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Department of Food and Nutrition, Molecular Nutrition Unit, Technical University of Munich, Freising-Weihenstephan, Germany
Received February 26, 2003; accepted September 2, 2003.
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Abstract |
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-nitro-L-arginine methyl ester in NCOL-1 cells or using HT-29 cells that intrinsically have low NO levels enabled flavone to trigger the apoptosis pathway. In conclusion, our studies demonstrate that the intracellular levels of nitric oxide significantly change the apoptotic response to antineoplastic agents in colonic cells.
). The ability of tumor cells to resist apoptosis in response to death signals has become a central theme in the understanding of tumor development (Hanahan and Weinberg, 2000), and one of the major goals in cancer therapy is to restore the sensitivity of transformed cells toward apoptotic signals and the execution of apoptotic cell death (Nicholson, 1996a,b). However, several escape mechanisms have evolved that enable a tumor cell to survive (Pitti et al., 1998; Nakashima et al., 1999; Garrido and Algarra, 2001; Violette et al., 2002), stressing the importance of defining drug-gene interactions to design the best therapeutic concept possible. Indeed, because of genetic differences, the response to a compound can vary greatly between normal, preneoplastic, and transformed cells. For example, the green tea polyphenol epigallocatechin-3-gallate was shown to kill tumor cells much more efficiently than their nontransformed counterparts (Ahmad et al., 1997), and this specificity was associated with the inhibition of nuclear transcription factor 
B (NF-B) expression and the binding of NF-B to DNA-cis-regulatory elements at much lower concentrations in the cancer cells than in the nontransformed cells (Ahmad et al., 2000).
We have shown formerly that the flavonoid flavone, occurring in cereal grains and in dill weed (Middleton and Kandaswami, 1993), induces apoptosis very efficiently in human colon cancer cells without affecting nontransformed primary murine colonocytes (Wenzel et al., 2000). Because the antitumor drug camptothecin potently promotes apoptosis in both cell types (Wenzel et al., 2000), it demonstrates that the apoptotic response of a cell varies depending not only on the genetic background but also on the apoptosis-inducing agent.
In the present study, we first investigated whether the selectivity of the apoptotic action of flavone in comparison to camptothecin could be seen also in NCOL-1 cells, a human colonic epithelial cell line derived from a nontransformed human biopsy specimen (Deveney et al., 1996). By using a semiquantitative RT-PCR approach and Western blotting, we assessed whether apoptosis-inducing activities of flavone and camptothecin were reflected at the transcript and protein levels of apoptosis-relevant gene products. With the use of confocal microscopy, we also determined the role of oxygen radicals and nitric oxide (NO) in the apoptotic response of the cells to flavone and camptothecin.
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Materials and Methods |
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Cell Culture. NCOL-1 cells (passage 50) were a kind gift of Professor Clifford W. Deveney and Dr. Michael J. Rutten (School of Medicine, Oregon Health & Science University, Portland, OR). Cells were cultured and passaged in Dulbecco's modified Eagle's medium/HEPES/glutamine supplemented with 10% fetal calf serum, modified Eagle's medium amino acids, bovine mammary epithelial vitamin solution, and 1 nM human recombinant epidermal growth factor. Antibiotics added to the media were 200 U/ml penicillin, 100 µg/ml streptomycin, 12.5 µg/ml gentamicin, and 1 µg/ml fungizone. 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.
Apoptosis Assays. Caspase-3-like activity was measured as described previously (Wenzel et al., 2000) with the use of the method described by Nicholson et al. (1995). Briefly, NCOL-1 cells were seeded at a density of 5 x 105 per well onto six-well plates and allowed to adhere for 24 h. Cells were then exposed for the times indicated to the test compounds. Subsequently, cells were trypsinized, cell numbers were determined, and then the cells were centrifuged at 2500g for 10 min. Cytosolic extracts were prepared by adding 750 µl of a buffer containing 2 mM EDTA, 0.1% CHAPS, 5 mM dithiotreitol, 1 mM phenylmethylsulfonyl 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 centrifuged at 100,000g at 4°C for 30 min, and the cytosolic supernatant was incubated with the fluorogenic caspase-3 tetrapeptide substrate Ac-DEVD-AMC at a final concentration of 20 µM. Cleavage of the caspase-3 substrate was followed by the determination of emission at 460 nm after excitation at 390 nm using a multiwell plate reader (Fluoroskan Ascent; Labsystem, Bornheim-Hersel, Germany).
Changes in membrane permeability as an early apoptosis marker were assessed by incubating 3 x 104 NCOL-1 cells on 24-well plates with the test compounds or cell-culture medium alone (control) for the times indicated. Cells were stained with 1 µg/ml Hoechst 33342, and the rate of accumulation of the dye in early apoptotic cells (Elstein and Zucker, 1994) was detected using an inverted fluorescence microscope (DMIL; Leica, Wetzlar, Germany) equipped with an band-pass excitation filter of 340 to 380 nm and a long-pass emission filter of 425 nm.
Mitochondrial membrane potential was assessed using MitoCapture. Therefore, NCOL-1 cells were grown on glass slides placed into Quadriperm wells and incubated with or without effectors for the times indicated. Subsequently, the cells were stained with MitoCapture according to the manufacturer's instructions. In healthy cells, MitoCapture accumulates and aggregates in mitochondria, giving off a bright red fluorescence that was viewed under a TCS SP2 laser scanning confocal microscope (Leica) using an excitation of 543 nm and measuring emission at 590 to 650 nm. In apoptotic cells, Mito-Capture cannot aggregate in the mitochondria because of the altered mitochondrial transmembrane potential, and thus it remains in the cytosol in its monomer form, fluorescing green, which was analyzed at excitation of 488 nm and emission of 500 to 530 nm.
Nuclear fragmentation as a late marker of apoptosis was determined by staining of DNA with Hoechst 33258. NCOL-1 cells (3 x 104) were incubated with the test compounds for the times indicated, and thereafter, cells were washed with phosphate-buffered saline, allowed to air dry for 30 min, and then were fixed with 2% para-formaldehyde before staining with 1 µg/ml Hoechst 33258 and visualization under the inverted fluorescence microscope.
Short-Term Cytotoxicity. Cytotoxicity was determined as described previously (Kuntz et al., 1999). Briefly, 5 x 104 adherent NCOL-1 cells were exposed for 3 h to the various test compounds. The percentage of viable cells in the cell population was determined by SYTOX fluorescence after cell lysis by 1% Triton X-100 (total cell counts) in relation to fluorescence measured before lysis of the cells (dead cells). Cell counts were determined from a calibration curve (Kuntz et al., 1999).
Semiquantitative RT-PCR. RNA from NCOL-1 cells was isolated at the times indicated in the figures according to the method described by Chomczynski and Sacchi (1987) with slight modifications (Wenzel et al., 2000). Reverse transcription was performed with 5 µg of isolated RNA. First-strand cDNA synthesis was accomplished with an oligo-(dT)15 primer. Amplification of sequence-specific fragments 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 glyceraldehyd-3-phosphate dehydrogenase (GAPDH) 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 (SPSS Inc., Chicago, IL). No products were obtained for all genes without reverse transcription indicating the specificity of mRNA determination. A 
-DNA/EcoRI plus HindIII marker was used in all polymerase chain reaction experiments as a size control of the amplified products. Primers used are shown in Table 1.
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Immunoblotting. NCOL-1 cells were incubated in 75-cm2 flasks with or without effectors for 24 h and scraped off in 250 mM sucrose. Cells were pelleted subsequently by centrifugation for 10 min at 2500g. The pelleted cells were permeabilized through a syringe with a 24-gauge needle using 0.025% digitonin in a buffer containing 250 mM sucrose, 2.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, complete miniprotease inhibitor cocktail, and 20 mM HEPES/Tris, pH 7.2 (Hausmann et al., 2000). To allow 95 to 100% of the cells to be permeabilized, the cells were incubated for 10 min on ice by gentle agitation, and completeness of permeabilization was assessed by Trypan blue exclusion. Separation of organelles and cytosol was achieved by centrifugation at 13,000g for 2 min at 4°C. The supernatant (cytosol) was carefully removed, and the pellet, containing mitochondria, was solubilized in a buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, protease inhibitor cocktail, and 50 mM Tris-HCl, pH 8.0 (Hausmann et al., 2000). Protein content was determined by the Bradford reaction (Bio-Rad). The homogenate, cytosol, and pellet fractions were resolved by SDS-polyacrylamide gel electrophoresis according to the method described by Schagger and von Jagow (1987) and were electroblotted onto polyvinylidene difluoride membranes. The control of protein transfer and identification of the molecular mass marker proteins was achieved by Ponceau Red staining. Thereafter, the blotting membranes were blocked for 1 h with Tris-buffered saline/Tween 20 and then incubated with the primary antibody (anti-NF-B p65, sc-372; anti-bcl-XL, sc-7195; anti-cytochrome c, sc-7159; anti-actin, sc-1615) for 1 h in a 1:1000 dilution. Bound antibodies were detected after 1-h incubation with horseradish peroxidase-conjugated secondary reagents (sc-2020 for anti-actin and sc-2004 for all other primary antibodies) and 3-amino-9-ethylcarbazole and H2O2 as substrates. Intensities of the bands were analyzed with use of the SigmaGel software.
Detection of Superoxide Radicals. NCOL-1 cells were loaded with 50 µM proxyl fluorescamine for 2 h in modified Krebs buffer containing 200 µM cysteine to yield an increase in fluorescence emission because of the reduction of the fluorophore nitroxide to its corresponding hydroxylamine in the presence of superoxide (Pou et al., 1993). The cells were washed before the application of compounds, and the emission of proxyl fluorescamine was measured at 460 nm after excitation at 390 nm using the fluorescence plate reader. All runs containing camptothecin were corrected for the autofluorescence occurring when camptothecin is excited at 460 nm.
Production of superoxide radicals in mitochondria was visualized by laser scanning microscopy. Therefore, cells were grown on glass slides placed in Quadriperm wells and loaded with 50 µM proxyl fluorescamine for the last 2 h of incubation. For the staining of mitochondria, cells were loaded with 500 nM MitoTracker for the last 30 min of incubation. Superoxide radicals and mitochondria were detected after excitation with the UV laser or at 543 nm and at emissions of 440 to 480 and 590 to 650 nm, respectively.
Detection of NO. NCOL-1 cells were loaded with 10 µM DAF-2DA for 45 min in modified Krebs buffer. The cells were washed and the fluorescence of DAF-2 (excitation, 485 nm; emission, 538 nm) was followed in the presence or absence of effectors using the microtiter plate reader.
Calculations and Statistics. Variance analysis between groups was performed by one-way analysis of variance, and significance of differences between groups was determined by a nonpaired Student's t test (Prism; GraphPad Software Inc., San Diego, CA). For each variable, at least three independent experiments were carried out. Data are given as the mean ± S.E.M.
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Results |
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). Apoptosis induction was associated with a several-fold increased caspase-3-like activity, with plasma membrane disintegration and finally chromatin condensation as a late apoptosis event (Wenzel et al., 2000). When the effects of flavone and camptothecin were assessed in nontransformed NCOL-1 human colonocytes, camptothecin proved to be a very potent apoptosis inducer, whereas flavone failed to affect apoptosis (Fig. 1). These results were obtained at concentrations of 150 µM flavone and 25 µM camptothecin, which in both cases represents the highest concentration that does not induce necrosis as determined by SYTOX-Green nucleic acid stain exclusion (Kuntz et al., 1999). NCOL-1 cells turned out to be very sensitive to camptothecin-induced apoptosis, as indicated by an almost 30-fold activation of caspase-3-like activity after 24 h that resulted in almost 80% of the cells undergoing apoptosis (Fig. 1). In HT-29 cells, for comparison, camptothecin and flavone induce caspase-3-like activity only 7-fold, and only 40% of the cell population showed late signs of apoptosis (Wenzel et al., 2000).
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Effects of Flavone and Camptothecin on Apoptosis-Relevant Transcript Levels in NCOL-1 Cells. To gain insights into the underlying mechanisms of the apoptosis induction by camptothecin in NCOL-1 cells and the selective triggering of apoptosis by flavone in transformed colonocytes only, we performed semiquantitative RT-PCR to determine changes in the transcript levels of 13 genes with relevance for apoptosis and/or cell-cycle control in colonocytes (Reed, 1996; Martinez et al., 1997; Zhan et al., 1999; Hellin et al., 2000; Greco et al., 2001; Sturm et al., 2001; Zvibel et al., 2001; Cao and Prescott, 2002). Relative mRNA levels of the following genes have been determined: bcl-XL, bax, bak, p16, p21, p53, COX-2, cyclin B, cyclin E, erb-B2, c-myc, nm-23, and NF-B. Special emphasis was put on those gene products that showed flavone-dependent alterations in HT-29 (Wenzel et al., 2000) but not in NCOL-1 cells but that changed expression in response to camptothecin in NCOL-1 cells.
Among the transcripts analyzed, neither bax, p16, p53, COX-2, erb-B2, c-myc, nor nm23 mRNAs showed any changes in response to camptothecin or flavone in NCOL-1 cells (data not shown). The mRNA levels of the proapoptotic bak were selectively increased after camptothecin treatment, but this increase in bak mRNA became significant only after 48 h of exposure (data not shown), a time point when almost all cells exposed to camptothecin already showed signs of the late apoptosis (Fig. 1C). This suggests that a direct involvement of bak in the initiation of apoptosis by camptothecin is very unlikely. An increase in the mRNA levels of the cyclin-dependent kinase inhibitor p21 and a concomitant decrease in transcript levels of the cell-cycle regulators cyclin B and cyclin E were seen after flavone as well as after camptothecin treatment. Thus, these gene products cannot be responsible for the induction of apoptosis in NCOL-1 cells by camptothecin. However, the growth-inhibitory potencies of flavone and camptothecin observed in NCOL-1 cells (data not shown) might involve the changes in mRNA levels of these gene products.
Both bcl-XL and NF-B have been shown to possess anti-apoptotic activities in colonocytes (Hirose et al., 1997; Wang et al., 1999). Previously, we demonstrated that both factors are efficiently down-regulated at the mRNA level in HT-29 cells in response to flavone (Wenzel et al., 2000). Similarly, in NCOL-1 cells, these mRNA levels are reduced by camptothecin at 8 and 24 h, respectively, whereas flavone did not affect bcl-XL or NF-B mRNA levels at all (Table 2). When the effects of camptothecin on the protein level were assessed at 24 h of incubation, a time point when a large number of cells already underwent late stages of apoptosis (Fig. 1C), bcl-XL was significantly reduced by camptothecin, whereas the NF-B protein levels remained unaltered compared with controls (Fig. 2). Flavone changed neither bcl-XL nor NF-B protein levels at 24 h of incubation (Fig. 2).
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Effects of Camptothecin and Flavone on the Mitochondrial Apoptosis Pathway. bcl-XL serves as an anti-apoptotic protein that remains constantly attached to mitochondria to prevent the release of cytochrome c by mechanisms that are still under debate (Hengartner, 2000). One important factor that is frequently associated with the down-regulation of bcl-XL gene expression is reactive oxygen species. Their generation is suggested to precede the loss of mitochondrial membrane potential, the release of cytochrome c, and finally the activation of caspase-3 (Herrera et al., 2001).
We observed that the down-regulation of bcl-XL in camptothecin-treated NCOL-1 cells was associated with the release of cytochrome c into the cytosol (Fig. 3A) and the dissipation of the mitochondrial inner membrane potential (Fig. 3B), which preceded the cytochrome c release by approximately 4 h (data not shown).
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By confocal microscopy, we observed an early increase of superoxide anion production in the mitochondria of NCOL-1 cells only 6 h after exposure to camptothecin, whereas flavone-treated cells did not respond (Fig. 4A). Because camptothecin, in contrast to flavone, is a potent producer of superoxide anions in the cytosol (Fig. 4B), the activation of the mitochondrial apoptosis pathway could be a direct consequence of superoxide anions entering the mitochondria. We have validated this hypothesis by analyzing the effects of menadion on apoptosis induction. Menadion is a classic producer of superoxide anions in the cytosol, but in contrast to camptothecin, menadion inhibited caspase-3-like activity in NCOL-1 instead of increasing it (data not shown). It therefore seems that other mitochondrial mechanisms that lead to the one-electron reduction of O2 are the cause of apoptosis triggering by camptothecin, and those are not directly dependent on superoxide anions released into the cytosol.
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NO Inhibits the Mitochondrial Apoptosis Pathway by Scavenging O2- NO is an endogenously generated radical that reacts very fast with 
to form peroxynitrite (Squadrito and Pryor, 1998). In this regard, NO functions as a potent antioxidant (Chiueh, 1999) with the capability to scavenge produced in the cytosol or mitochondria. When analyzing the NO levels in NCOL-1 cells in response to camptothecin, we observed a decline in endogenous free NO concentration (Fig. 5A), which obviously prevents a quenching of later on in the mitochondria (Fig. 4A). When we applied the NO donor SNP, a sustained, increased NO level in camptothecin-treated NCOL-1 cells resulted (Fig. 5A), and this efficiently prevented the camptothecin-induced generation of mitochondrial (Fig. 5B). Moreover, when NO levels were reduced by the NO-synthase inhibitor L-NAME (Fig. 5A), even in flavone-treated cells, an increased production of in mitochondria was seen after 6 h of exposure (Fig. 5B). Flavone alone did not affect the NO level (Fig. 5A). That the NO level inside NCOL-1 cells is strictly related to the mitochondrial apoptosis pathway was also confirmed by measuring mitochondrial membrane potential (Fig. 6A), levels of bcl-XL (Fig. 6B), and the release of cytochrome c (Fig. 6C). All of these parameters indicated that camptothecin as well as flavone can activate the mitochondrial apoptosis pathway, but NO levels inside the cells seem to select whether apoptosis is executed or not. When NO levels decline, as observed in the presence of camptothecin or in the presence of a combination of flavone and L-NAME, apoptosis can occur. When NO levels are maintained or increased, as in the case of flavone or the combination of camptothecin with SNP, the mitochondrial apoptosis pathway is efficiently blocked. These effects of the NO level on the mitochondrial apoptosis parameters were completely mirrored by its actions on the early or late stages of apoptosis (Fig. 7).
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The selectivity of NO action on apoptosis in NCOL-1 cells is further stressed by the fact that high doses of flavone (150, 250, and 500 µM) do not change NO levels (Fig. 8C) but clearly cause concentration-dependent cell death (Fig. 8A) in the absence of an activation of caspase-3 (Fig. 8B). When using camptothecin at higher concentrations (25, 50, and 150 µM), NO levels are reduced to a similar extent (Fig. 8C), caspase-3-like activity is increased to similar levels at all concentrations, whereas cell death is strictly dose-dependent (Fig. 8A). When caspase-3-like activity was measured after 3 h of exposure to the test compounds, no changes over that seen in control cells was observed (data not shown), which strongly suggests that the simultaneously observed cytotoxicity of high doses of flavone or camptothecin are not linked to apoptosis.
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Discussion |
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). Several escape mechanisms are described which enable a tumor cell to survive, and those are derived from genetic differences between the parent nontransformed cells and the malignant cancer cell (Pitti et al., 1998; Nakashima et al., 1999; Garrido and Algarra, 2001; Violette et al., 2002). The identification of molecular markers that determine whether apoptosis in a cell occurs or not is consequently crucial for the development of strategies that specifically target cancer cells. In previous studies, we collected evidence that the flavonoid flavone is capable of submitting selectively tumor cells to apoptosis and of overcoming the escape mechanisms. It efficiently induces apoptosis in HT-29 human colon cancer cells but not in primary murine nontransformed colonocytes (Wenzel et al., 2000). This selectivity was not found for camptothecin, a classic apoptosis-inducing antitumor drug. In search of the underlying mechanisms for the selectivity of flavone, we compared its effects with those of camptothecin in a human nontransformed colonocyte line derived from a human biopsy specimen (Deveney et al., 1996). Although NCOL-1 cells were found not to form colonies in a soft-agar assay, suggesting that the long-term culturing did not cause these cells to become transformed (Deveney et al., 1996), they should be regarded as preneoplastic because of their growth-dependent selection. We observed that flavone did not induce apoptosis in NCOL-1 cells. Camptothecin, in contrast, was a very potent apoptosis inducer in NCOL-1 cells, as evidenced by its effects on caspase-3-like activity, on plasma membrane disintegration and nuclear fragmentation.
Taken from a screening of the transcript levels of 13 apoptosis-relevant genes in NCOL-1 cells, only the antiapoptotic factors bcl-XL and NF-B showed a down-regulation by camptothecin but not flavone treatment. In HT-29 colon cancer cells, however, flavone also reduced the expression of bcl-XL and NF-B mRNAs (Wenzel et al., 2000). At the protein level, bcl-XL but not NF-B was down-regulated by camptothecin, and this could be observed when the majority of NCOL-1 cells showed signs of late apoptosis. These data demonstrate the prominent role of bcl-XL in the regulation of apoptosis in colonocytes and consequently in the development of colonic cancers (Maurer et al., 1998).
Because reactive oxygen species produced in mitochondria were found to be responsible for the down-regulation of bcl-XL in hepatocytes followed by a loss of the mitochondrial membrane potential, the release of cytochrome c, and finally the activation of the effector caspase-3 (Herrera et al., 2001), we concluded that flavone and camptothecin could differ in their ability to generate mitochondrial superoxide anions in NCOL-1 cells. Indeed, only camptothecin initiated the generation of in the mitochondria of NCOL-1 cells followed by dissipation of the mitochondrial membrane potential, release of cytochrome c, caspase-3 activation, plasma membrane disintegration, and finally nuclear fragmentation. Because HT-29 cells do not generate detectable amounts of NO endogenously (Ambs et al., 1998b) but respond to flavone with apoptosis, we suggested that NO could act as an antioxidant in NCOL-1 cells and interfere with the mitochondrial apoptosis pathways. Here we show that camptothecin produces cytosolic and thereby scavenges free NO with a concomitant decline of cellular NO levels that could allow apoptosis to occur. When NO levels in camptothecin-treated NCOL-1 cells were increased by the coadministration of the NO donor SNP, all proapoptotic processes induced by camptothecin were blocked. When NO levels were reduced in NCOL-1 cells by using the NO-synthase inhibitor L-NAME, flavone was enabled to induce apoptosis via the mitochondrial pathway. The product of the reaction between NO and is peroxynitrite, which is known to induce apoptosis in various cell types as well (Ambs et al., 1998a). To elucidate whether peroxynitrite contributes to camptothecin-mediated apoptosis in NCOL-1 cells, we applied 100 µM SIN-1, a peroxynitrite generator. Neither SIN-1 alone nor in combination with camptothecin affected caspase-3-like activity or other indicators of apoptosis, but SIN-1 possessed necrotic effects, which were not seen under any of the experimental conditions with camptothecin or flavone (data not shown). This indicates that the amount of peroxynitrite formed has an impact on its effects as well.
In conclusion, our comparative analysis of flavone and camptothecin effects in NCOL-1 and HT-29 cells demonstrates that superoxide anions produced in mitochondria regulate apoptosis in colonocytes by affecting the expression level of the antiapoptotic factor bcl-XL. By its ability to scavenge the cellular concentration of NO decisively controls the occurrence of apoptosis. In HT-29 cells, the intrinsic low level of NO allows flavone to efficiently induce apoptosis, and this is prevented when cellular NO levels are increased (Wenzel et al., 2003). Moreover, our studies demonstrate that small genetic differences between cells can have a great impact on the therapeutic responsiveness to drug exposure, stressing the importance of investigating drug-gene interactions.
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Footnotes |
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B, nuclear transcription factor B; AMC, amino-4-methylcoumarin; CHAPS, 3-[(cholamidopropyl)-dimethyl-ammonium]-1-propane-sulfonate; COX-2, cyclooxygenase-2; DAF-2DA, 4,5-diamino-fluorescein diacetate; GAPDH, glyceraldehyd-3-phosphate dehydrogenase; L-NAME, N-nitro-L-arginine methyl ester; proxyl fluorescamine, 5-(2-carboxyphenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrrolin-4-one, potassium salt; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, sodium nitroprusside; SIN-1, 3-morpholinosydnonimine. Address correspondence to: Dr. Uwe Wenzel, Department of Food and Nutrition, Molecular Nutrition Unit, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany. E-mail: uwenzel{at}wzw.tum.de
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ahmad N, Gupta S, and Mukhtar H (2000) Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells. Arch Biochem Biophys 376: 338-346.[CrossRef][Medline]
Ambs S, Merriam WG, Bennett WP, Felley-Bosco E, Ogunfusika MO, Oser SM, Klein S, Shields PG, Billiar TR, and Harris CC (1998a) Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res 58: 334-341.
Ambs S, Merriam WG, Ogunfusika MO, Bennett WP, Ishibe N, Hussain SP, Tzeng EE, Geller DA, Billiar TR, and Harris CC (1998b) p53 and vascular endothelial growth factor regulate tumor growth of NOS2-expressing human carcinoma cells. Nat Med 4: 1371-1376.[CrossRef][Medline]
Cao Y and Prescott SM (2002) Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J Cell Physiol 190: 279-286.[CrossRef][Medline]
Chiueh CC (1999) Neuroprotective properties of nitric oxide. Ann N Y Acad Sci 890: 301-311.[CrossRef][Medline]
Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159.[Medline]
Deveney CW, Rand-Luby L, Rutten MJ, Luttropp CA, Fowler WM, Land J, Meichsner CL, Farahmand M, Sheppard BC, Crass RA, et al. (1996) Establishment of human colonic epithelial cells in long-term culture. J Surg Res 64: 161-169.[CrossRef][Medline]
Elstein KH and Zucker RM (1994) Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations. Exp Cell Res 211: 322-331.[CrossRef][Medline]
Garrido F and Algarra I (2001) MHC antigens and tumor escape from immune surveillance. Adv Cancer Res 83: 117-158.[Medline]
Greco C, Alvino S, Buglioni S, Assisi D, Lapenta R, Grassi A, Stigliano V, Mottolese M, and Casale V (2001) Activation of c-MYC and c-MYB proto-oncogenes is associated with decreased apoptosis in tumor colon progression. Anticancer Res 21: 3185-3192.[Medline]
Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57-70.[CrossRef][Medline]
Hausmann G, O'Reilly LA, van Driel R, Beaumont JG, Strasser A, Adams JM, and Huang DC (2000) Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L). J Cell Biol 149: 623-634.
Hellin AC, Bentires-Alj M, Verlaet M, Benoit V, Gielen J, Bours V, and Merville MP (2000) Roles of nuclear factor-kappaB, p53 and p21/WAF1 in daunomycin-induced cell cycle arrest and apoptosis. J Pharmacol Exp Ther 295: 870-878.
Hengartner MO (2000) The biochemistry of apoptosis. Nature (Lond) 407: 770-776.[CrossRef][Medline]
Herrera B, Alvarez AM, Sanchez A, Fernandez M, Roncero C, Benito M, and Fabregat I (2001) Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes. FASEB J 15: 741-751.
Hirose Y, Yoshimi N, Suzui M, Kawabata K, Tanaka T, and Mori H (1997) Expression of bcl-2, bax and bcl-XL proteins in azoxymethane-induced rat colonic adenocarcinomas. Mol Carcinog 19: 25-30.[CrossRef][Medline]
Kuntz S, Wenzel U, and Daniel H (1999) Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity and apoptosis in human colon cancer cell lines. Eur J Nutr 38: 133-142.[CrossRef][Medline]
Martinez R, Venturelli D, Perrotti D, Veronese ML, Kastury K, Druck T, Huebner K, and Calabretta B (1997) Gene structure, promoter activity and chromosomal location of the DR-nm23 gene, a related member of the nm23 gene family. Cancer Res 57: 1180-1187.
Maurer CA, Friess H, Buhler SS, Wahl BR, Graber H, Zimmermann A, and Buchler MW (1998) Apoptosis inhibiting factor Bcl-xL might be the crucial member of the Bcl-2 gene family in colorectal cancer. Dig Dis Sci 43: 2641-1648.[CrossRef][Medline]
Middleton E and Kandaswami C (1993) The impact of plant flavonoids on mammalian biology: implications for immunity, inflammation and cancer, in The Flavonoids: Advances in Research Since 1986 (Harborne JB ed) pp 619-652, London, Chapmann & Hall.
Nakashima M, Sonoda K, and Watanabe T (1999) Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1. Nat Med 5: 938-942.[CrossRef][Medline]
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, et al. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature (Lond) 376: 37-43.[CrossRef][Medline]
Nicholson DW (1996a) From bench to clinic with apoptosis-based therapeutic agents. Nature (Lond) 407: 810-816.
Nicholson DW (1996b) ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis. Nat Biotechnol 14: 297-301.[CrossRef][Medline]
Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, Huang A, Donahue CJ, Sherwood SW, Baldwin DT, et al. (1998) Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature (Lond) 396: 699-703.[CrossRef][Medline]
Pou S, Huang YI, Bhan A, Bhadti VS, Hosmane RS, Wu SY, Cao GL, and Rosen GM (1993) A fluorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils. Anal Biochem 212: 85-90.[CrossRef][Medline]
Reed JC (1996) Mechanisms of Bcl-2 family protein function and dysfunction in health and disease. Behring Inst Mitt 97: 72-100.
Schagger H and von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379.[CrossRef][Medline]
Squadrito GL and Pryor WA (1998) Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite and carbon dioxide. Free Radic Biol Med 25: 392-403.[CrossRef][Medline]
Sturm I, Petrowsky H, Volz R, Lorenz M, Radetzki S, Hillebrand T, Wolff G, Hauptmann S, Dorken B, and Daniel PT (2001) Analysis of p53/BAX/p16 (ink4a/CDKN2) in esophageal squamous cell carcinoma: high BAX and p16(ink4a/CDKN2) identifies patients with good prognosis. J Clin Oncol 19: 2272-2281.
Violette S, Poulain L, Dussaulx E, Pepin D, Faussat AM, Chambaz J, Lacorte JM, Staedel C, and Lesuffleur T (2002) Resistance of colon cancer cells to long-term 5-fluorouracil exposure is correlated to the relative level of Bcl-2 and Bcl-X(L) in addition to Bax and p53 status. Int J Cancer 98: 498-504.[CrossRef][Medline]
Wang CY, Cusack JC Jr, Liu R, and Baldwin AS Jr (1999) Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB. Nat Med 5: 412-417.[CrossRef][Medline]
Wenzel U, Jambor de Sousa U, Kuntz S, and Daniel H (2003) Nitric oxide suppresses apoptosis in human colon cancer cells by scavenging mitochondrial superoxide anions. Int J Cancer 106: 666-675.[CrossRef][Medline]
Wenzel U, Kuntz S, Brendel MD, and Daniel H (2000) Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. Cancer Res 60: 3823-3831.
White MK and McCubrey JA (2001) Suppression of apoptosis: role in cell growth and neoplasia. Leukemia 15: 1011-10121.[CrossRef][Medline]
Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, Harris CC, and Fornace AJ Jr (1999) Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene 18: 2892-2900.[CrossRef][Medline]
Zvibel I, Brill S, Halpern Z, Moskovitz S, Yayon A, and Papa M (2001) Soluble and matrix-associated heparan sulfate proteoglycans increase expression of erb-B2 and erb-B3 in colon cancer cell lines. Int J Cancer 91: 316-321.[CrossRef][Medline]
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, P < 0.05; 
B in cells exposed to flavone or camptothecin
