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Dietary Flavone Is a Potent Apoptosis Inducer in Human Colon Carcinoma Cells

Institute of Nutritional Sciences, University of Giessen, 35392 Giessen [U. W., S. K., H. D.], and Third Department of Internal Medicine, University of Giessen, 35385 Giessen [M. D. B.], Germany

	ABSTRACT

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Flavonoids are polyphenolic compounds that occur ubiquitously in plants. They are discussed to represent cancer preventive food components in a human diet that is rich in fruits and vegetables. To understand the molecular basis of the putative anticancer activity of flavonoids, we investigated whether and how the core structure of the flavones, 2-phenyl-4H-1-benzopyran-4-one (flavone) affects proliferation, differentiation, and apoptosis in HT-29 human colon cancer cells. Moreover, the effects of flavone in transformed epithelial cells were compared with those obtained in nontransformed primary mouse colonocytes. Proliferation, differentiation, and apoptosis in transformed as well as nontransformed colon cells were measured by fluorescence-based techniques. Apoptosis was also determined by changes in membrane permeability, FACScan analysis, and detection of DNA fragmentation. Semiquantitative reverse transcription PCR was performed to assess the effects of flavone on transcript levels. Flavone was found to reduce cell proliferation in HT-29 cells with an EC₅₀ value of 54.8 ± 1.3 µM and to potently induce differentiation as well as apoptosis. The flavonoid proved to be a stronger apoptosis inducer than the clinically established antitumor agent camptothecin. The effects of flavone in HT-29 cells were associated with changed mRNA levels of cell-cycle- and apoptosis-related genes including cyclooxygenase-2 (COX-2), nuclear transcription factor B (NF-B), and bcl-X_L. Moreover, flavone, but not camptothecin, displayed a high selectivity for the induction of apoptosis and of growth inhibition only in the transformed colonocytes.

In conclusion, the plant polyphenol flavone induces effectively programmed cell death, differentiation, and growth inhibition in transformed colonocytes by acting at the mRNA levels of genes involved in these processes. Because these genes play a crucial role in colon carcinogenesis, flavone may prove to be a potent new cytostatic compound with improved selectivity toward transformed cells.

	INTRODUCTION

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Although a diet rich in fruits and vegetables is generally recognized as preventive with regard to the development of colorectal cancer, the dietary compounds responsible for this biological effect have not been identified as yet (1, 2) . Flavonoids are a class of more than 4000 phenylbenzopyrones that occur in many edible plants, like fruits and vegetables. These polyphenolic compounds display a remarkable spectrum of biochemical activities including those that might be able to influence processes that are dysregulated during cancer development. These include, e.g., antioxidant activities (3) , the scavenging effect on activated carcinogens and mutagens (4, 5) , the action on proteins that control cell cycle progression (6) , and altered gene expression (7) . Because the biochemical activities are dependent on the individual flavonoid structure, each compound needs to be studied systematically to assess its individual biological potency. As part of a screening program, we have recently (8) shown for more than 30 flavonoids of the flavone, flavonol, flavanone, and isoflavone subgroups in three cancer cell lines that the potency and selectivity of the antiproliferative activities are strongly dependent on the particular structure of the compound. Several members of the flavone subgroup displayed strong antiproliferative activity in the human colon carcinoma cell line HT-29. Here, we tested whether the core structure of the flavone subgroup, 2-phenyl-4H-1-benzopyran-4-one (flavone), is able to potently inhibit proliferation of HT-29 as well. To get insight into the mechanism of growth-inhibition, we assessed whether flavone affects differentiation and apoptosis, which are impaired during colon cancer development (9, 10) . A semiquantitative RT² -PCR approach was used to associate the effects of flavone on cell cycle progression, differentiation, and apoptosis with altered transcript levels of genes having central functions in these processes. Alterations of m-RNA levels were determined for COX-2, NF-B, p53, p21, cyclin E and B as well as for bak, bax, and bcl-X_L as members of the apoptosis-associated bcl-2 gene family. To establish that the effects of flavone are carcinoma-cell-specific, we investigated whether proliferation, differentiation, and apoptosis are also altered by the flavonoid in nontransformed mouse colonocytes in culture.

	MATERIALS AND METHODS

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Animals
C57BL/6JOlaHsd inbred 9-week-old mice were purchased from Harlan Winkelmann (Borchen, Germany) and were housed under standard conditions with free access to food and water.

Cell Culture
HT-29 Cells.
HT-29 cells (passage 106) were provided by American Type Culture Collection and were used between passage 150 and 200. Cells were cultured and passaged in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine (all from Life Technologies, Inc., Eggenstein, Germany). Antibiotics added were 100 units/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). The cultures were maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Cells were passaged at preconfluent densities by the use of a solution containing 0.05% trypsin and 0.5 mM EDTA (Life Technologies, Inc.).

Murine Colonocytes.
Cultures of colonic primary epithelial cells from adult mice were established based on the method described by Booth et al. (11) with slight modifications. Therefore, the colons of seven mice that were at least 12 weeks old were cut into pieces of approximately 2 mm² and washed five or six times with 10 ml of DMEM, containing 25 mM HEPES, 1% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml gentamicin (all from Life Technologies, Inc.), by vigorous shaking and subsequent removal of the supernatant after sedimentation of the fragments. The colon pieces were minced to a pulp and digested in the DMEM with 150 units/ml collagenase (Sigma, Deisenhofen, Germany) and 20 µg/ml dispase (Boehringer, Mannheim, Germany) for 1–3 h at 37°C with gentle shaking until isolated crypts were visible under the microscope. The suspensions were transferred into 50-ml tubes and centrifuged for 3 min at 400 x g to separate crypts and digestive enzymes. The pellets obtained were resuspended in 10 ml of DMEM, containing HEPES, 10% FCS, antibiotics, and 1.6% sorbitol, and the large crypt fragments were allowed to settle under gravity for 1 min, whereas single crypts remained in solution. The upper 5 ml of the suspensions were carefully taken by a pipette and tranferred into new 50-ml tubes. The remaining loose pellet was extracted twice with 6–10 ml of sorbitol containing medium by vigorous agitation. The collected crypts were centrifuged for 5 min at 60 x g, and the supernatants containing bacteria and fibroblasts were removed. The extraction and centrifugation steps were repeated 4–5 times until the supernatant was clear. The final crypt pellet was suspended in 100 ml of DMEM containing 25 mM HEPES, 2.5% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, 25 µg/ml gentamicin, and 0.25 units/ml human insulin (Hoechst Marion Roussel, Bad Soden, Germany). Crypt numbers were determined by pipetting 10 µl on a cover slide and counting the crypts by using the microscope. Crypts were seeded onto culture plates and cultured in a humidified atmosphere of 91% air and 9% CO₂ at 37°C. The culture plates were coated with collagen type I (Vitrogen 100; 3.1 mg/ml from Collagen GmbH, Ismaning, Germany) by diluting the collagen with 50 mM sterile filtered acetic acid at a ratio 1:9 and pipetting 200 µl of this solution into each well. The solution was allowed to evaporate by placing the plates, uncovered, in a laminar flow hood overnight.

	Cell Proliferation and Acute Cytotoxicity

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For determination of proliferation, HT-29 cells were seeded at a density of 5 x 10³ per well onto 24-well cell culture plates (Renner, Dannstadt, Germany) and allowed to adhere for 24 h. Mouse colonic crypts were seeded at a density of 50 crypts/well on collagen-coated 24-well plates and allowed to adhere for 48 h. Thereafter, medium was replaced by fresh culture medium containing the test compounds, and cells were allowed to grow for another 72 h. In case of the crypt cells only one-half of the medium was removed to maintain the growth factors that were secreted by the cells and that are necessary for optimal growth. Flavone and camptothecin (both from Sigma) were applied in DMSO, and the solvent reached a concentration not higher than 2% in all experiments. Controls were always treated with the same amount of DMSO as used in the corresponding experiments. Total cell counts were determined by SYTOX-Green (Bioprobes, Leiden, Netherlands), which becomes fluorescent after DNA binding. Therefore, cells were lysed by 1% Triton X-100 in isotonic NaCl, and cell numbers were determined based on a calibration curve. The calibration curve was measured using cell numbers between 1 x 10³ and 1.5 x 10⁵ cells, which had been adjusted after the determination of cell numbers in a Neubauer chamber, and fluorescence of the corresponding cell numbers was measured at 538 nm after excitation at 485 nm using a fluorescence multiwell-plate reader (Fluoroskan Ascent, Labsystems, Bornheim-Hersel, Germany).

Acute cytotoxicity was assessed by SYTOX-fluorescence with 5 x 10⁴ adherent HT-29 or crypt cells per 24 wells, which had been exposed for 3 h to test compounds (150 µM). The percentage of dead cells in a cell population was determined by SYTOX-fluorescence prior to cell lysis in relation to the fluorescence measured after the solubilization of cells.

	Differentiation Assay

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After having reached 40% confluency on 25-cm² culture flasks (Renner), HT-29 cells were incubated for 72 h with the test compounds. Crypts were seeded at a density of 250 per well on 24-well plates and were incubated with the test compounds for 72 h after 24 h of adherence. Cell numbers were determined after cells had been washed twice with PBS and trypsinized, and the harvested cells were pelleted for 10 min at 1500 x g. The pellets were resuspended in 550 µl of 1 M diethanolamine buffer (pH 9.8) with 0.5 mM MgCl₂ and were homogenized. Homogenate (500 µl) was mixed with 500 µl of 0.1 mM fluorescein-diphosphate (Bioprobes) in diethanolamine buffer, and AP activity as a marker for differentiation was determined by the release of fluorescein using the multiwell plate reader with excitation at 485 nm and emission at 538 nm. Fluorescein (Sigma) was used as the standard to determine AP-activity on the basis of the two phosphate residues released by cleavage of one fluorescein-diphosphate molecule.

	Determination of Apoptosis Markers

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Early Markers

Apopain Activity.
Apopain activity was measured as described previously (12) . In brief, HT-29 cells were seeded at a density of 5 x 10⁵ per well and crypts at a density of 1000 per well onto 6-well plates (Renner) and allowed to adhere for 24 h. Cells were then exposed to the test compounds for the times indicated in Fig. 2 A and 6C. Subsequent to the determination of cell numbers, cells were centrifuged at 2500 x g for 10 min. Cytosolic extracts were prepared by adding 750 µl of a buffer containing 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 1 mM phenyl-methyl-sulfonyl-fluoride, 10 µg/ml pepstatin A, 20 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 mM HEPES/KOH (pH 7.4) to each pellet and homogenizing by 10 strokes. The homogenate was centrifugated at 100,000 x g at 4°C, and the supernatant was incubated with the fluorogenic caspase-3 tetrapeptide-substrate Ac-DEVD-amino-4-methylcoumarin (Calbiochem, Bad Soden, Germany) at a final concentration of 20 µM. Cleavage of the apopain substrate was followed by determination of emission at 460 nm after excitation at 390 nm using the fluorescence plate reader. 7-amino-4-methylcoumarin (Bioprobes) served as standard for the determination of the rate of hydrolysis of the substrate cleaved by apopain.

View larger version (14K): [in this window] [in a new window]   Fig. 2. A, time-dependent apopain-activity in HT-29 cells incubated with medium alone () or containing in addition 150 µM flavone (•) or 50 µM camptothecin (). Cells (5 x 105) were incubated for the time indicated before the cytosol of the cells was prepared, and apopain-activity was determined by the use of the fluorogenic substrate Ac-DEVD-amino-4-methylcoumarin. B, apopain-activity as a function of flavone (•) and camptothecin () concentration as determined after 24-h incubation. Apopain-activity in control cells was 4.5 ± 1.6 pmol/min x 106 cells.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Determination of early and late apoptosis events by HT-29 cell-staining with Hoechst 33342 (A–D) and Hoechst 33258 (E–H). Cells (3 x 104) were seeded onto 24-well plates and exposed to flavone or camptothecin for the times given in graphs D and H. A–C, cells stained with Hoechst 33342 after 16 h of exposure; E–G, cells stained with Hoechst 33258 after 24 h of incubation either with medium alone (A, E), with 150 µM flavone (B, F), or with 50 µM camptothecin (C, G). Cells that accumulated Hoechst 33342 (D) or that displayed nuclear fragmentation (arrows) by Hoechst 33258 staining (H) were counted and expressed as the percentage of apoptotic cells under control conditions (), after flavone exposure (•), or after camptothecin treatment ().

	Late Markers

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DNA Fragmentation Assay.
DNA fragmentation served as a late apoptosis marker. HT-29 cells at 40% confluency grown on 25-cm² flasks were incubated with the effectors for 24 or 48 h and trypsinized; 1 x 10⁶ cells of each experiment were centrifuged for 5 min at 500 x g, and pellets were lysed by the addition of 2 ml of DNA-pure (Peqlab, Erlangen, Germany). DNA was precipitated by the addition of 1 ml of ethanol. The probe was inverted 5–8 times and was left for 3 min at room temperature before it was pelleted by centrifugation at 5000 x g and 4°C for 5 min. The DNA precipitate was washed by removal of the supernatant, the addition of 95% ethanol, and centrifugation at 1000 x g and 4°C for 2 min. The pellet was dissolved in aqua bidest and adjusted to 0.2–0.3 µg DNA/µl by measuring the UV absorbance at 260 and 280 nm. The DNA was separated on a 1.5% agarose gel and visualized by UV after ethidium-bromide staining.

	Cell Cycle Analysis

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HT-29 cells were seeded at a density of 1 x 10⁶ onto 25-cm² culture flasks and were incubated for 24 or 48 h in the presence or the absence of the test compounds after having reached 40% confluency. The cells were trypsinized, pelleted by centrifugation at 500 x g for 5 min, washed twice with PBS, and adjusted to 2 x 10⁶ cells/ml PBS. Subsequently the cells were treated with 1unit of DNase free RNase (MBI Fermentas, Heidelberg, Germany) per ml of PBS for 30 min at 37°C before they were centrifuged again. The cells were fixed by suspending the pellet for 1 h at 4°C in ethanol. Thereafter, the suspension was centrifuged in a microfuge for 0.5 min, and the pellet was resuspended in 0.9% NaCl containing 1 µM SYTOX-Green. After a 15-min incubation, cell cycle analysis was performed using a FACScan (Becton Dickinson, San Jose, CA) and the Lysis II Ver. 1.1 software.

	Semiquantitative RT-PCR

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RNA from HT-29 cells was isolated at the times indicated in Figs. 7–10 according to the method described by Chomczynski and Sacchi (13) with slight modifications. Reverse transcription was done with 5 µg of isolated RNA. First-strand cDNA synthesis was accomplished with an oligodeoxythymidine₁₅ primer (MBI Fermentas). Amplification of sequence-specific fragments (Taq polymerase was from Sigma) was performed with 30 cycles (95°C-denaturation for 1 min, 55°C-hybridization for 2 min, 72°C-extensions for 2 min; Personal Cycler; Biometra, Göttingen, Germany). RT-PCR products were separated on a 1% agarose gel and visualized by ethidium bromide. The amount of first strand used to amplify specific sequences was derived from the linear range of amplification. The amplified GAP-DH sequence was used as a constitutively expressed control. The amplified products were photographed, and the intensity of the bands was analyzed by the SigmaGel software. No products were obtained for any genes without reverse transcription indicating the specificity of mRNA determination. A -DNA/EcoRI + HindIII marker (MBI Fermentas) was used in all PCR experiments as a size control of the amplified products. Amplified cDNA sequences (primers were custom-synthesized by Eurogentec, Seraing, Belgium) were: GAP-DH, bp 558-1010; COX-2, bp 1366–1870; NF- B, bp 2832–3401; p53, bp 18788–19376 (bp 189–777 of exon 11); p21, bp 95–562; cyclin B,bp 140–893; cyclin E, bp 42–732; bax, bp 40–570; bak, bp 320–763; and bcl-X_L, bp 255–674.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Semiquantitative determination of COX-2 (A) and NF-B (B) mRNA levels in HT-29 cells by use of RT-PCR. The cells were incubated for the indicated time points with or without (control) 150 µM flavone, and subsequently RNA was isolated and reversely transcribed; cDNA sequences of COX-2, NF-B, and GAP-DH (as a constitutively expressed standard gene) were amplified by specific primers. Left, the amplified cDNA products (representative experiment); right, the ratio of target gene to standard gene in control () or flavone treated (•) cells (n = 4). *, P < 0.05; **, P < 0.01

View larger version (56K): [in this window] [in a new window]   Fig. 8. Semiquantitative determination of p53 (A) and p21 (B) transcript levels. Left, the products of GAP-DH (control gene) and p53 or p21, respectively, as obtained by RT-PCR after the indicated incubation times with or without (control) 150 µM flavone. Right, the target gene:GAP-DH ratios from control () and flavone (•)-treated cells are displayed (n = 4). *, P < 0.05; **, P < 0.01.

View larger version (57K): [in this window] [in a new window]   Fig. 9. Determination of cyclin B (A) and cyclin E (B) mRNAs. HT-29 cells were treated for the indicated times with or without (control) flavone. Right, cyclin Band E:GAP-DH ratios from control () and flavone (•)-treated cells (n = 4). *, P < 0.05; **, P < 0.01.

	Calculation and Statistics

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	RESULTS

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Effects of Flavone on Proliferation, Differentiation, and Apoptosis in HT-29 Cells.
Flavone reduced proliferation of the human colon carcinoma cell line HT-29 dose-dependently with an EC₅₀ value of 54.8 ± 1.3 µM. Under the same experimental conditions, the antitumor agent camptothecin led to one-half of a maximal growth inhibition at a concentration of 20.5 ± 1.0 µM (Fig. 1A ). At concentrations at which cell growth was completely abolished, i.e., at about 50 µM camptothecin and 100 µM flavone, neither compound revealed any cytotoxic effects (95.3 ± 5.7% living cells at 50 µM camptothecin; 102.4 ± 6.9% living cells at 100 µM flavone). However, camptothecin at concentrations of >50 µM proved to be cytotoxic (86.9 ± 4.1% living cells at 75 µM, 45.5 ± 8.5% at 100 µM; 32.6 ± 7.5% at 150 µM). In contrast, flavone at a concentration of 150 µM was not toxic (97.4 ± 1.0% living cells) and exerted only moderate cytotoxic effects at concentrations 250 µM (82.9 ± 1.1% living cells at 250 µM; 73.4 ± 4.5% living cells at 500 µM). When applied at the highest nontoxic concentrations (150 µM flavone, 50 µM camptothecin) flavone was found to cause a 6-fold increase of AP-activity serving as a marker for cell differentiation (Fig. 1B ), whereas camptothecin increased AP activity only by 2-fold (Fig. 1B ). At a concentration of 250 µM, flavone caused an additional increase of AP activity to reach 853.8 ± 47.3% of control cells (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1. A, proliferation of HT-29 cells as a function of flavone (•) and camptothecin () concentration. Cells (5 x 103 per well) were incubated for 72 h with medium alone (control) or containing the compounds at various concentrations, and cell numbers were determined subsequently using SYTOX-Green nucleic acid stain. Cell counts for controls were 73,500 ± 4,500. B, AP activity after 72-h incubation with medium alone () or containing 150 µM flavone () or 50 µM camptothecin (). Forty % confluent cells were incubated with or without the compounds before AP activity was determined using fluorescein-diphosphate as a substrate. AP activity in control cells was determined as 25.2 ± 1.4 pmol/min x 106 cells. *, P < 0.05; ***, P < 0.001 (n = 3).

That the apoptotic signal given by activation of apopain is transmitted to the end point of the apoptotic pathway, i.e., the fragmentation of DNA, could be demonstrated for both compounds. Whereas nuclear fragmentation after 24 h of exposure of cells to camptothecin or flavone was detectable in about 25% of the cells (Fig. 3, E–H) , it was more prominent at 48 h (Fig. 3H ). When DNA was isolated from flavone- or camptothecin-treated cells, DNA fragmentation became evident in adherent cells and even more pronounced in floating apoptotic cells only after 48 h (Fig. 4A ).

View larger version (42K): [in this window] [in a new window]   Fig. 4. A, DNA-fragmentation in HT-29 cells as a consequence of flavone or camptothecin incubation. Cells were incubated with 150 µM flavone or 50 µM camptothecin, and DNA was isolated at the indicated time points as described in the "Materials and Methods" section. B, flow cytometric analysis of HT-29 cells incubated without (control) or with 150 µM flavone for 24 and 48 h. DNA was stained by SYTOX-Green.

Effects of Flavone on Proliferation, Differentiation, and Apoptosis in Nontransformed Murine Colonocytes.
To assess the cell specificity of flavone, we compared its effects on proliferation, differentiation, and apoptosis in HT-29 cells with colonic cells of a nontransformed genetic background. Isolation of crypts from mouse colon and growth of the isolated colonocytes are shown in Fig. 5 .

View larger version (117K): [in this window] [in a new window]   Fig. 5. Colonocyte culture derived from isolated murine crypts. Crypts from adult mice were isolated as described in the "Materials and Methods" section and digested by collagenase/dispase. The panels show the separation of single crypts during the digestion procedure at 30 min (A), 45 min (B), 60 min (C), 75 min (D), and 190 min (E). Single colonocytes detach from the crypts 8 h postseeding (F) and proliferate (G) until they form intact monolayers (H).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effects of flavone and camptothecin on proliferation (A), differentiation (B), and apoptosis (C) in primary colonocytes. Crypts were seeded at densities of 50, 250, and 1000 per well for the determination of proliferation, differentiation, and apoptosis, respectively. In A, to determine the effects on proliferation, flavone (•) and camptothecin () were applied at various concentrations for 72 h. Control cell counts were 63,200 ± 6,600. In B, AP activity as a marker for differentiation was assessed after 72-h incubation either in the presence of medium alone () or in medium containing 150 µM flavone () or 50 µM camptothecin () respectively. AP activity in the murine control cells was 1.08 ± 0.06 nmol/min x 106 cells. In C, apopain activity as a marker for apoptosis was determined after 24-h and 48-h incubation with medium alone (), 150 µM flavone (), or 50 µM camptothecin (). Apopain-activity of the control cells was 3.4 ± 0.2 pmol/min x 106 cells at 24 h and 2.2 ± 0.2 pmol/min x 106 cells at 48 h of incubation. **, P < 0.01; ***, P < 0.001 (n = 3).

Induction of apoptosis by flavone was found to be associated also with altered transcript levels of pro- and antiapoptotic genes. Whereas transcript levels of the pro-apoptotic bax was not affected during the 48 h of flavone exposure of cells (Fig. 10A ), mRNA levels of the pro-apoptotic bak were increased at 48 h of incubation by 3-fold (Fig. 10B ). The mRNA levels of the antiapoptotic bcl-X_L showed a rapid and significant reduction to 25% of that in control cells (Fig. 10C ).

View larger version (58K): [in this window] [in a new window]   Fig. 10. Amplification of cDNA sequences of bax(A) bak (B) and bcl-XL (C) and GAP-DH(constitutively expressed control) subsequent to incubation of HT-29 cells with or without (control) flavone for the indicated times. Bottom panels, the target gene:GAP-DH ratios from control () and flavone (•)-treated cells (n = 4). *, P < 0.05.

	DISCUSSION

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Flow-cytometric analysis revealed that flavone can arrest HT-29 cells in a post-G₁-phase before apoptosis occurs or differentiation is initiated in apoptosis-resistant cells. This inhibition of cell-cycle progression was associated with an altered expression of cell-cycle-relevant genes including the CDK-inhibitor p21. Transcript levels of p21 were found to be significantly increased by treating HT-29 cells with flavone. Development of sporadic tumors is generally associated with reduced expression of p21 (27) , mainly as a result of loss-of-function mutations of p53 (28, 29) . Moreover, p21 expression is directly related to terminal differentiation (28) and increased expression of p21 has been demonstrated to inhibit proliferation of malignant cells in vitro and in vivo (30) . However, the role of p21 in the control of apoptosis is controversial. Whereas Chinery et al. (31) demonstrated that increased p21 expression promoted apoptosis in colorectal cancer cells, others reported p21 to be responsible for cell-cycle arrest and differentiation but not for apoptosis (30) . Prevention of apoptosis induced by antitumor drugs has even been attributed to increased expression of p21 (32) . However, Polyak et al. (33) showed that although the p21-mediated growth-arrest can protect cells from apoptosis, certain trans-acting factors can overcome these protective effects.

That the increased p21-expression contributes directly to the observed effects of flavone on apoptosis in our studies is unlikely because apoptosis attributable to increased p21 levels is generally preceded by a G₁ arrest of cells (30) , whereas flavone was found to arrest HT-29 cells in G₂-M. Moreover, this G₂-M arrest, already shown to occur by camptothecin in HT-29 cells (34) , seems to be crucial for apoptosis induction in cells that do not express a functional p53, such as HT-29 (29, 35) .

Nevertheless, flavone may prove to be a valuable tool for inhibition of CDKs in colorectal tumors for the following reasons: (a) induction of p21 by flavone was found to be independent of p53 mRNA levels, an effect of flavone that has recently been described in human lung adenocarcinoma cells (36) ; and (b) the inhibition of CDK2 by p21 may be amplified by the simultaneous decrease of cyclin E because S-phase entry in mammalian cells is induced by CDK2 complexed with S-phase cyclins such as cyclin E (37) .

Not only cyclin E but also cyclin B has been shown to control cell-cycle progression in colon cancer cells (38, 39) and a reduction of cyclin B and cyclin E expression is associated with an improved cell-cycle control and cell-growth inhibition (40) . In contrast to the decreased transcript levels of cyclin E, reduction in cyclin B-mRNA may contribute to the observed cell-cycle arrest induced by flavone because cyclin B complexed with CDK1 is important for the transition of G₂-M phases (40) .

Another gene product that has been linked to the loss of growth-control of colorectal tumors is COX-2 (41) . This enzyme is overexpressed in almost 90% of all colorectal tumors (41) and has been shown to contribute to diminished apoptosis in colon cancer cells (42) . Consequently, COX-2 has become the key target of pharmacotherapy (17) . COX-2 mRNA levels were strongly reduced in cells exposed to flavone. Similarly, flavone exposure reduced the transcript levels of NF-B, a transcription factor that is able to inhibit apoptosis in cancer cells and, thereby, contributes significantly to chemoresistance (18) .

Besides the possibility of promoting apoptosis by down-regulation of NF-B, flavone may prevail over apoptotic processes also by altering expression of other apoptosis-relevant genes such as bak, bax, and bcl-X_L. bcl-X_L has especially been associated with a reduced sensitivity to apoptotic signals in developing colon cancers (43) , and its mRNA levels change rapidly. Because the mRNA of the pro-apoptotic bak was found to be up-regulated by flavone exposure only after 48 h when a large number of cells already had undergone apoptosis, it seems unlikely that bak is significantly involved in the apoptotic pathway activated by flavone. Nevertheless, it could contribute to apoptosis control because bak levels are generally decreased in primary colorectal adenocarcinomas (44) .

As for all of the agents used or developed for cancer treatment, selectivity toward cancer cells is an important criterion. We, therefore, compared the flavone effects on HT-29 cells with that on primary murine colonocytes. Whereas the classical antitumor agent camptothecin induced apoptosis also in nontransformed cells, flavone failed to significantly affect caspase-3 activation in mouse colonocytes. This suggests that flavone is much more selective than campothecin and may have the potential for reducing side effects compared with drugs that possess limited selectivity and that can cause mucosal damage (45) . Moreover, the restricted selectivity of classical apoptosis-inducing antitumor drugs and, thereby, the increased apoptosis rates in normal cells of the human colorectum, can accompany neoplastic transformation (46) .

In conclusion, we have demonstrated that the naturally occurring plant polyphenol, flavone, restores apoptosis sensitivity and differentiation in the human colon cancer cell line HT-29. The effects are likely to occur in vivo as well, because flavone has been shown to accumulate in intestinal cells (47) . Flavone exerts its effects most likely by changing expression of genes known to play a crucial role in apoptosis, differentiation, and proliferation. Despite directing the dysregulated gene expression of HT-29 cells toward an expression pattern of nontransformed colonic cells, it needs to be elucidated which of the genes are affected causally and which are regulated secondarily. Moreover, it remains to be determined whether all of the gene products remain unaffected in nontransformed cells or whether some are also responsive in the primary colonic cells without altering the phenotype.

	ACKNOWLEDGMENTS

 
We greatly acknowledge Prof. Dr. K. Gietzen and Dr. L. Nei for teaching us the method of murine crypt preparation.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Institute of Nutritional Sciences, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany. Phone: 49-8161-71-3997; Fax: 49-8161-71-3999; E-mail: uwenzel{at}pollux.weihenstephan.de

² The abbreviations used are: Ac-DEVD, acetyl-aspartyl-glutamyl-valyl-aspartate; AP, alkaline phosphatase; CHAPS, 3-[(cholamidopropyl)-dimethylammonium]-1-propansulfonate; CDK, cyclin-dependent kinase; COX-2, cyclooxygenase-2; FACS, fluorescence activated cell sorter; GAP-DH, glyceraldehyde-3-phosphate dehydrogenase; NF-B, nuclear transcription factor B; RT, reverse transcription.

Received 12/20/99. Accepted 5/16/00.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS Cell Proliferation and Acute... Differentiation Assay Determination of Apoptosis... Late Markers Cell Cycle Analysis Semiquantitative RT-PCR Calculation and Statistics RESULTS DISCUSSION REFERENCES

 

1. Potter J. D., Slattery M. L., Bostick R. M., Gapstur S. M. Colon cancer: a review of the epidemiology. Epidemiol. Rev, 15: 499-545, 1993.[Free Full Text]
2. Witte J. S., Longnecker M. P., Bird C. L., Lee E. R., Frankl H. D., Haile R. W. Relation of vegetable, fruit, and grain consumption to colorectal adenomatous polyps. Am. J. Epidemiol, 144: 1015-1027, 1996.[Abstract/Free Full Text]
3. Duthie S. J., Dobson V. L. Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro. Eur. J. Nutr, 38: 28-34, 1999.[Medline]
4. Williamson G., Faulkner K., Plumb G. W. Glucosinolates and phenolics as antioxidants from plant foods. Eur. J. Cancer Prev, 7: 17-21, 1998.[Medline]
5. Calomme, M., Pieters, L., Vlietinck, A., and Vanden Berghe, D. Inhibition of bacterial mutagenesis by citrus flavonoids. Planta Med., 62: 222–226, 1996.
6. Plaumann B., Fritsche M., Rimpler H., Brandner G., Hess R. D. Flavonoids activate wild-type p53. Oncogene, 13: 1605-1614, 1996.[Medline]
7. Gerritsen M. E. Flavonoids: inhibitors of cytokine induced gene expression. Adv. Exp. Med. Biol, 439: 183-190, 1998.[Medline]
8. Kuntz S., Wenzel U., Daniel H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity and apoptosis in human colon cancer cell lines. Eur. J. Nutr, 38: 133-142, 1999.[Medline]
9. Chang W. C., Chapkin R. S., Lupton J. R. Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis (Lond.), 18: 721-730, 1997.[Abstract/Free Full Text]
10. Morin P. J., Vogelstein B., Kinzler K. W. Apoptosis, and APC in colorectal tumorigenesis. Proc. Natl. Acad. Sci, 93: 7950-7954, 1996.[Abstract/Free Full Text]
11. Booth C., Patel S., Bennion G. R., Potten C. S. The isolation and culture of adult mouse colonic epithelium. Epithelial Cell Biol, 4: 76-86, 1995.[Medline]
12. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A., Munday N. A., Raju S. M., Smulson M. E., Yamin T-T., Yu V. L., Miller D. K. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature (Lond.), 376: 37-43, 1995.[Medline]
13. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem, 162: 156-159, 1987.[Medline]
14. Hoshi T., Sasano H., Kato K., Yabuki N., Ohara S., Konno R., Asaki S., Toyota T., Tateno H., Nagura H. Immunohistochemistry of Caspase3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis. Anticancer Res, 18: 4347-4353, 1998.[Medline]
15. Droin N., Dubrez L., Eymin B., Renvoize C., Breard J., Dimanche-Boitrel M. T., Solary E. Upregulation of CASP genes in human tumor cells undergoing etoposide-induced apoptosis. Oncogene, 16: 2885-2894, 1998.[Medline]
16. Ormerod M. G., Sun X. M., Snowden R. T., Davies R., Fearnhead H., Cohen G. M. Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. Cytometry, 14: 595-602, 1993.[Medline]
17. Elder D. J., Paraskeva C. COX-2 inhibitors for colorectal cancer. Nat. Med, 4: 392-393, 1998.[Medline]
18. Wang C. Y., Cusack J. C., Jr., Liu R., Baldwin A. S., Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-B. Nat. Med, 5: 412-417, 1999.[Medline]
19. Pellegata N. S., Antoniono R. J., Redpath J. L., Stanbridge E. J. DNA damage and p53-mediated cell cycle arrest: a reevaluation. Proc. Natl. Acad. Sci. USA, 93: 15209-15214, 1996.[Abstract/Free Full Text]
20. Witte J. S., Longnecker M. P., Bird C. L., Lee E. R., Frankl H. D., Haile R. W. Relation of vegetable, fruit, and grain consumption to colorectal adenomatous polyps. Am. J. Epidemiol, 144: 1015-1025, 1996.
21. Knekt P., Jarvinen R., Seppanen R., Hellovaara M., Teppo L., Pukkala E., Aromaa A. Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am. J. Epidemiol, 146: 223-230, 1997.[Abstract/Free Full Text]
22. Deschner E. E., Ruperto J., Wong G., Newmark H. L. Quercetin and rutin as inhibitors of azoxymethanol-induced colonic neoplasia. Carcinogenesis (Lond.), 12: 1193-1196, 1991.[Abstract/Free Full Text]
23. Tanaka T., Makita H., Kawabata K., Mori H., Kakumoto M., Satoh K., Hara A., Sumida T., Tanaka T., Ogawa H. Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis (Lond.), 18: 957-965, 1997.[Abstract/Free Full Text]
24. Middleton E., Kandaswami C. The impact of plant flavonoids on mammalian biology: implications for immunity, inflammation and cancer Harborne J. B. eds. . The Flavonoids: Advances in Research since 1986, : 619-652, Chapmann & Hall London 1993.
25. Cunningham D., Glimelius B. A Phase III study of irinotecan (CPT-11) versus best supportive care in patients with metastatic colorectal cancer who have failed 5-fluorouracil therapy. V301 Study Group. Semin. Oncol, 26: 6-12, 1999.
26. Whitacre C. M., Zborowska E., Willson J. K., Berger N. A. Detection of poly(ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness. Clin. Cancer Res, 5: 665-672, 1999.[Abstract/Free Full Text]
27. Sinicrope F. A., Roddey G., Lemoine M., Ruan S., Stephens L. C., Frazier M. L., Shen Y., Zhang W. Loss of p21^WAF1/Cip1 protein expression accompanies progression of sporadic colorectal neoplasms but not hereditary nonpolyposis colorectal cancers. Clin. Cancer Res, 4: 1251-1261, 1998.[Abstract]
28. Doglioni C., Pelosio P., Laurino L., Macri E., Meggiolaro E., Favretti F., Barbareschi M. p21/WAF1/CIP1 expression in normal mucosa and in adenomas and adeno-carcinomas of the colon: its relationship with differentiation. J. Pathol, 179: 248-253, 1996.[Medline]
29. Rodrigues N. R., Rowan A., Smith M. E., Kerr I. B., Bodmer W. F., Gannon J. V., Lane D. P. p53 mutations in colorectal cancer. Proc. Natl. Acad. Sci. USA, 87: 7555-7559, 1990.[Abstract/Free Full Text]
30. Yang Z. Y., Perkins N. D., Ohno T., Nabel E. G., Nabel G. J. The p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity in vivo. Nat. Med, 1: 1052-1056, 1995.[Medline]
31. Chinery R., Brockman J. A., Peeler M. O., Shyr Y., Beauchamp R. D., Coffey R. J. Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPß. Nat. Med, 3: 1233-1241, 1997.[Medline]
32. Waldman T., Zhang Y., Dillehay L., Yu J., Kinzler K., Vogelstein B., Williams J. Cell-cycle arrest versus cell death in cancer therapy. Nat. Med, 3: 1034-1036, 1997.[Medline]
33. Polyak K., Waldman T., He T. C., Kinzler K. W., Vogelstein B. Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev, 10: 1945-1952, 1996.[Abstract/Free Full Text]
34. Shao R. G., Cao C. X., Shimizu T., O’Connor P. M., Kohn K. W., Pommier Y. Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function. Cancer Res, 57: 4029-4035, 1997.[Abstract/Free Full Text]
35. Arita D., Kambe M., Ishioka C., Kanamaru R. Induction of p53-independent apoptosis associated with G₂-M arrest following DNA damage in human colon cancer cell lines. Jpn. J. Cancer Res, 88: 39-43, 1997.[Medline]
36. Bai F., Matsui T., Ohtani-Fujita N., Matsukawa Y., Ding Y., Sakai T. Promoter activation and following induction of the p21^WAF1 gene by flavone is involved in G₁ phase arrest in A549 lung adenocarcinoma cells. FEBS Lett, 437: 61-64, 1998.[Medline]
37. Sherr C. J. Cancer cell cycles. Science (Washington DC), 274: 1672-1677, 1996.[Abstract/Free Full Text]
38. Yasui W., Kuniyasu H., Yokozaki H., Semba S., Shimamoto F., Tahara E. Expression of cyclin E in colorectal adenomas and adenocarcinomas: correlation with expression of Ki-67 antigen and p53 protein. Virchows Arch, 429: 13-19, 1996.[Medline]
39. Qiao L., Shiff S. J., Rigas B. Sulindac sulfide alters the expression of cyclin proteins in HT-29 colon adenocarcinoma cells. Int. J. Cancer, 76: 99-104, 1998.[Medline]
40. Ji C., Rouzer C. A., Marnett L. J., Pietenpol J. A. Induction of cell cycle arrest by the endogenous product of lipid peroxidation, malondialdehyde. Carcinogenesis (Lond.), 19: 1275-1283, 1998.[Abstract/Free Full Text]
41. Hao X., Bishop A. E., Wallace M., Wang H., Willcocks T. C., Maclouf J., Polak J. M., Knight S., Talbot I. C. Early expression of cyclooxygenase-2 during sporadic colorectal carcinogenesis. J. Pathol, 187: 295-301, 1999.[Medline]
42. Sheng H., Shao J., Morrow J. D., Beauchamp R. D., DuBois R. N. Modulation of apoptosis and Bcl-2 expression by prostaglandin E₂ in human colon cancer cells. Cancer Res, 58: 362-366, 1998.[Abstract/Free Full Text]
43. Hirose Y., Yoshimi N., Suzui M., Kawabata K., Tanaka T., Mori H. Expression of bcl-2, bax, and bcl-XL proteins in azoxymethane-induced rat colonic adenocarcinomas. Mol. Carcinog, 19: 25-30, 1997.[Medline]
44. Krajewska M., Moss S. F., Krajewski S., Song K., Holt P. R., Reed J. C. Elevated expression of Bcl-X and reduced Bak in primary colorectal adeno-carcinomas. Cancer Res, 56: 2422-2427, 1996.[Abstract/Free Full Text]
45. Van Huyen J. P., Bloch F., Attar A., Levoir D., Kreft C., Molina T., Bruneval P. Diffuse mucosal damage in the large intestine associated with Irinotecan (CPT-11). Dig. Dis. Sci, 43: 2649-2651, 1998.[Medline]
46. Sinicrope F. A., Roddey G., McDonnell T. J., Shen Y., Cleary K. R., Stephens L. C. Increased apoptosis accompanies neoplastic development in the human colorectum. Clin. Cancer Res, 2: 1999-2006, 1996.