permeabilized and stained with propidium-iodine (10 µg/ml) in the following solution: 0.1% Triton X-100, 0.1% Na3-citrate and 0.01% RNase. After incubation for 30 min at room temperature in dark, the cells were analyzed on a FACSCalibur cytofluorimeter (Becton Dickinson, Bedford, MA, USA) using CELLQuest and/or Modfit software programs (Becton Dickinson, Bedford, MA, USA). For Annexin V labeling Jurkat cells were washed twice with PBS and resuspended in binding buffer (0.01 M HEPES, 0.14 M NaCl and 2.5 mM CaCl2). 

Fluorescein isothiocyanate (FITC) conjugated Annexin V (Pharmingen, San Diego, CA, USA) and propidium-iodine (10 µg/ml) were added to the cells for 15 min in dark, at room temperature. After washing, the cells were analyzed on FACSCalibur cytofluorimeter. Measurement of cell proliferation. Proliferation assays were carried out in flat-bottomed 96-well tissue culture plates in RPMI supplemented with 5% FCS. The rate of PBMC proliferation was monitored by the incorporation of tritiated thymidine ([3H]TdR) (Amersham, Little Chalfout, UK) into the DNA of the cells in triplicate samples. PBM cells were seeded at 105 cells/well with increasing concentrations of MSC and PHA in a final volume of 200 µl and incubated for 72 h. For the last 16 h of culturing, cells were pulsed with 1 µCi/well = 37kBq/well of [3H]TdR, then harvested for scintillation counting. Analysis of the downregulation of cell surface MHC class I expression. Jurkat, Raji or U937 tumor cells were suspended in 5x105 cells/ml concentration in cell culture medium (RPMI1640 supplemented with 5% FCS) and incubated in 24-well tissue culture plates with 2 mg/ml MSC or 0.6 µg/ml DMBQ. After 4 h at 37˚C the cells were washed with FACS-buffer (PBS supplemented with 1% FCS and 0.1% NaN3), and incubated with anti-MHC I monoclonal antibody (TMB6-5, produced in our laboratory) for 1 h at 4˚C followed by goat anti-mouse IgG-FITC for 30 min at 4˚C. The fluorescence intensity was measured on FACSCalibur (Becton Dickinson, Bedford, MA, USA). The MHC I level was calculated as follows: MHC I level=10[(Ch#TMB6-5-Ch#ctl)/ChD)], where Ch#TMB6-5 = median channel number of anti-MHC I labeled sample; Ch#ctl = median channel number of negative control; ChD = number of channels per decade. 

Results Specific tyrosine phosphorylation in tumor T and B cell lines after MSC treatment. One of the earliest intracellular responses for extracellular signals is the tyrosine phosphorylation of intracellular signal proteins. The newly tyrosine phosphorylated proteins are involved in and many times determine the subsequent biochemical events and therefore the eventual cell response. Short-term exposure of Jurkat cells to the wheat germ extract resulted in reproducible tyrosine phosphorylation of specific proteins with molecular weights of 76, 63 and 38 kDa (Fig. 1A). A protein of 50 kDa was constitutively phosphorylated in the untreated samples, as well. The pattern of MSC induced tyrosine phosphorylation was largely different from the TCR stimulated tyrosine phosphorylation indicating that MSC used distinct signaling pathways from that of TCR stimulation. The B cell line, BL-41 was stimulated with MSC under similar conditions to that of Jurkat cells. In BL-41 an increase in the phosphorylation of several proteins was detected upon MSC treatment, compared to the unstimulated control. The pattern of phosphorylation induced by MSC was different from the BCR stimulated (Fig. 1B). A 63 kDa protein was preferentially phosphorylated in both T and B cell tumors indicating a specific role of this protein in MSC stimulated cell response. CD45 tyrosine phosphatase activity decreases after MSC treatment. CD45, an abundant heavily glycosylated cell surface receptor with intracellular phosphatase activity is expressed on all nucleated leukocytes. It has a pivotal role in leukocyte signaling (17), therefore we analyzed the effect of MSC on the phosphatase activity of CD45.

 As it is shown in Fig. 2, the phosphatase activity decreased in Jurkat cells upon treatment with wheat germ extract. A similar effect of MSC was detected in B cell line, BL-41 (data not shown). To test whether modulation of phosphatase activity was a direct effect of the lectin present in MSC, we also used purified wheat germ agglutinin (WGA) for cell stimulation. WGA was added to the cells in a concentration that was comparable to its concentration in the MSC dilution used in the experiment (5 mg/ml). WGA treatment resulted in a similar decrease in phosphatase activity than was achieved by MSC treatment (Fig. 2) indicating that the component was responsible for the MSC induced modulation of CD45 activity.Stimulation of Jurkat cells with the wheat germ extract caused an early and transient 3-fold elevation of intracellular Ca2+ concentration. This Ca2+ entirely came from the extracellular environment, since addition of EGTA, a Ca2+ chelator, to the extracellular space blocked the increase of intracellular Ca2+ (Fig. 3A). The mechanism and the degree of Ca2+ elevation were compared to that induced via the T cell receptor with anti-TCR mAb, OKT3. Anti-TCR triggered a much greater increase (15-fold) in intracellular Ca2+ than MSC did and that was composed of the release of intracellular Ca2+ (could not be blocked with EGTA) and influx from the extracellular space (blocked with addition of EGTA) (Fig. 3B). MSC triggers apoptosis in tumor T and B cell lines but does not induce it in healthy peripheral blood mononuclear cells. Incubation of T cell line, Jurkat with MSC at 1 mg/ml concentration for 24 h resulted in apoptosis as demonstrated by cytofluorimetric analysis of the DNA content (Fig. 4A). 

Tumor B cell line, BL-41 responded to MSC treatment with a similar degree of apoptosis (data not shown). In contrast, MSC treatment of peripheral blood mononuclear cells (PBMC) obtained from healthy donors did not induce apoptosis (Fig. 4A). Early marker of the apoptotic process - e.g. exposure of phosphatydilserine on the outer face of the plasma membrane - was detected on Jurkat cells after 12 h of MSC treatment using Annexin V labeling (Fig. 4B). DMBQ, used in a concentration comparable to its concentration in 1 mg/ml MSC, induced a similar degree of apoptosis in Jurkat to that induced by MSC (Fig. 4A). To determine the role of the early cell responses in the apoptotic process including tyrosine phosphorylation of intracellular substrates and elevation of the intracellular Ca2+ concentration, we used Na-vanadate and EGTA to modulate apoptosis. Addition of vanadate to block tyrosine phosphatase activity resulted in an increase of MSC induced apoptosis (Fig. 5). In contrast, the presence of EGTA, which blocked the influx of Ca2+ from the extracellular space decreased MSC-induced cell death (Fig. 5). Two mutant variants of the Jurkat cell line, J45.01 (deficient in CD45 receptor tyrosine phosphatase) and JCam (deficient in p56lck) were also analyzed to prove or exclude the role of CD45 and p56lck, two indispensable enzymatic factors of lymphocyte activation (17,18), in MSC-induced apoptosis. The lack of either CD45 or p56lck did not influence the apoptotic process, indicating that these factors may not be directly involved in the MSC-induced apoptosis (data not shown). 

WGA, a lectin with immunomodulatory properties, present in wheat germ did not induce apoptosis of the tumor cells (data not shown). The prominent anti-proliferative effect of MSC was demonstrated by the dose-dependent inhibition of PBM proliferation induced by a polyclonal mitogen, phytohemagglutinine (PHA) (Fig. 6).