FWGE (MSC) is a nontoxic fermented wheat germ extract demonstrated to have antitumor effects. FWGE has the potential to significantly improve the survival rate in patients suffering from malignant colon tumors. We studied its effects in the HT-29 human colon carcinoma cell line. FWGE had an inhibiting effect on colonies of HT-29 cells with an IC50 value of 118 lg/ml (7 days of incubation); this value could be decreased to 100 and 75 lg/ml in the presence of vitamin C. In the cell line examined, FWGE induced both necrosis and apoptosis, as demonstrated by Hoechst/propidium iodide staining. The incubation of cells with 3200 lg/ml FWGE for 24 hrs caused necrosis in 28% and the induction of apoptosis in 22% of the cells. FWGE inhibited the cell-cycle progression of HT-29 cells in the G1 phase of the cell cycle. In addition, FWGE inhibited the activity of the key enzyme of de novo DNA synthesis, ribonucleotide reductase. In addition, we determined the effects of FWGE on the activity of cyclooxygenase-1 and -2. Both enzymes were significantly inhibited by FWGE with IC50 values of 100 and 300 lg/ml, respectively. We outline new explanations for its antitumor activity, which might serve as the basis for further studies using FWGE .

The in vitro and in vivo effects of a fermented wheat germ extract, invented by the Hungarian biochemist Mate Hidve´gi, were recently FWGE . FWGE (MSC) is an extract standardized to methoxy-substituted benzoquinones, and has been demonstrated to induce apoptosis in pancreatic carcinoma cells, T and B lymphocytic tumor cell lines, and leukemia cells in vitro (1–3). In lymphoid tumor cells, apoptosis was selectively induced via tyrosine phosphorylation and Ca2þ influx (3). In addition, FWGE was shown to have a selective inhibitory effect on glycolysis and pentose-cycle enzymes, and to cause the down-regulation of major histocompatibility complex class I proteins in tumor cells (1–3). FWGE also has metastasisinhibiting effects and is capable of synergistically enhancing the metastasis-inhibiting effect of 5-fluorouracil (5-FU) and dacarbazine (DTIC) under experimental conditions when applied in combination with these cytostatics (4–6). The down-regulation of auto-antibody production following treatment with FWGE  was observed in a mouse model for systemic lupus erythematosus (SLE), indicating that FWGE can ameliorate the clinical manifestation of experimental SLE (7). It has also been reported that FWGE is able to inhibit experimental azoxymethane-induced colon carcinogenesis in F-344 rats. The administration of FWGE decreased the number of animals that developed experimentally induced colon tumors by 46% (8). In addition, the number of colonic tumors per animal was significantly decreased by 57%, from 2.3 to 1.3. FWGE is distributed in various countries as a dietary supplement. 

The effects of FWGE in patients suffering from colon carcinoma were also evaluated. The oral coadministration of FWGE with conventional treatments helped to improve the clinical outcome of colon cancer treatment when compared with treatment with conventional regimens alone (9, 10) and, at the same time, demonstrated no signs of toxicity. A multicenter study using 170 patients with colorectal cancer who received a dose of 9 g of FWGE once per day demonstrated that the coadministration of FWGE with other treatments significantly improved disease progression, incidence of metastasis, and survival rate. These results prompted us to further investigate the biochemical effects involved in the antitumor activity of FWGE in HT-29 human colon carcinoma cells. We determined the effects of this compound on the clonogenic efficacy of HT-29 cells. In addition, we investigated the induction of necrosis and apoptosis by FWGE , its effects on the cell-cycle phase distribution of HT-29 cells, and also whether FWGE inhibits ribonucleotide reductase (RR; EC 1.17.4.1), a key enzyme in malignant cells. Furthermore, we examined the effects of FWGE on cyclooxygenase (COX)- 1 and -2 activity, because these enzymes play an important role in inflammation and the development of colon cancer. We hope that our results may help to elucidate the molecular mechanisms of FWGE effect as a biologically active wheat germ extract on colon tumor cells. Materials and Methods Chemicals. Ascorbic acid (vitamin C) and radioactive [14C]cytidine were purchased from Sigma (Vienna, Austria). 

FWGE was a gift from Fresenius-Kabi Inc., (Graz, Austria). All other reagents used were commercially available and of the highest purity. Cell Culture. The human colon tumor cell line HT-29 was purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in a RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2. Cell counts were determined using a CC-108 micro-cell counter (Sysmex, Kobe, Japan). Cells in a logarithmic phase of growth were used for all the studies described below. Clonogenic Assay. HT-29 cells (1 3 103 cells/well) in the logarithmic phase of growth were plated in 24-well Costar plates (Costar, Cambridge, MA) and incubated with increasing concentrations of FWGE , and also FWGE  in coadministration with vitamin C (50 and 100 lM; Sigma, St. Louis, MO) for 7 days at 378C under cell culture conditions. After crystal violet staining, colonies (.40 cells) were counted using an inverted microscope. All experiments were performed in triplicate and were repeated three times. Determination of Necrosis and Apoptosis. HT29 cells (0.2 3 106 /ml) were incubated with FWGE (800, 1600, 2400, and 3200 lg/ml) for 24 hrs. Then, 0.5 3 106 /ml cells were stained with Hoechst 33258 (HO; Sigma) and propidium iodide (PI; Sigma) stain for 1 hr at 378C. The cells were centrifuged at 600–750 g for 5 mins. Cells were then examined using a Leica (Wetzlar, Germany) DMR XA fluorescence microscope equipped with the appropriate filters for Hoechst 33258 and PI. The cells were photographed with a COHU (San Diego, CA) high-performance CCD camera using Leica Q-fish software. 

This method makes it possible to distinguish between early apoptosis, late apoptosis, and necrosis. Cells were judged according to their morphology and the integrity of their cell membranes, which can easily be seen after PI staining. Cell-Cycle Phase Distribution Analysis. HT-29 cells (0.4 3 106 /ml) were incubated with various concentrations of FWGE (200, 400, and 800 lg/ml) at 378C under cell culture conditions. After 24 hrs, the cells were harvested and suspended in 5 ml of cold phosphate-buffered saline (PBS), centrifuged (600 g for 5 mins), resuspended, and fixed in 3 ml of cold ethanol (70%) for 30 mins at 48C. After two washing steps in cold PBS, RNase A and PI were added to a final concentration of 50 lg/ml each and incubated at 48C for 60 mins before measurement. Cells were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) and cell-cycle phase distribution was calculated using ModFit LT software (Verity Software House, Topsham, ME). Determination of Ribonucleotide Reductase (RR) In Situ Activity. Exponentially growing HT-29 cells (5 3 106 ) were incubated with 400, 800, and 1600 lg/ ml FWGE for 24 hrs at 378C in a humidified atmosphere containing 5% CO2 to assess changes in RR in situ activity. The cells were then pulsed with [14C]cytidine (Sigma, Vienna, Austria; 3.125 ll in a 5-ml cell suspension) for 30 mins at 378C. The cells were collected by centrifugation (1200 g for 5 mins), washed twice with PBS, and processed to extract the total genomic DNA. We then measured the radioactivity of the DNA samples through the measurement of converted cytidine.

 Determination of Deoxyribonucleoside Triphosphates (dNTPs). HT-29 cells were seeded for 24 hrs in 175 cm2 flasks (4 3 107 ) to ensure attachment and then incubated with 400, 800, and 1600 lg/ml of FWGE for another 24 hrs. The cells were then centrifuged at 1800 g for 5 mins, resuspended in 100 ll of PBS, and extracted with 10 ll of trichloracetic acid (90%). The lysate was allowed to rest on ice for 30 mins and neutralized by the addition of 1.5 volumes of freon containing 0.5 mol/l tri-noctylamin. Concentrations of dNTPs were then determined using the method described by Garrett and Santi (11). Aliquots (120 ll) of the sample were analyzed using a Merck ‘‘La Chrom’’ high-performance liquid chromatography (HPLC) system (Merck, Darmstadt, Germany) equipped with a D-7000 interface, L-7100 pump, L-7200 autosampler, and a L-7400 UV detector. Detection time was set at 80 mins, with the detector operating on 280 nm for 40 mins and then switched to 260 nm for another 40 mins. Samples were eluted with a 3.2 M ammonium phosphate buffer (pH 3.6, adjusted by the addition of 3.2 M H3PO4) containing 20 M acetonitrile using a 4.6 3 250 mm PARTISIL 10 SAX column (Whatman Ltd., Kent, UK). Separation was performed at constant ambient temperature and a flow rate of 2 ml per minute. The concentration of each dNTP was calculated as a percentage of the total area under the curve for each sample. The concentrations of dCTP, dTTP, and dATP in untreated, exponentially growing HT-29 cells were 2.23, 4.71, and 0.71 lM per 106 cells, respectively.COX Inhibitor Assay. An enzyme immunoassay of IBL Products (Hamburg, Germany) was used for the determination of COX-1 and COX-2 activities. 

The assay quantitatively determines prostaglandins F, E, and D and thromboxane B-type prostaglandins produced in COX reactions. COX-1 and COX-2 activity was determined and expressed as an IC50 value (i.e., the concentration of FWGE that results in a 50% inhibition of the activity of each enzyme). The IC50 values were determined after calculation of a dose-response curve by the Prism 3.03 software package (GraphPad, San Diego, CA). Statistical Calculations. Dose-response curves were calculated using the Prism 3.03 software package, and statistical significance was determined through an unpaired t test. Results Clonogenic Assay. FWGE inhibited the growth of cell colonies when a clonogenic assay with an IC50 value (i.e., a 50% inhibition of cell colonies when compared with the untreated cell colonies) of 118 lg/ml was employed. The coadministration of vitamin C decreased this IC50 value to 100 lg/ml when 50 lM/L vitamin C was added, and to 75 lg/ml when 100 lM/L vitamin C was added. Vitamin C administered alone inhibited the number of colonies to 87% (50 lM) and 84% (100 lM) of control cells, respectively. Determination of Necrosis and Apoptosis. The effect of FWGE on the necrosis and apoptosis of HT-29 human colon carcinoma cells is presented in Table 1. Cells were incubated with increasing concentrations of FWGE (800–3200 lg/ml) for 24 hrs. Then cells were doublestained with a Hoechst/PI stain and judged according to their morphology. FWGE (800–2400 lg/ml) caused the induction of necrosis, with only a few cells undergoing the changes associated with apoptosis. At a concentration of 3200 lg/ml, necrosis could be established in 28% of the cells and apoptosis in 22% of the cells.

 Cell-Cycle Phase Distribution After FWGE Incubation. Figure 1 shows the effect of FWGE on the cell-cycle phase distribution in HT-29 cells. FWGE incubation in solutions up to 400 lg/ml had hardly any effect on the cell-cycle distribution of tumor cells; however, incubation in a solution of 800 lg/ml for 24 hrs led to an arrest in the G1 phase, causing a depletion of cells in the S and G2-M phases of the cell cycle. RR In Situ Activity Assay. HT-29 cells were treated with FWGE (400–1600 lg/ml) for 24 hrs and then incubated with radioactive [14C]cytidine for 30 mins. DNA was then extracted and the radioactivity in the DNA samples was determined as a measure of RR in situ activity. The incorporation of the label into the DNA decreased in a concentration-dependent manner. Incubation with 1600 lg/ ml FWGE decreased the incorporation of the radiolabeled cytidine into DNA to 13.5% of control values, as depicted in Figure 2. Determination of dNTPs. HT-29 cells were incubated with 400, 800, and 1600 lg/ml of FWGE for 24 hrs. Pool sizes of dNTPs were then determined using the HPLC method described above in the Materials and Methods section. The concentration of dGTP pools was below the detection limit of the method. All three other dNTP pool sizes (dATP, dCTP, and dTTP) significantly decreased after incubation with FWGE . The most pronounced effects were observed for dCTP concentrations, which decreased to 45% of control values after incubation with 400 lg/ml FWGE . 

Increasing FWGE concentrations further decreased dCTP pools. A significant decrease was also observed for dTTP after incubation with 800 lg/ml, and for dATP after exposing the tumor cells to 400 lg/ml FWGE . The dATP pool further decreased to 19% of control values after cells were incubated with 1600 lg/ml FWGE . Results are shown in Figure 3. Effect of FWGE on COX Activities. COX-1 and COX-2 enzymes were incubated with increasing concentrations of FWGE for 2 mins at 378C using a cell-free enzyme. The inhibition of COX-1 and COX-2 in the presence of FWGE was then determined, and IC50 values (FWGE concentration resulting in 50% enzyme inhibition) were calculated. For COX-1 activity the IC50 was 100 lg/ ml, while a concentration of 300 lg/ml inhibited COX-2 activity to 50% of the control. These results demonstrate the COX-inhibiting capacity of FWGE ; and no selectivity toward one of the COX enzymes could be observed. Discussion FWGE is a fermented wheat germ extract introduced as a nontoxic dietary supplement with anticarcinogenic effects. Various authors describe its beneficial in vitro and in vivo effects. In particular and most recently, Jakab and coauthors (10) conducted a clinical study using FWGE in patients with colorectal cancer. They demonstrated that the coadministration of FWGE produced a statistically significant overall survival benefit when compared with results in patients who received conventional chemotherapy alone. As a result, we decided to investigate the biochemical mechanism of the action of FWGE in a human colon tumor cell line.

 First, we determined the IC50 values of FWGE in HT29 cells employing a clonogenic assay and demonstrated that the compound inhibits tumor cell colonies at an IC50 of 118 lg/ml. It was previously demonstrated that the coadministration of vitamin C can influence the effects of FWGE on metastasis in experimental animals (4). We therefore tested the combined effects of FWGE and vitamin C in HT-29 cells and demonstrated their dose-dependent, synergistic clonogenic effects. The synergism observed might be the result of free radical scavenging effects or vitamin C’s protection of the active ingredient of FWGE from oxidation. Because FWGE had been demonstrated to induce apoptosis in a number of cell lines, we investigated the apoptosis-inducing effects in HT-29 human colon carcinoma cells as well. Both effects—necrosis and the induction of apoptosis—were observed, depending on the concentrations used. Morphologic analysis after doublestaining with a Hoechst/PI stain revealed that lower FWGE concentrations resulted mainly in necrosis, whereas apoptotic changes could be observed with higher compound concentrations. We were also able to confirm previous findings regarding the cell-cycle phase-specific action of FWGE in colon cells. As shown by Comin-Anduix and coworkers in Jurkat cells, FWGE stopped the cell-cycle transition of HT29 cells in the G1 phase of the cell cycle, resulting in the depletion of S and G2-M phase cells (2). Several of FWGE effect mechanisms have previously been described. For instance, a cancer cell–specific inhibition of glycolysis and pentose cycle enzymes was caused by FWGE in Jurkat cells, pinpointing one mechanism of action of the compound in leukemia cells. 

Comin-Anduix and coworkers (2) also speculated that the decreased oxidative ribose synthesis might limit the leukemia cells’ metabolic needs for the reduction of ribonucleotides to dNTPs. Another mechanism could be the free radical scavenging effects of FWGE or components of FWGE . RR is responsible for the conversion of ribonucleotides to deoxyribonucleoside triphosphates, which are precursors of DNA synthesis. RR was demonstrated to be significantly up-regulated in tumor cells to meet the increased need for dNTPs of these rapidly proliferating cells for DNA synthesis (12, 13). The enzyme was therefore indicated as being an excellent target for cancer chemotherapy, and various inhibitors of RR have entered clinical practice or are under preclinical or clinical development. The enzyme consists of two subunits, the effector binding and the nonheme iron subunits. The inhibition of the nonheme iron subunit can be caused, for instance, by iron chelation or by the free radical scavenging of a free tyrosine radical, which is needed for iron stabilization. To determine whether FWGE action in HT-29 cells involves such RR inhibition, we first employed an in situ enzyme assay. Radiolabeled cytidine has to be reduced by RR to be incorporated into DNA. We were able to demonstrate that the in situ RR activity of HT-29 cells can be inhibited by FWGE in a concentration-dependent manner.