Pancreas Vol. 22, No. 1, pp. 1–7 © 2001 Lippincott Williams & Wilkins, Inc., Philadelphia
Genistein Inhibits Nonoxidative Ribose Synthesis in MIA Pancreatic Adenocarcinoma Cells: A New Mechanism of Controlling Tumor Growth Laszlo G. Boros, Sara Bassilian, Shu Lim, and Wai-Nang Paul Lee Harbor-UCLA Research and Education Institute, UCLA School of Medicine, Torrance, California, U.S.A.
Summary: Genistein is a plant isoflavonoid bearing potent tumor growth–regulating characteristics. This effect of genistein has been attributed partially to its tyrosine kinase– regulating properties, resulting in cell-cycle arrest and limited angiogenesis. Genistein has been used in chemotherapyresistant cases of advanced leukemia with promising results. Here we demonstrate that genistein primarily affects nucleic acid synthesis and glucose oxidation in tumor cells using the [1,2-13C2]glucose isotope as the single tracer and gas chromatography/mass spectrometry to follow various intracellular glucose metabolites. The ribose fraction of RNA demonstrated a rapid 4.6%, 16.4%, and 46.3% decrease in isotope uptake through the nonoxidative branch of the pentose cycle and a sharp 4.8%, 24.6%, and 48% decrease in 13CO2 release from
glucose after 2, 20, and 200 mol/L genistein treatment, respectively. Fatty acid synthesis and the 13C enrichment of acetyl units were not significantly affected by genistein treatment. De novo glycogen synthesis from media glucose was not detected in cultured MIA cells. It can be concluded from these studies that genistein controls tumor growth primarily through the regulation of glucose metabolism, specifically targeting glucose carbon incorporation into nucleic acid ribose through the nonoxidative steps of the pentose cycle, which represents a new paradigm for the antiproliferative action of a plant phytochemical. Key Words: Pentose cycle—Ribose synthesis— Genistein—Nonoxidative glucose metabolism—Glucose oxidation.
Genistein, the isoflavonoid of the soy plant, has potent tumor growth–regulating characteristics (1–3). Genistein has exhibited tyrosine kinase (4) and protein kinase (PK) (5) inhibiting properties, resulting in cell-cycle arrest (6) and limited angiogenesis (7) in several tumor models. Genistein also enhances the effect of various plant phytochemicals including limonene, curcumin, epigallocatechin gallate, or sulforaphene (8). Genistein counteracts the growth-promoting effect of many human growth factors through their signaling pathways that include signal coupling to transcription factors that depend on triggering of Met-receptor and protein kinase transducers (9). The invasive transformation of several human epithelial carcinoma cell lines in response to transforming growth
factor  (TGF-) treatment (10) is characterized by increased nucleic acid ribose synthesis through the nonoxidative reactions of the pentose cycle (11). Experimental studies strongly indicate that genistein inhibits cell growth by modulating TGF- signaling pathways specifically (12). Genistein has recently been reported as a clinically effective and well-tolerated anticancer drug in advanced chemotherapy-resistant cases of acute childhood lymphoblastic leukemia, as well as adult chronic lymphocytic leukemia (13). Pancreatic tumor cells also respond to genistein treatment, as stimulated growth and p42 activation were inhibited by genistein in MIA pancreatic adenocarcinoma cell cultures (14,15). Because genistein provides the basis for an effective treatment strategy for therapy-resistant human malignancies including pancreatic cancer, we were interested in the mechanism of how genistein regulates nucleic acid, amino acid, lipid syntheses, and glucose oxidation in pancreatic adenocarcinoma cells. This was accomplished using biologic mass spectrometry of important metabo-
Manuscript received March 1, 2000; revision accepted April 26, 2000. Address correspondence and reprint requests to Dr. L. G. Boros, Harbor-UCLA Research and Education Institute, UCLA School of Medicine, 1124 West Carson street RB1, Torrance, CA 90502, U.S.A. E-mail:
[email protected]
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lites formed from a uniquely labeled glucose molecule in tumor cell cultures in the presence of increasing doses of genistein. METHODS Cell line and culture MIA pancreatic adenocarcinoma cells (American Type Culture Collection) were grown in minimum essential medium (MEM) in the presence of 10% fetal bovine serum (FBS), at 37°C in 95% air/5% CO2. To compare glucose utilization rates, ribose synthesis, lactate production, and glutamine oxidation, 75% confluent cultures of MIA cells were incubated in [1,213 C2]glucose-containing media (180 mg/dL, 50% isotope enrichment). Cultures for the study were selected with the same cell number (6 × 107), which was achieved using standard cell-counting techniques. Media glucose and lactate levels were measured using a Cobas Mira chemistry analyzer (Roche). Glucose oxidation was measured by media 13C/12C ratios in released CO2 by a Finnegan Delta-S ion ratio mass spectroscope (GC/C/ IRMS). 13CO2 release was used to estimate glucose carbon utilization through oxidation by the cell lines and expressed as atom percentage excess (APE), which is the percentage of 13C produced by the cultured cells above background in calibration standard samples (16). RNA ribose was isolated by acid hydrolysis of cellular RNA after Trizol purification of cell extracts. Ribose was purified using a tandem set of Dowex1/Dowex50 ionexchange columns (Sigma). Ribose was derivatized to its aldonitrile acetate form using hydroxyl amine in pyridine and acetic anhydrate. We monitored the ion cluster around the m/z256 (carbons 1–5 of ribose, chemical ionization; CI), m/z217 (carbons 3–5 of ribose), and m/z242 (carbons 1–4 of ribose, electron-impact ionization; EI) to find molar enrichment and positional distribution of 13C labels in ribose (17,18). Stable [1,2-13C2]D-glucose isotope was purchased with >99% purity and 99% isotope enrichment for each position (Isotec, Inc., Miamisburg, OH, U.S.A.). For isotope-incubation and drug-treatment studies, fibroblasts were seeded in T-75 tissue-culture flasks after adjusting the number of cells to the values reported earlier. During the study, the cultures were supplied with 50% [1,213 C2]glucose dissolved in otherwise glucose and sodium pyruvate–free DMEM with 10% FBS. The final glucose concentration was adjusted to 180 mg/100 mL. Glucose mass isotope analysis of the medium before cell incubations showed that the actual labeled glucose enrichment was 48% in the culture media, and this number was used for further calculations to determine maximal labeled Pancreas, Vol. 22, No. 1, 2001
glucose enrichment in the molecules we studied. Singly labeled ribose molecules (m1) recovered from RNA on the first carbon position were used to measure the ribose molar fraction produced by direct oxidation of glucose through the G6PD pathway, after subtracting the fraction of the singly labeled product that came from the TK pathway, calculated by the published m1 = m2(m3/m4 formula (18). Doubly labeled ribose molecules (m2) on the first two carbon positions were used to measure the molar fraction produced by transketolase. Doubly labeled ribose molecules (m2) on the fourth and fifth carbon positions were used to measure the molar fraction produced by triose phosphate isomerase and TK. Isotopomers with three labels were used to estimate ribose production by combining recycled products of the G6PD reaction through the TK and transaldolase reactions. Isotopomers with four labels (m4) were used to estimate synthesis through TK and triose phosphate isomerase. Figure 1a shows the possible rearrangement of labels from glucose to ribose as detected by gas chromatography/mass spectrometry. Lactate from the cell-culture media (0.2 mL) was extracted by ethyl acetate after acidification with HCl. Lactate was derivatized to its propylamine-HFB form, and the m/z328 (carbons 1–3 of lactate, chemical ionization; CI) was monitored for the detection of m1 (recycled lactate through the PC) and m2 (lactate produced by the Embden–Meyerhoff–Parnas pathway) for the estimation of pentose cycle activity (17). Fragmental lactate analysis was not necessary because the [1,2-13C2]glucose tracer labels lactate on the third (m1) or the second and third (m2) carbon positions, which are clearly distinguished in the molecular ion, as shown in Fig. 1b. Glutamate tissue culture medium was first treated with 6% perchloric acid. Proteins were removed by centrifugation, and the supernatant was neutralized with potassium hydroxide. The neutralized supernatant was passed through a 3-mL Dowex-50 (H+) column. Amino acids were eluted from the Dowex-50 column with 15 mL 2N ammonium hydroxide, and the solution was evaporated to dryness by blowing air. To separate glutamate further from glutamine, the amino acid mixture was passed through a 3-mL Dowex-1 (acetate) column. Glutamine was washed with 10 mL water, and glutamate was collected with 15 mL 0.5N acetic acid. Glutamate fraction from tissue culture medium was converted to its trifluoroacetyl butyl ester (TAB) (19,20). Under EI conditions, ionization of TAB-glutamate gives rise to two fragments, m/z198 and m/z152, corresponding to C2–C5 and C2–C4 of glutamate. Glutamate labeled on the 4–5 carbon positions indicates pyruvate dehydrogenase activity, whereas glutamate labeled on the 2–3 carbon positions
CHANGES AFTER GENISTEIN IN MIA CELLS
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The m2/m1 ratio in glutamate is proportional with the activity of glucose oxidation as 13CO2 is released from ␣-ketoglutarate during each completed cycle. Anaplerotic flux is calculated based on the m2/m1 ratios of glutamate (20). Fatty acids were extracted after saponification of cell pellets in 30% KOH and 100% ethanol using petroleum ether. Fatty acids were converted to their methylated derivative using 0.5N methanolic-HCL. Palmitate was monitored at m/z270, and the enrichment of acetyl units as well as the synthesis of the new lipid fraction in MIA cells in response to genistein treatment was determined using the mass isotopomer distribution analysis (MIDA) approach of different isotopomers of palmitate, as reported previously (21). Glycogen glucose was extracted after sonication of cell pellets and digestion with amyloglucosidase from Aspergillus niger (Boehringer, Mannheim, Germany). Glucose was purified using a tandem set of Dowex1/ Dowex50 ion-exchange columns and then derivatized to its aldonitrile-acetate form. Glucose molecular ion was detected in the m/z328 ion cluster. Electron-impact ionization gives rise to two glucose fragments, m/z187 (carbons 3–6 of glucose) and m/z242 (carbons 1–4 of glucose), to determine positional distribution of 13C labels (22). Gas chromatography/mass spectrometry (GC/MS) Mass spectral data were obtained on the HP5973 mass selective detector connected to an HP6890 gas chromatograph. The settings are 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) was used for glucose, ribose, glutamate, and lactate analysis. A Bpx70 column (25 m length, 220 m diameter, 0.25 m film thickness; SGE Incorporated, Austin, TX, U.S.A.) was used for fatty acid analysis with specific temperature programming for each compound studied. FIG. 1. Possible 13C rearrangement in intermediates of the pentose cycle (A), lactate (B), or glutamate (C) using [1,2-13C2]glucose as the single tracer. For measuring the activity of each synthesis pathway, the ratio of 12C- versus 13C-labeled molecules and the position of 13Clabeled carbons is determined using mass isotopomer analysis by gas chromatography/mass spectrometry. Absolute glucose utilization and lactate production also are measured from the cell-culture media to obtain absolute values for glucose carbon utilization through the specific pathways of intermediate metabolism.
indicates pyruvate carboxylase activity for the entry of glucose carbons to the TCA cycle. The TCA cycle metabolite ␣-ketoglutarate is in equilibrium with glutamate, which is released by the cells into the medium (Fig. 1c).
Data analysis and statistical methods In vitro experiments were carried out using three cultures each time for each treatment regimen. 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
