This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. FWGE exhibits about a 50-fold higher IC50 (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.The preventive and therapeutic potential of two natural wheat products, wheat bran and fermented wheat germ (FWGE ), in experimental carcinogenesis has recently been described (1, 2). Although no chemical constituents are yet isolated and tested experimentally, it is likely that benzoquinones and wheat germ agglutinin in wheat germ and fiber and lipids and phytic acid in wheat bran play a significant role in exerting anti-carcinogenic effects. In a recent report utilizing intracellular carbon flow studies with a 13C-labeled isotope of glucose and biological mass spectrometry (GC/MS),1 it was demonstrated that the crude powder of fermented wheat germ dosedependently inhibits nucleic acid ribose synthesis primarily through the nonoxidative steps of the pentose cycle while increasing direct glucose carbon oxidation and acetyl-CoA utilization toward fatty acid synthesis in pancreatic adenocarcinoma cells (3). These metabolic changes indicate that fermented wheat germ exerts its anti-proliferative action through altering metabolic enzyme activities, which primarily control glucose carbon flow toward nucleic acid synthesis.

This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. FWGE exhibits about a 50-fold higher IC50 (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.The preventive and therapeutic potential of two natural wheat products, wheat bran and fermented wheat germ (FWGE ), in experimental carcinogenesis has recently been described (1, 2). Although no chemical constituents are yet isolated and tested experimentally, it is likely that benzoquinones and wheat germ agglutinin in wheat germ and fiber and lipids and phytic acid in wheat bran play a significant role in exerting anti-carcinogenic effects. In a recent report utilizing intracellular carbon flow studies with a 13C-labeled isotope of glucose and biological mass spectrometry (GC/MS),1 it was demonstrated that the crude powder of fermented wheat germ dosedependently inhibits nucleic acid ribose synthesis primarily through the nonoxidative steps of the pentose cycle while increasing direct glucose carbon oxidation and acetyl-CoA utilization toward fatty acid synthesis in pancreatic adenocarcinoma cells (3). These metabolic changes indicate that fermented wheat germ exerts its anti-proliferative action through altering metabolic enzyme activities, which primarily control glucose carbon flow toward nucleic acid synthesis.

 In vivo, FWGE has a marked inhibitory effect on metastasis formation in tumor-bearing animals (4), and this effect is attributed to its immune-restorative properties (5), which result in a decreased survival time of skin grafts and reduced cell proliferation while enhancing apoptosis. FWGE  remarkably inhibits tumor metastasis formation after chemotherapy and surgery in clinically advanced colorectal cancers. Patients receiving standard surgical and chemopreventive therapies for their advanced colorectal cancers developed significantly less new metastases during the 9-month follow-up period when treated with additional 9 g/day FWGE  daily (6, 7). In a recent randomized clinical study report FWGE significantly prolonged (doubled) time-to-progression in high-risk melanoma patients (8). Many anticancer drugs have been shown to induce cell death through the induction of apoptosis. It is well known that apoptosis is a well controlled process by a programmed set of cellular events partially mediated by caspases. A large number of substrates for caspases have been reported, including poly(ADP-ribose) polymerase (PARP), a 116-kDa nuclear DNA repair enzyme that is cleaved during apoptosis by caspases-3 and -7 (9, 10). Powerful and selective reversible and irreversible peptide-based inhibitors are also available to better characterize and understand the mechanism(s) of how caspases regulate apoptosis. The tripeptide benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk) is a broadly used general caspase inhibitor that blocks apoptosis in many cell types, including human leukemic Jurkat T cells (10, 11). Here we report the effect of fermented wheat germ on cell cycle regulation, proliferation, and apoptosis induction in Jurkat leukemia cell cultures. 

Our results confirm strong tumor growth inhibitory properties of FWGE and additionally reveal its cell cycle-regulating characteristics. FWGE decreases G6PDH and transketolase activities that are key enzymes involved in glucose conversion into the five-carbon nucleotide precursor ribose pool. Stable isotope studies indicate that FWGE  is a powerful inhibitor of de novo nucleic acid synthesis. This likely is the underlying mechanism of the anti-proliferative tumor growth-controlling and apoptosis-inducing potential of fermented wheat germ in leukemia tumor cells. On the contrary, FWGE has no toxic biological effects on PBLs in the doses that affect tumor cells in an adverse manner.

MATERIALS AND METHODS

Chemicals—Ribose 5-phosphate, xylulose 5-phosphate, MgCl2, triose-phosphate isomerase, NADH, thiamine pyrophosphate (TPP), glucose 6-phosphate, dithiothreitol, NADP
, propidium iodide (PI), Igepal CA-630, Ponceau S, and vincristine were purchased from Sigma Co. and Tris from ICN Pharmaceuticals Inc (Costa Mesa, CA). The Bio-Rad protein assay was purchased from Bio-Rad and the BCA protein assay from Pierce. Fetal bovine serum, RPMI 1600 medium was purchased from Invitrogen (Carlsbad, CA). Dulbeccos phosphate-buffered saline (PBS), typsin-EDTA and solution C (0.05% trypsin and EDTA (1:500) in PBS) were purchased from Biological Industries (Kibbutz Beit Haemek, Israel).

Nitrocellulose strips were purchased from Schleicher & Schuell (Postach, Dasell, Germany). Annexin V was purchased from Bender MedSystems (Vienna, Austria), PARP from BD PharMingen cat. 66391 A and clone 7D3-6) and the secondary antibody anti-mouse immunoglobulin from DAKO (Copenhagen, Denmark). ECL was purchased from Amersham Biosciences. FK-109 Z-VAD.fmk were from Enzyme Sysytems Products (Livermore, CA).  FWGE was kindly provided by Biromedicina, Co. (Budapest, Hungary) through a material and chemical transfer agreement. Cell Culture—Jurkat cells (acute lymphoid T-cell leukemia) were purchased from ATCC and cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, and antibiotics: 100 units/ml penicillin and 100
g/ml streptomycin (Invitrogen). Cells were grown in an isolated 37 °C, 5% CO2 tissue incubator compartment. Cells were plated in 0.2–1 105 cells/ml density for the enzyme kinetics experiments in T75 culture flasks. For apoptosis, necrosis, and cell cycle studies cells were seeded into 6-well plates at 5 105 cells/well density. FWGE was added to the cultures after 1 h of equilibration in the cell culture chambers after seeding. FWGE was dissolved in Dulbecco’s PBS for all experiments. Control cultures were treated with an equal volume of PBS as the FWGE cultures. 

Jurkat cell cultures used in this study were free of mycoplasm infection as shown by the Gen-probe rapid mycoplasm detection system prior to treatments with FWGE . Peripheral blood mononuclear cells from healthy donors were isolated from buffy coat cells using the Ficoll gradient method (12); further purification of lymphocytes from peripheral blood mononuclear cells was performed by depletion of contaminating cells by adherence to plastic plates for 4 h. PBL were used as non-dividing and non-tumor cells to test the apoptosis-inducing effects of FWGE in a control cell system. Cell Cycle Analysis—Jurkat cells were harvested after FWGE treatment and stained in Tris-(hydroxymethyl)aminomethane-buffered saline containing PI (50
g/ml), ribonuclease A (10
g/ml), and Igepal CA-630 (0.1%) for 1 h at 4 °C. DNA content was analyzed by fluorescence-activated cell sorting (FACS). Fluorescence of 12,000 Jurkat cells was acquired for each histogram and then analyzed using the Multicycle program interface (Phoenix Flow Systems, San Diego, CA). Flow cytometry DNA histograms were collected in triplicates on an XL flow cytometer (Coulter Corporation, Hialeah, FL).Cell Viability Assay—Cell number was determined by the MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (13). 20,000 Jurkat cells per well were incubated in 96-well plates in the presence or in the absence of FWGE at different concentrations. Vincristine was used as a positive control for apoptosis induction. The blue MTT formazan precipitate was dissolved in 100
l of Me2SO, and the absorbance values at 550 nm were determined on a multiwell plate reader. 

For peripheral blood cells, 500,000 cells were seeded in 12-well plates in the presence or in the absence of FWGE at different concentrations. Viability was estimated by a MultiziserIIL Coulter (Beckman Coulter, Fullerton, CA) to count the cells and by FACS analysis adding 18
g/ml PI (Sigma Co.) staining method without cell permeabilization. The fluorescence of cells was analyzed by flow cytometry using an Epics XL flow cytometers (Beckman Coulter, Fullerton, CA). Only non-viable cells are PI positives as indicated by previous studies (14). Assessment of Apoptosis by Flow Cytometry and LSC—Jurkat cells after FWGE treatment were washed once in binding buffer (10 mM HEPES, sodium hydroxide, pH 7.4, 140 mM sodium chloride, 2.5 mM calcium chloride) and resuspended in the same buffer at 106 cells ml1 in the presence of 0.5
l of annexin V-FITC. After 30 min of incubation at room temperature, PI was added at 0.05
g ml1 (11). The fluorescence of cells was analyzed by FC and LSC. Approximately 3 104 cells were tested for each histogram for FC and 1500 cells for LSC. Gel Electrophoresis and Immunoblotting of PARP—To analyze PARP, SDS-page electrophoresis (15) and immunoblotting were performed as previously described (16). Briefly, 30
g of the protein extract was used on an 8% polyacrylamide gel and transferred to Protean membranes (Schleicher & Schuell, GmbH, Postach, Dasell, Germany). Monoclonal antibodies either against PARP were used at a 1:1000 dilution. As a control of protein loading the blot membrane was stained with Red Ponceau.

 The reaction was visualized with a secondary antibody (anti-mouse immunoglobulin, DAKO) conjugated to horseradish peroxidase diluted 1:1000 in bovine serum albumin/Tween-20/PBS and the enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences). PARP immunoblotting was performed after 48 h of incubation with the FWGE and vincristine. The protein concentration of cell extracts was determined by the BCA protein assay. Measurements of Enzyme Activities—Jurkat cells treated with increasing doses of FWGE were lysed in 1 ml of 20 mM Tris buffer (pH 7.5) containing 1 mM dithioerythreitol and 0.2 mM phenylmethylsulfonyl fluoride, 1 mM K-EDTA, 0.2 g/liter Triton X-100, and 0.2 g/liter sodium deoxycholate.

 Cell extracts were stored at 20 °C for 24 h. The homogenates were then defrosted in an ice bath, sonicated in a Brason– 2000 cell disintegrator for 5 min, ultracentrifuged at 100,000 g for 1 h, and the supernatant used for enzyme activity assays as described below. Transketolase (EC 2.2.1.1) activity was determined using the enzyme-linked method of De La Haba et al. (17). 1-ml aliquots of transketolase free buffer were measured in spectrophotometry cuvettes containing 50 mM Tris-HCl, pH 7.6, 2 mM ribose 5-phosphate, 1 mM xylulose 5-phosphate, 5 mM MgCl2, 0.2 units/ml triose-phosphate isomerase/-glyceraldehyde-3-phosphate dehydrogenase, 0.2 mM NADH, and 0.1 mM thiamine pyrophosphate. The transketolase reaction was initiated by the addition of 25 and 50
l of cell extract at 37 °C. The oxidation of NADH, which is directly proportional to transketolase activity, was measured by the decrease in 340-nm absorbance. Transketolase activity is expressed as nmol/min/million cells. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity was measured as described by Tian et al. (18). Briefly, cuvettes were prepared with a 50 mM Tris-HCl, pH 7.6 buffer, containing 2 mM glucose 6-phosphate and 0.5 mM NADP
. Reactions were initiated by the addition of 25 and 50
l of cell extract at 37 °C. The reduction of NADP, which is directly proportional to G6PDH activity, was quantified by the increase in 340-nm absorbance, and G6PDH activity is expressed as nmol/min/million cells. Lactate dehydrogenase (LDH; EC 1.1.1.27) activity was measured as described by Mommsen et al. (19). The assay medium for lactate dehydrogenase contained 50 mM Tris-HCl buffer, pH 7.6, 0.2 mM NADH, and 5 mM pyruvate (omitted for control).

 The oxidation of NADH, which is directly proportional to lactate dehydrogenase activity, was measured by the decrease in 340-nm absorbance. LDH activity is expressed as nmol/min/million cells. Hexokinase (HK; EC 2.7.1.1) activity was measured by the enzymelinked method of Grossbard and Schimke (20). Briefly, cuvettes were prepared with a 50 mM Tris-HCl, pH 7.6 buffer, containing 10 mM glucose, 1 mM NADP
,2mM ATP, 10 mM magnesium chloride, and 1 unit of G6PDH. Reactions were initiated by the addition of 50 and 100
l of cell extract at 37 °C. The reduction of NADP, which is directly proportional to HK activity, was quantified by the increase in 340-nm absorbance, and HK activity is expressed as nmol/min/million cells. Stable Isotope Incorporation into RNA Ribose—In order to measure actual substrate carbon flow in the pentose cycle and glycolysis, which are controlled by the enzymes listed above, we utilized stable isotopebased metabolic profiling as introduced for drug effect studies in cancer (21). Jurkat cell continuous S phase-independent nucleic acid synthesis rates were measured by the incorporation of [1,2-13C2]glucose into RNA ribose as the single tracer and biological mass spectrometry. 13C label accumulation into RNA was determined by measuring the molar enrichment (ME) of ribose using chemical ionization methods, which is capable of determining both total activity (mn) and positional distribution of 13C labels in nucleic acid ribose as described previously (22, 23). Stable Isotope Incorporation into Lactate—Lactate from the cell culture media (0.2 ml) was extracted by ethyl acetate and derivatized to its propylamine-HFB form.

 The m/z 328 (carbons 1–3 of lactate, chemical ionization) ion cluster was monitored for the detection of m1 (recycled lactate through the pentose cycle) and m2 (lactate produced by glycolysis) for the estimation of the pentose cycle activity relative to glycolysis (23). Gas Chromatography/Mass Spectrometry—Mass spectral data were obtained on the HP5973 mass selective detector connected to an HP6890 gas chromatograph. The settings were as follows: GC inlet, 230 °C; transfer line, 280 °C; MS source, 230 °C; MS Quad, 150 °C. An HP-5 capillary column (30-m length, 250-
m diameter, 0.25-
m film thickness, Supelco) was used for ribose analysis at the ion cluster m/z 256 and for lactate analysis (23). Data Analysis and Statistical Methods—Experiments in vitro were carried out using three cultures each time for each treatment regimen and then repeated twice. Mass spectral analyses were carried out by three independent automatic injections of 1-
l samples by the automatic sampler and accepted only if the standard sample deviation was less than 1% of the normalized peak intensity. Enzyme activity measurements were determined after correction for total protein content in cell extracts. Statistical analysis was performed using the parametric unpaired, two-tailed independent sample Student’s t test with 99% confidence intervals (
 2.58) and p  0.01 was considered to indicate significant differences in glucose carbon metabolism and enzyme activities with increasing doses of FWGE . Because of the human cell line involved, a clearance was obtained from the Institutional Review Boards (IRB) of both Harbor-UCLA and The University of Barcelona for the use of these commercially available cells for the experiments reported.

At concentrations of 0.7 and 1 mg/ml FWGE , even after 24 h, a broad peak appeared in the sub-G1 region with a significant decrease in the S cycle phase. The sub-G1 region is indicative of apoptosis (Fig. 2, black arrows). Although lower concentrations of FWGE (0.1 and 0.3 mg/ml) induced only minor changes in the cell cycle distribution of Jurkat cells, they were still effective in controlling cell growth as there was a significant decrease in formazan-accumulating Jurkat cells as shown in Fig. 1A. Induction of Apoptosis—FWGE triggered prominent apoptosis at 0.5 mg/ml dose after 72 h of culturing as demonstrated by FACS analysis. Increasing doses of FWGE induced more prominent apoptosis, which also appeared earlier (Fig. 3A). In order to discriminate between late apoptotic and necrotic cells, we investigated PI and annexin V-FITC positive cells using LSC analyses. We observed that all cells with PI
/FITC
characteristics presented pycnotic nuclei, which is a definite sign of apoptotic cell formation after treatment with 1 mg/ml FWGE (72 h). The portion of normal cell figures with LSC was only 5.5%, whereas early apoptotic cells showed 64.5% and late apoptotic cells 29.3% frequency (Fig. 4). All FWGE -treated Jurkat cells inside the PI/FITC
region presented the typical green appearance of early apoptosis caused by the labeling of annexin V by FITC. Involvement of Caspases in the Apoptotic Effect of FWGE —Decreased apoptosis-related phosphatidylserine externalization by specific caspase inhibitors is a routinely used method to reveal the presence of caspase cascades in the cell death process. 

In order to assess the involvement of caspases in the apoptotic effect of FWGE , we studied whether the caspase inhibitor Z-VAD.fmk could prevent FWGE -induced phosphatidylserine externalization. Jurkat cells incubated for 72 h with 1 mg/ml of FWGE in the presence or absence of 100
M Z-VAD.fmk showed severely decreased phosphatidylserine externalization in both early (annexin V-FTIC
/PI) and late (annexin V-FTIC
/PI
) apoptotic cells (Fig. 3B). We also investigated whether incubation of Jurkat cells with different doses of FWGE induced proteolytic cleavage of PARP, which is considered to be a hallmark of activation of caspase-3 like proteases during apoptosis (24, 25). Incubation of Jurkat cells for 48 h with 0, 0.3, 0.5, and 0.7 mg/ml of FWGE induced prominent cleavage of PARP at a concentration of 0.5 mg/ml or higher (Fig. 3C). Transketolase and G6PDH Enzyme Activities—G6PDH and transketolase are two key enzymes that regulate carbon flow in the pentose cycle because of their high substrate flux coefficients and thus regulate ribose synthesis and NADPH production for proliferating cells (24–26). FWGE inhibited G6PDH activity at concentrations of 0.7 mg/ml and higher after 48 h of treatment, and G6PDH was completely inhibited after 72 h (Fig. 5A). Transketolase was significantly inhibited with 0.7 and 1 mg/ml FWGE after 72 h of treatment (Fig. 5B). HK and LDH Enzyme Activities—HK and LDH are two of the key enzyme in the regulation of glycolytic flux. FWGE inhibited LDH and HK at concentrations of 0.3 mg/ml or higher after 48 h of treatment as shown on Fig. 6.13C Label Accumulation in Lactate—We observed a decrease in m2 and m1 13C label in lactate in FWGE -treated Jurkat cells, which is indicative of decreased glucose uptake and glycolysis.

 FWGE is the first fermented and concentrated wheat germ extract produced by an optimized process to yield 0.4 mg/g (on dry matter basis) 2,6-dimethoxy-p-benzoquinone and given as a nutritional supplement for cancer patients. The suspicion that wheat germ contains powerful cancer-fighting chemicals is not new; in his later life, the Nobel laureate biochemist Albert Szent-Gyo¨rgyi studied various extracts of the wheat plant extensively for their anti-carcinogenic effects. This study investigates the complex responses to FWGE treatment, a potent natural fermented wheat germ extract with anticarcinogenic properties, of Jurkat T-progeny leukemia cells in culture. Using flow and laser scanning cytometry techniques, direct enzyme activity measurements, carbon substrate flow measurements with a 13C-labeled glucose tracer has enabled us to study a broad range of cellular response mechanisms, such as cell cycle progression, apoptosis, cell proliferation, and their dose-response to this cancer growth-modifying agent. Activity changes of four important metabolic enzymes involved in direct glucose oxidation (G6PDH), non-oxidative glucose utilization (transketolase) toward nucleic acid synthesis, glycolysis (LDH), and glucose activation (HK) are herein also reported. Our studies revealed profound differences and a dose-dependent response of Jurkat leukemia cells that directly affected metabolic enzyme activities, metabolic pathway substrate flow, apoptosis formation, and cell proliferation in response to FWGE .