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Carcinogenesis Advance Access originally published online on March 7, 2007
Carcinogenesis 2007 28(7):1446-1454; doi:10.1093/carcin/bgm040
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The suppression of aberrant crypt multiplicity in colonic tissue of 1,2-dimethylhydrazine-treated C57BL/6J mice by dietary flavone is associated with an increased expression of Krebs cycle enzymes

Isabel Winkelmann, Daniela Diehl1, Doris Oesterle2, Hannelore Daniel and Uwe Wenzel3,*

Molecular Nutrition Unit, Department of Food and Nutrition, Technical University of Munich, Am Forum 5, D-85350 Freising, Germany
1 Institute of Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilian-University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany
2 Institute of Toxicology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany
3 Molecular Nutrition Research, Interdisciplinary Research Center, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany

* To whom correspondence should be addressed. Tel: +49 641/99-39220; Fax: +49 641/99-39229; Email: uwe.wenzel{at}ernaehrung.uni-giessen.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colorectal cancer is the second leading cause of cancer deaths worldwide with diet playing a prominent role in disease initiation and progression. Flavonoids are secondary plant compounds that are suggested as protective ingredients of a diet rich in fruits and vegetables. We here tested whether flavone, a flavonoid that proved to be an effective apoptosis inducer in colon cancer cells in culture, can affect the development of aberrant crypt foci (ACFs) in C57BL/6J mice in vivo when preneoplastic lesions were induced by the carcinogen 1,2-dimethylhydrazine (DMH). Flavone applied at either a low dose (15 mg/kg body wt per day) or a high dose (400 mg/kg body wt per day) reduced the numbers of ACFs significantly, independent of whether it was supplied simultaneously with the carcinogen (blocking group) or subsequent to the tumor induction phase (suppressing group). Proteome analysis performed in colonic tissue samples revealed that flavone treatment increased the expression of a number of Krebs cycle enzymes in the suppressing group and this was associated with reduced crypt multiplicity. It suggests that mitochondrial substrate oxidation is increased by flavone in colonic cells in vivo as already observed in HT-29 cells in vitro as the prime mechanism underlying tumor cell apoptosis induction by flavone. In conclusion, flavone reduces the number of ACFs in DMH-treated mice at doses that can be achieved for flavonoids by a diet rich in fruits and vegetables. Moreover, reduction in crypt multiplicity by flavone is most probably due to the preservation of a normal oxidative metabolism.

Abbreviations: AC, aberrant crypt; ACF, aberrant crypt focus; DMH, 1,2-dimethylhydrazine; IPG, immobilized pH gradient; ROS, reactive oxygen species


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Colorectal cancer is still the second leading cause of cancer-related deaths for both men and women in Western countries (1). Although epidemiological findings on the relation between foods and colon cancer are inconsistent (2), evidence emerging from many different types of experimental and observational studies supports the concept that dietary habits, such as a high intake of fruits and vegetables, can reduce the risk of developing colorectal cancer (3,4). Flavonoids are considered to act as chemopreventive agents in such a diet by targeting the molecular pathways that can terminate proliferation, induce apoptosis or inhibit the spread of tumor cells (5–7). Since a failure in execution of the apoptotic program promotes cancer development (8,9), apoptotic cell death has emerged as a target for drug treatment at various stages of tumor progression (10). We previously observed that flavonoids are able to induce apoptosis in colonic cancer cells in vitro (11) and flavone proved a very potent inducer of apoptosis in the HT-29 human colon cancer cell model (12). Moreover, flavone displayed a high selectivity of action toward the transformed cells, since it failed to induce apoptosis in non-transformed colonocytes (13). Mechanistically, the generation of reactive oxygen species (ROS) was shown to be the crucial step for apoptosis initiation that was followed by changes in gene expression of a variety of apoptosis-relevant proteins (14,15). The increase in ROS production by exposure of cells to flavone was due to an increased uptake of oxidizable substrates, such as lactate or pyruvate, into the mitochondria of colon cancer cells followed by increased rates of respiration (16,17).

In the present study we assessed whether flavone can act as a chemopreventive ingredient in vivo as well. Aberrant crypt foci (ACFs), which are considered to represent preneoplastic changes in the intestine (18), were induced in C57BL/6J mice using the colon carcinogen 1,2-dimethylhydrazine (DMH) (19). Flavone was applied to animals by gavage at either 15 mg/kg body wt per day or 400 mg/kg body wt per day. Whereas the high dose represents amounts usually used in chemoprevention studies (20,21), the low dose represents an amount that may represent a daily intake of flavonoids provided by a diet rich in fruits and vegetables (22). Flavone at both doses was either supplied together with DMH to test its potency as a blocking agent or subsequent to DMH application to assess its suppressing effects. ACFs were determined by light microscopy after methylene blue staining of fixed gut specimens. A proteomic approach was finally used for identifying molecular targets of flavone action in the intestinal tract of mice.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
Female C57BL/6J mice were purchased from Harlan Winkelmann (Borchen, Germany) at 4 weeks of age and were divided into six groups (Figure 1). Tap water and diet (V1534, Ssniff, Soest, Germany) were supplied ad libitum and the animals were kept on a 12:12 h light–dark cycle in a controlled temperature and humidity room with four to five animals per cage. All mice were weighed weekly and before killing. The test chemicals were applied by gavage in 4 ml/kg body wt vehicle. For inducing ACFs, all animals were treated with 40 mg/kg body wt DMH once a week for 6 weeks. For application of flavone by gavage, flavone (Sigma–Aldrich, Deisenhofen, Germany) was vigorously pulverized and suspended in 0.9% saline solution containing 0.1% of the co-surfactant Myrj 53 (Sigma–Aldrich). The ‘blocking group’ animals received flavone either at 15 mg/kg body wt (low dose, n = 23) or at 400 mg/kg body wt (high dose, n = 23) 5 days a week over 6 weeks in parallel to the DMH application (Figure 1). The ‘suppressing group’ animals received low (n = 23) or high doses of flavone (n = 23) five times a week over 4 weeks starting 1 week after the last DMH application (Figure 1). Control animals received the vehicle only (n = 23 for each control group). All animals were killed 5 weeks after the last injection of DMH, always between 9 and 11 AM. The mice were killed by cervical dislocation under ether anesthesia. Animal handling and experimentation were performed in accordance to the German Animal Protection Law and approved by the Animal Care und Use Committee of Bavaria (AZ 211-2531-37/00).


Figure 1
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  		Fig. 1. Schema describing treatment of animals in the blocking and suppressing groups. DMH was applied once a week at doses of 40 mg/kg body wt and flavone at 5 days a week either at low dose (15 mg/kg body wt) or at high dose (400 mg/kg body wt). Vehicle consisted of 0.9% saline solution containing 0.1% of the co-surfactant Myrj 53. All groups consisted of 23 animals.

 
Determination of the number of ACFs
ACFs were counted in flat preparations of the colon in 17 animals from each group. For this purpose, the gut was removed and rinsed with ice-cold Tris buffer (pH 7.4). Thereafter, the colon was cut in parts matching the length of a slide. Before placing on slides, a stirring rod was inserted and each piece of the colon was dissected along the longitudinal axis. After placing flat on a microscopic slide with the mucosal side up, the colon was covered with a filter paper and fixed in 10% neutral-buffered formalin for 6 h. The colonic crypts were stained with 2 g/l of methylene blue in phosphate-buffered saline for ~10–15 min. The number of ACFs and the aberrant crypt (AC) multiplicity were determined by light microscopy at 25-fold magnification.

Calculations and statistics
Variance analysis between groups was performed by one-way analysis of variance and significance of differences between control and treated cells was determined by a Tukey's multiple comparison test (GraphPadPrism, San Diego, CA). For each variable at least three independent experiments were carried out. Data are given as the mean ± standard error of mean.

Sample preparation for two-dimensional polyacrylamide gel electrophoresis
Colon tissues of six mice of each group served for proteome analysis. Tissue samples were immediately snap-frozen in liquid nitrogen after killing of mice, removal of colons, rinsing them with Tris buffer and crushing in a mortar in the presence of liquid nitrogen. Samples were stored in aliquots at –80°C until further use.

For protein extraction, 0.1 g of the colon samples were mixed with 1 ml of lysis buffer containing urea (7 M), thiourea (2 M), dithiothreitol (65 mM), 3-[(cholamidopropyl)-dimethyl-ammonium]-1-propane-sulfonate (2%), protease inhibitor cocktail tablets (Complete Mini, Roche, Mannheim, Germany) and pharmalyte, pH 3–10 (10%) (Amersham Biosciences, Freiburg, Germany). The resultant cell lysate was sonicated for 6 x 10 s (Hielscher ultrasonic processor, amplitude 45) and centrifuged at 14 000 g for 45 min to collect the supernatant. Lysates were precipitated with acetone overnight and dialyzed using the Mini Dialysis Kit (Amersham Biosciences) at 4°C according to the manufacturer's instructions. The concentration of the solubilized proteins was determined using the Bradford method with the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany).

Two-dimensional polyacrylamide gel electrophoresis
For the first dimension, a total protein amount of 500 µg was applied by cup-loading at the anodic end of an 18 cm long immobilized pH gradient (IPG) strip with immobilized broad range pH gradients (linear IPG strips, pH 3–10, Amersham Biosciences), which were rehydrated overnight in solubilization buffer containing 8 M urea, 2% CHAPS, 2%, pharmalyte and 13 mM dithiothreitol. Proteins were focused using the Ettan IPG Phor II from Amersham Biosciences as described (23) with minor modifications. Focusing was achieved at the following conditions: 1 min at 500 V (gradient), 90 min up to 4000 V (gradient) and for a total of 25 000 Vh at 8000 V (step-n-hold). The gel strips with the focused proteins were either frozen at –80°C or directly processed for second dimension. After equilibration with a buffer containing 6 M urea, 30% glycerol, 0.4% sodium dodecyl sulfate, 50 mM Tris buffer (pH 8.8) and either dithiothreitol or iodacetamide, the IPG strips were transferred onto a 12.5% acrylamide gel for the second dimension. One millimeter thick 12.5% sodium dodecyl sulfate–polyacrylamide gels were cast according to the method of Laemmli (24) and were run using an Amersham Biosciences Ettan-Dalt II System employing the following conditions: 4 mA per gel for 1 h, and then 12 mA per gel.

The proteins in the gels were fixed in 40% (v/v) ethanol and 10% (v/v) acetic acid for 5 h. Gels were then stained overnight in Coomassie solution containing 10% (w/v) (NH4)2SO4, 2% (v/v) phosphoric acid, 25% (v/v) methanol and 0.625% (w/v) Coomassie brilliant blue G250. Gels were destained in double distilled water until the background was completely clear.

Analysis of proteins using the ProteomWeaver software
Gels stained with Coomassie were scanned using an Umax scanner Power Look III (software: Magic Scan version V4.5, UMAX) and spots detected by the ProteomWeaver software (Definiens, Munich, Germany). Background subtraction and volume normalization were made automatically by the software. After spot detection, all gels were matched to each other. Six gels derived from individual mice were grouped and compared with the gels derived from six control mice. Spots differing significantly (P < 0.05, Mann–Whitney test) in their intensities were picked for MALDI-TOF mass spectrometry analysis.

Enzymatic digestion of protein spots and MALDI-TOF mass spectrometry
Selected Coomassie-stained spots were excised from the two-dimensional gels with a PROTEINEER spII spot picker (Bruker Daltonics, Leipzig, Germany). Destaining, drying and digestion were performed with the PROTEINEER dpTM workstation using the calibration and digestion kits by following the manufacturer's instructions (Bruker Daltonics). Subsequently, samples were spotted automatically by the workstation either onto an AnchorChip MALDI target 800/384 by using {alpha}-cyano-4-hydroxy-trans-cinnamic acid as matrix, acidified by using an aqueous 0.1% trifluoroacetic acid as washing solution and air-dried at room temperature, or onto a prespotted AnchorChip target PAC384 with {alpha}-cyano-4-hydroxy-trans-cinnamic acid matrix for 384 samples and 96 calibration spots. Analysis was performed with an Autoflex MALDI-TOF mass spectrometer (Bruker Daltonics), operating in reflectron mode with a 20 kV accelerating voltage and a 130 ns delayed extraction. Mass spectra were acquired in the automatic mode using the AutoXecute module of FlexControl software version 2.4 (Bruker Daltonics). Spectra of identical protein spots from at least four independent gels and from different treatment groups were processed with flexAnalysis 2.4 (Bruker Daltonics), by using the smoothing option and calibrating both external, and internally with the autoproteolysis peptide of trypsin (m/z 2211.10). Background peaks like keratin, Coomassie, etc, were removed and a signal to noise threshold (S/N) of 3 was applied for the samples and 6 for the peptide calibration standard (1000–4000 Da, Bruker Daltonics). Peptides were selected in the mass range of 800–3500 Da. The resulting mass list was evaluated using Bio Tools 3.0 with the search engine Mascot (version 1.9.00, http://www.matrixscience.com) and the MSDB database. The criteria for positive identification of proteins were set as follows: ±50–150 p.p.m. peptide mass tolerance, 0 or 1 missed cleavage, carbamidomethyl modification of cysteine as global and methionine oxidation as variable modification and charged state as MH+. A protein was seen as validated when three samples satisfactorily showed the same results with a probability-based mowse score being significant (P < 0.05) and theoretical molecular weight and pI showing at least similar results as in the gels from which they were picked.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Flavone in low and high dose acts as a blocking and suppressing agent by reducing the total number of ACF in colonic sections of DMH-treated mice
DMH treatment caused on average ~40 ACF per animal in colonic tissue of female C57BL/6J mice at 5 weeks after the last intra-peritoneal DMH application (Figure 2). Mice not treated with DMH did not develop any ACF (data not shown). Flavone, applied at low (15 mg/kg body wt) or high (400 mg/kg body wt) doses by gavage, either during the DMH induction phase or thereafter, reduced the formation of ACF significantly, independent on the dose and on the time of application (Figure 2). Flavone may therefore be regarded as a blocking as well as a suppressing agent in the development of preneoplastic ACF caused by DMH.


Figure 2
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  		Fig. 2. Flavone at low or high dose acts as a blocking and suppressing agent on formation of ACF in colonic tissue of mice undergoing DMH treatment. Female C57BL/6J mice received flavone at 15 mg/kg body wt (low dose) or at 400 mg/kg body wt (high dose) either during the DMH treatment phase (blocking) or thereafter (suppressing) as described in Materials and Methods. Colonic tissues were stained by methylene blue and ACF were counted under a light microscope. *P <0.05 and **P <0.01 versus control.

 
Flavone suppresses potently ACFs with high crypt multiplicity
DMH treatment of mice caused also the formation of ACF consisting of different numbers of ACs (Figure 3A). ACs usually arise during the early stages of chemically induced colorectal cancers as a consequence of accumulation of somatic mutations (25) and crypt multiplicity is generally increased in more advanced stages of carcinogenesis (26,27). Flavone applied either at low or high dose reduced especially these ACFs with a higher multiplicity and appeared more effective here as a suppressing agent (Figure 3B). However, ACFs consisting of greater than or equal to five ACs were reduced in number significantly in both the suppressing and the blocking groups (Figure 3B). When ACFs were divided into small ACFs, consisting of one to two AC, or large ACFs, consisting of three to greater than or equal to five ACs, it became obvious that large ACFs were reduced by flavone only in the suppressing group (Figure 3C) but this potent reduction in the numbers of ACFs with high multiplicity was observed at both flavone doses (Figure 3C).


Figure 3
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  		Fig. 3. Flavone suppresses more potently ACF formation with higher crypt multiplicity. (A) ACF induced by treatment of mice with DMH with different numbers of ACs shown by arrows. (B) ACF in mice colonic tissue were counted and grouped by crypt multiplicity in the blocking and suppressing groups after treatment with 15 mg/kg body wt flavone (low dose), 400 mg/kg body wt flavone (high dose) or with vehicle alone (control). (C) ACF with low (consisting of one to two AC) or high crypt multiplicity (consisting of three to greater than or equal to five ACs) in control and animals treated with either low- or high-dose flavone in the blocking and suppressing groups. *P <0.05, **P <0.01 and ***P <0.001 versus control.

 
Flavone alters the protein steady-state levels of numerous proteins including Krebs cycle enzymes
For a comprehensive analysis of the effects of flavone on the proteome in colonic cells of DMH-treated mice, proteins were isolated from tissues and submitted to two-dimensional polyacrylamide gel electrophoresis. More than 600 protein spots were resolved on the gels from the total tissue lysate. In the blocking group, flavone at low dose was found to alter 17 proteins significantly in steady-state levels of which 15 were identified by MALDI-TOF analysis, whereas flavone at high dose altered levels of 17 proteins significantly of which 16 could be identified (Table I and Figure 4). Interestingly, the vast majority of proteins was regulated in a similar manner and magnitude by both the low and high flavone doses in the blocking trial. Only annexin 4 was reduced in level by the low dose but not the high dose, whereas T-complex protein 1 was increased only in the high-dose flavone group (Table I and Figure 4).


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  		Table I. Proteins regulated in steady-state level by low or high doses of flavone in murine colon tissue samples when co-administered with DMH (blocking group)

 


Figure 4
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  		Fig. 4. Two-dimensional polyacrylamide gel electrophoresis of proteins from colonic tissue extracts of mice treated with low-dose or high-dose flavone or with vehicle alone (control) during the phase of DMH treatment (blocking group). Proteins were separated on a linear, pH 3–10, IPG strip in the first dimension and on a 12.5% sodium dodecyl sulfate–polyacrylamide gel in the second dimension. (A) Two-dimensional gel with separated proteins from the colons of flavone high-dose mice. (B) Enlargements from identical sections of gels derived from separations of colon proteins from control mice or mice treated with low- or high-dose flavone.

 
Similarly to the blocking experiment, regulation of proteins found in the suppressing group by low and high doses of flavone were essentially identical (Table II and Figure 5). Out of 36 proteins altered significantly by the low dose of flavone and 35 proteins altered significantly by the high dose, 30 regulated proteins were identified in both groups (Table II and Figure 5). Of those, only five proteins were not regulated significantly by both doses applied. Only heterogeneous nuclear ribonucleoprotein A2/B1 and proteasome subunit beta type 6 (Precursor) were found as regulated only by the low dose, whereas glutathione synthetase, aldo-keto reductase and NG,NG-dimethylarginine dimethylaminohydrolase were only altered by the high dose (Table II and Figure 5). In contrast to these similarities in regulation independent of the flavone dose used, proteins identified as affected by flavone in the blocking group differed vastly from those in the suppressing group (Tables I and II and Figures 4 and 5). Only creatine kinase B-type was altered in both groups by flavone but completely different depending on the time of flavone administration. Whereas both flavone doses caused an increase in creatine kinase level in the suppressing group, they showed the opposite effect in the blocking group (Tables I and II and Figures 4 and 5). Interestingly, most of the proteins regulated by flavone treatment in the suppressing group represent enzymes of the intermediary metabolism and especially of the Krebs cyle such as citrate synthase, isocitrate dehydrogenase or succinate dehydrogenase (Table II and Figure 5).


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  		Table II. Proteins regulated in steady-state level by low or high doses of flavone in murine colonic tissues when administered subsequently to initiation with DMH (suppressing group)

 


Figure 5
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  		Fig. 5. Two-dimensional polyacrylamide gel electrophoresis of proteins from colonic tissue of mice treated with low- or high-dose flavone or with vehicle alone (control) subsequent to the phase of DMH treatment (suppressing group). Identical sections of gels from the control and low- or high-dose flavone treatment are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Colorectal cancer ranks as the second most common cause of cancer death in western societies (1). About 90% of human colorectal cancers are of sporadic origin (28). It is suggested that changes in lifestyle including dietary habits could prevent disease development in most cases (29). Among the dietary factors a high intake of fruits and vegetables appears inversely associated with the mortality of various cancers including colorectal carcinomas (3,4). A large number of intervention studies mainly in rodent models with chemically induced colon tumors showed that distinct components of plant foods including flavonoids are able to inhibit colon cancer development (21,22,30). We have previously demonstrated in human colon cancer cells a unique property of the flavonoid flavone as a selective and potent inhibitor of tumor cell growth and inducer of apoptosis (12,13). This feature of a selective induction of apoptosis only in transformed but not normal colonocytes is highly preferable for a chemopreventive but also therapeutic agent since apoptosis induction in normal cells can lead to transformation whereas apoptosis induction in cancer cells is difficult to achieve by the increased resistance of these cells to death signals (8).

The earliest identifiable morphological changes in colonic mucosa in the chronology of cancer development are ACs (18,26,27). They represent lesions that can also be characterized by genetic and biochemical alterations (27). In more advanced stages of carcinogenesis, the multiplicity of ACs within an ACF increases and correlates particularly well with the incidence of colorectal adenomas (26,27,31). Others, however, found that in rats neither the numbers of total ACFs nor numbers of large ACFs did correlate with colorectal cancer development (32). In the present study, DMH applied to female C57BL/6J mice induced a large number of ACFs in colonic tissues and flavone proved to be effective in reducing ACFs formation both when given together with DMH or after tumor initiation. Moreover, flavone displayed similar blocking and suppressing activities when given at low doses of 15 mg/kg body wt or at high doses of 400 mg/kg body wt per day. However, when crypt multiplicity was taken into account, flavone appeared particularly effective at both doses under suppressing condition with increasing effectiveness as more ACs were present in controls in which DMH could cause more ACFs without interference by flavone. This suggests that ACs of the same multiplicity in the blocking and suppressing groups are different and that flavone especially targets cells that might have crossed a certain stage of transformation. This assumption is further supported by the fact that flavone did not affect proliferation or apoptosis in colonic tissues of control animals, as assessed by immunohistological staining of tissue sections for 5-bromo-2'-deoxyuridine and cleaved caspase-3, respectively (data not shown). It appears to target primarily cells that are in the process of transformation. The lack of apoptosis induction by flavone in non-transformed colonic tissue is further stressed by our proteomics data. Increased levels of lamin A in the blocking group and unaltered levels of the same protein in the suppressing group were found when mice were exposed to flavone. A critical role of lamin A cleavage in apoptotic signaling in colonocytes has been demonstrated (33,34) and our proteome data suggest this not to happen under flavone treatment. The selective action of flavone is currently best explained by our previous analysis on its mode of action in human colon cancer cells. We showed that flavone can drastically enhance the uptake of monocarboxylates—mainly of lactate—into mitochondria (16) followed by its oxidation. Most cancer cells obtain their ATP predominantly from converting glucose to lactate even in the presence of abundant oxygen (35–37). This so-called Warburg effect, describing aerobic glycolysis, is overcome by flavone in tumor cells and the increased delivery of energy substrates to the respiratory chain in turn increases the production of O2–· radicals in mitochondria that promotes a very efficient route of apoptosis induction (12,14–17). Our findings strongly suggest that alterations in the energy state of transformed cells are crucially linked to apoptosis induction and may also help to explain why non-transformed cells are not responsive to flavone that reverses alterations in energy metabolism of tumor cells to a phenotype characteristic for normal cells.

The effects of flavone on the colonic tissue proteome in mice are also characterized by changes in protein levels of a variety of enzymes crucially involved in intermediary metabolism. These alterations in enzyme protein levels were more prominent in the suppressing group in which flavone was also more efficient in suppressing ACF formation. Moreover, flavone affected mainly different protein entities when analyzed in the blocking and suppressing group suggesting that it predominantly acts to block DMH effects on the proteome that appear to differ in early and later phases of tumor development.

As we observed increased levels of several Krebs cycle enzymes such as citrate synthase, isocitrate dehydrogenase and succinate dehydrogenase in colon tissue of animals of the suppressing group when flavone was supplied, one could assume that an enhanced flux of intermediates through the citric acid cycle in mitochondria results. Interestingly, in human colon cancer cells, Krebs cycle enzyme levels changed in the opposite direction when cells were exposed to flavone for 24 h and this led to low ATP levels and apoptosis induction (38). In an earlier phase, however, the same cells displayed increased ATP levels suggesting a down-regulation of Krebs cycle enzymes as a feedback adaptation to prevent cell death (38). The up-regulation of the citric acid cycle enzymes found here in vivo in mice may represent therefore the response of a normal mucosa that at the same time may prevent cells from being transformed. An increased delivery of energy substrates to mitochondrial citric acid cycle in the presence of flavone may be further indicated by the increased levels of fatty acid-binding protein as observed in the blocking group. We have shown previously that colon cancer cells are unable to import fatty acids into mitochondria and therefore fatty acids are not used as energy substrates via ß-oxidation and oxidation in the respiratory chain. However, when colon cancer cells are forced to utilize fatty acids as substrates for energy metabolism, they undergo apoptosis by the increased burden of ROS produced in the respiratory chain (39). The observed up-regulation of adenosine kinase by flavone could further promote mitochondrial oxidative metabolism since increased ADP levels, besides preventing toxic concentrations of adenosine to build up, would allosterically activate isocitrate dehydrogenase as one of the key enzymes of the Krebs cycle (40). Glycerol-3-phosphate dehydrogenase and ornithine aminotransferase present two of the few metabolic enzyme proteins for which lower levels were observed in mice treated with flavone. Whereas the diminished levels of glycerol-3-phosphate dehydrogenase could cause a reduction of flow of precursors through the gluconeogenetic chain and provide more substrates for glycolysis and mitochondrial oxidation, changes in ornithine aminotransferase prevented by flavone are of particular interest. Ornithine aminotransferase showed increased enzyme levels in transformed colonic tissues and was defined as a robust marker to distinguish neoplastic sections from normal mucosa (41). We detected two protein spots representing ornithine aminotransferase as down-regulated which may refer to two isoforms differing slightly in their isoelectric points and molecular weight, suggesting that this protein could be regulated by post-translational modifications such as phosphorylation as well.

Notably, especially in the suppressing group important proteins involved in detoxification processes such as glutathione synthetase and glutathione-S-transferase were found at increased levels under flavone treatment. This may be mediated by electrophile-responsive element-mediated gene transcription, which involves the release of the transcription factor Nrf2 from a complex with Keap1 (42) and may also be taken as an indicator of an increased capability of the colon mucosa for clearance of xenobiotcs and ROS handling.

In conclusion, our study provides evidence that dietary flavone inhibits in vivo in mice the formation of ACFs and thus impairs progression toward colonic tumors. Our proteome analysis approach revealed that these functions of flavone are associated with increased levels of a number of enzyme proteins of intermediary metabolism. Based on previous studies in colon cancer cell models, we propose that these changes in citric acid cycle enzyme protein levels could increase the substrate flux through the cycle and in turn increase mitochondrial respiratory chain activity leading finally to apoptosis in transformed but not in normal colon epithelial cells.


   Acknowledgments
 
The authors greatly acknowledge the expert technical assistance of Mrs Carmen Spiller, Mrs Marianne Berauer and Mrs Beate Rauscher.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 23, 2006; revised February 5, 2007; accepted February 9, 2007.


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