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Carcinogenesis Advance Access originally published online on April 13, 2007
Carcinogenesis 2007 28(7):1575-1581; doi:10.1093/carcin/bgm080
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Chemopreventive effects of lupulone, a hop ß-acid, on human colon cancer-derived metastatic SW620 cells and in a rat model of colon carcinogenesis

Virginie Lamy1,2,3, Stamatiki Roussi1,2,3, Mehdi Chaabi4,5, Francine Gossé1,2,3, Nicolas Schall1,2,3, Annelise Lobstein4,5 and Francis Raul1,2,3,*

1 Inserm U682, Laboratoire de Prévention Nutritionnelle du Cancer, Strasbourg, F-67000 France
2 Faculté de Médecine, Université Louis Pasteur EA3430, Strasbourg, F-67000 France
3 IRCAD, Strasbourg, F-67000 France
4 CNRS UMR7081, Laboratoire de Pharmacognosie, Illkirch, F-67400 France
5 Faculté de Pharmacie, Université Louis Pasteur, Illkirch, F-67400 France

* To whom correspondence should be addressed. Tel: +33(0)388119023; Fax: +33(0)388119097; Email: francis.raul{at}ircad.u-strasbg.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The bitter acids of hops (Humulus lupulus L.) mainly consist of humulones or {alpha}-acids and lupulones or ß-acids. We aimed to evaluate the antiproliferative mechanisms of lupulones on a human metastatic colon carcinoma-derived cell line (SW620 cells) and to assess their chemopreventive effects in a model of colon carcinogenesis. SW620 cell growth was inhibited by 70% after a 48 h exposure to lupulones (40 µg/ml). Lupulones up-regulated the expression of Fas receptor (Fas) and Fas ligand (FasL) as well as TNF-related apoptosis inducing ligand (TRAIL)-R1 (DR4) and -R2 (DR5) receptor proteins, suggesting the involvement of Fas and TRAIL receptors-mediated pathways in lupulone-induced apoptosis. Lupulones also increased the mitochondrial membrane permeability. Colon carcinogenesis was initiated in Wistar rats by intra-peritoneal injections of azoxymethane (AOM), once a week for 2 weeks. One week after the last injection, rats received lupulones (0.001 or 0.005%) in drinking water, and AOM-control rats received the excipient. After 7 months of treatment, the colon of rats receiving 0.001 and 0.005% lupulones showed, respectively, a 30 and a 50% reduction (P < 0.05) of the number of preneoplastic lesions (aberrant crypt foci). In addition, we observed a drastic reduction (70–80%) of the total number of tumors in the colon of rats treated with lupulones when compared with the AOM control group. Lupulones induced apoptosis in SW620 colon-derived metastatic cells by activating both Fas and TRAIL death receptor signaling pathways, and antagonize at a low dose (4 mg/kg/day) colon cancer development. These observations suggest the use of lupulones for colon cancer chemoprevention trials.

Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; DMSO, dimethylsulfoxide; Fas, Fas receptor; FasL, Fas ligand; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TRAIL, TNF-related apoptosis-inducing ligand


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The bitter acids of hops are divided into two main categories, the humulones or {alpha}-acids and the lupulones or ß-acids. Both categories of bitter acids consist of a mixture of homologues, and lupulones are composed mainly of a mixture of n-, co- and ad-lupulones (1). To date, several reports have described various potent biological activities of {alpha}-acids, iso-{alpha}-acids or of a mixture of hop acids. In addition to their antibacterial action (2), these compounds present also anticancer properties, including inhibition of leukemia cell proliferation, induction of differentiation and apoptosis (2–5), inhibition of oxidation (6,7), inhibition of cyclooxygenase-2 expression and reduction of prostaglandin E2 secretion (8,9). It was also shown that humulones inhibit angiogenesis in vivo and in vitro (10). In contrast, no data exist on the anticancer properties of lupulones. In the present study, we aimed to evaluate the anti-proliferative mechanisms of lupulones on a human metastatic colon carcinoma-derived cell line (SW620 cells) and to evaluate their anticarcinogenic potential in vivo. We present evidence showing that lupulones trigger apoptosis by altering several death-related signaling pathways and that these compounds inhibit tumor formation in an experimental model of colon carcinogenesis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Isolation and purification of lupulones
For the specific isolation of lupulones, we used an industrial by-product that contains high amounts of ß-acids (Brasseries Kronenbourg, Strasbourg, France). One volume of this preparation was mechanically stirred at room temperature during 15 min, with two volumes of demineralized water. The ß-acids of hops were then separated from the aqueous solution by centrifugation (300g for 15 min). This step was repeated until the supernatant was clear. The water-insoluble pellets were combined, re-suspended in ethanol (96%) and centrifuged once again. The supernatant containing the ß-acids was filtered on a Buchner funnel and then dry-concentrated under reduced pressure, at 30°C. In order to preserve from photo-isomerization, all steps were conducted in the dark. The obtained mixture of ß-acids was analyzed by reversed phase-high-pressure liquid chromatography (250 mm x 4.6 mm, Nucleodur®, Macherey-Nagel, Hoerdt, France) recorded with diode-array detection (Varian Polychrom 9065, Les Ulis, France) in order to verify the absence of degradation and to check their relative purity. The elution was carried out with trifluoroacetic acid 0.01 M (phase A) and acetonitrile (phase B) solutions in the following conditions: from 100% (A) to 100% (B) for 15 min, and then elution was kept isocratic at 100% (B) for 30 min. A representative analytical reversed phase-high-pressure liquid chromatography of the hop ß-acids fraction is shown in Figure 1. The degree of purification of lupulones was 85–90% with the absence of oxidation-derived products. The lupulones fraction was constituted by 40% of co-lupulones (C25H36O4, molecular weight: 400.56) and 60% of n-lupulones (C26H38O4, molecular weight: 414.58). Stock solutions for in vitro studies were prepared in dimethylsulfoxide (DMSO, Sigma–Aldrich, Steinheim, Germany), whereas ethanol was used as a solvent vehicle for the in vivo experiments.


Figure 1
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  		Fig. 1. Representative analytical reversed phase-high-pressure liquid chromatography of ß-acids hops after separation on a Nucleodur® column (250 mm x 4.6 mm). UV detection at 270 nm. For details see Materials and Methods. Peaks 1 and 2 are identified as lupulone fractions containing 40% of co-lupulones and 60% of n-lupulones.

 
Cell culture
SW620 cells were purchased from the European Collection of Animal Cell Culture (Salisbury, UK). They were cultured in 75 cm2 Falcon flasks in Dulbecco's modified Eagle's medium containing 25 mM glucose and supplemented with 10% heat-inactivated (56°C) horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 1% non-essential amino acids (Invitrogen Corp., Cergy Pontoise, France). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2 and subcultured after trypsinization (0.5% trypsin/2.6 mM ethylenediaminetetraacetic acid). For all experiments, cells were seeded at 1 x 106 cells in culture dishes (100 mm internal diameter). The culture medium was Dulbecco's modified Eagle's medium supplemented with 3% heat-inactivated horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml transferrin, 5 ng/ml selenium, 10 µg/ml insulin and 1% non-essential amino acids (Invitrogen Corp.).

Cell growth
Cells were exposed for 48 and 72 h to different concentrations of lupulones varying from 0 to 60 µg/ml. The final concentration of DMSO in the culture medium was 0.1%. Culture medium was replaced every 48 h. At different time points, cells were harvested by trypsinization (0.5% trypsin/2.6 mM ethylenediaminetetraacetic acid) and centrifuged for 5 min at 900g (4°C), and the pellet was re-suspended in 0.1 M phosphate-buffered saline (PBS), pH 7.2. For estimation of cell survival and cell death, cells were stained with trypan blue dye (1/1, vol/vol) (Invitrogen Corp.). Only dead cells were stained and the number of stained and non-stained cells was determined by optical microscopy.

Detection of apoptosis
Cells were exposed to lupulones (40 µg/ml) for 48 and 72 h. At each time point, cells were harvested by trypsinization and washed twice with PBS. Apoptosis was quantified by measuring phosphatidylserine externalization using a flow cytometer as described previously (11). Assays were carried out with annexin-V–FLUOS staining kit (Roche Diagnostics GmbH, Mannheim, Germany). The cell pellet was re-suspended in 100 µl of solution containing staining buffer and annexin-V–fluorescein isothiocyanate (FITC) and incubated for 15–20 min at 15–25°C according to the manufacturer's instructions. Samples (10 000 cells per sample) were analyzed by flow cytometry using 488 nm excitation and green emission (FL-1, 515 nm) filter on cell populations from which debris were gated out. Histograms were analyzed by the CellQuest Software (FACScan, BD Biosciences, San Jose, CA, USA).

Mitochondrial membrane potential
Cells were cultured with lupulones (40 µg/ml) and harvested by trypsinization at 24, 38 and 72 h. Changes in mitochondrial membrane potential were assessed by using the MitoProbeTM DiOC2(3) assay kit (Invitrogen Corp.). The kit provides the cationic cyanine dye DiOC2(3) (3,3'-diethyloxacarbocyanine iodide) that is lipophilic and accumulates in mitochondria with active membrane potential and fluorescent emission increases due to dye stacking. The stain intensity decreases when agents disrupt mitochondrial membrane potential (12). Cells stained with DiOC2(3) can be visualized by flow cytometry with excitation at 488 nm and green (FL-1, 515 nm) or red emissions (FL-3, >600 nm) filters according to the manufacturer's instructions. This method allows to quantify the cells with depolarized mitochondria. Briefly, after trypsinization step, the cells were washed once in PBS and incubated with DiOC2(3) dye at 37°C for 30 min. The cells were washed twice and re-suspended in PBS for flow cytometric analysis (10 000 events per sample) and histograms were analyzed by the CellQuest Software (FACScan, BD Biosciences).

Flow cytometric analysis of Bcl-2 and Bax expression
Cells were harvested by trypsinization (0.5% trypsin/2.6 mM ethylenediaminetetraacetic acid) at different time periods (24, 48 and 72 h) after initial treatment with lupulones (40 µg/ml) and proceeded for the fixation and permeabilization step with the BD Cytofix/CytopermTM kit (BD Biosciences). According to the manufacturer's instructions, pellet was re-suspended on fixation/permeabilization solution for 20 min at 4°C and after washing twice in 1x BD Perm/WashTM buffer; cells were incubated with antibodies for the detection of Bcl-2 or Bax proteins. For Bcl-2 protein detection, cells were labeled directly with FITC-conjugated mouse anti-human Bcl-2 monoclonal antibody or FITC-conjugated mouse IgG1 monoclonal isotype control antibody (BD Biosciences). For Bax protein detection, cells were incubated with rabbit anti-human Bax polyclonal antibody (BD Biosciences) for 30 min at 4°C in the dark. After washing twice, FITC-conjugated swine anti-rabbit F(ab’)2/FITC antibody was added (Abcam plc, Cambridge, UK) for 30 min at 4°C. After washing twice in BD buffer, the fluorescence of 10 000 cells was analyzed using a FACScan flow cytometer (488 nm, FL-1: 515 nm) and CellQuest software (FACScan, BD Biosciences).

Analysis of Fas receptor and Fas ligand expression
For Fas receptor (Fas) and Fas ligand (FasL) detection, cells were stained with FITC-conjugated mouse anti-human Fas (CD95) monoclonal antibody (clone DX2) or with biotin-conjugated mouse anti-human FasL (clone NOK-1) antibody (BD Biosciences) for 45 min at 4°C according to manufacturer's instructions. For FasL detection, cells were washed twice with PBS and incubated with streptavidin–phycoerythrin (BD Biosciences) for 45 min at 4°C in the dark. FITC-conjugated mouse IgG1 (BD Biosciences) or phycoerythrin-conjugated mouse IgG1 (Immunotech Coulter, Marseille, France) monoclonal isotype control antibodies were used for isotype control (13). For cytometry analysis, cells were washed twice and re-suspended in PBS. FL-1 filter (515 nm) was used for Fas detection, whereas FasL expression required FL-2 filter (585 nm). The fluorescence of 10 000 events per sample was analyzed using FACScan flow cytometer and CellQuest Software (FACScan, BD Biosciences).

Detection of TNF-related apoptosis-inducing ligand receptors R1, R2, R3 and R4
Cell pellets were washed twice with PBS and incubated with FITC-conjugated mouse anti-human TNF-related apoptosis-inducing ligand (TRAIL) receptors from R1 to R4 monoclonal antibodies (Alexis Biochemicals, Lausen, Switzerland) or FITC-conjugated mouse IgG1 monoclonal isotype control antibody (BD Biosciences) for 30 min at 4°C in the dark. After the washing step, cells were re-suspended in PBS and the fluorescence (FL-1: 515 nm) of 10 000 events per sample was analyzed by flow cytometer and CellQuest Software (FACScan, BD Biosciences).

Animals and treatments
All animal experiments were performed in accordance with the institutional guidelines of the French Ethical Committee (authorization no. A67-480, French Ministry of Agriculture). Male Wistar rats (n = 18) obtained from Charles River Laboratories (Les Oncins, France) and weighing 240–250 g were housed under standardized conditions (22°C, 60% relative humidity, 12 h light/12 h dark cycle, 20 air changes/h) and fed a standard diet with free access to drinking water. All animals received intra-peritoneal injections of azoxymethane (AOM) (Sigma–Aldrich), 15 mg/kg body wt, once a week for 2 weeks. One week after the last injection of AOM, rats were randomly separated into two experimental and one control groups. The first experimental group (n = 6) received daily at 5 PM a freshly prepared mixture of either 0.001% of lupulones and 0.001% ascorbic acid in drinking water containing 0.5% ethanol, and the second experimental group (n = 6) received daily 0.005% lupulones under the same conditions. The AOM-treated control group (n = 6) received drinking water with 0.5% ethanol and 0.001% ascorbic acid. Ascorbic acid was used to protect lupulones from oxidation. Rats consumed daily 30–34 ml of the drinking fluid during the whole experimental period. The mean daily intake of lupulones per rat in the two experimental groups was, respectively, of 0.8 and 4 mg/kg.

Assessment of aberrant crypts and tumors in the colon
All animals were killed 7 months after the last AOM injection. The entire colon was collected, washed with saline buffer and cut out flat with the mucosa on the upper side in order to record the number, size and localization of tumors. The tumor volume was evaluated at autopsy based on the formula V = 4/3{pi} x (A/2)2 x B/2, where A is the width of the tumor and B the length in millimeters.

The determination of aberrant hyperproliferative crypts was performed on a 6 cm segment in length corresponding to the distal part of the colon. The segment was fixed in 10% buffered formalin and stained with 0.2% methylene blue for 5 min, rinsed in Krebs–Ringer buffer, placed onto a glass slide and examined microscopically using a low-power objective (x5) for the assessment of the number of hyperproliferative crypts and aberrant crypt foci (ACF). The criteria for the identification of hyperproliferative aberrant crypts were (i) an increased size, (ii) a thicker epithelial cell lining and (iii) an increased pericryptal zone relative to normal crypts.

Statistical analysis
Data are reported as mean ± SE. Statistical differences between control and treated groups were evaluated using the Student's t-test or the Student–Neuman–Keuls multiple comparison test. Differences between groups are considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of lupulones on SW620 cell growth and death
SW620 cells were exposed for 72 h to lupulones at concentrations varying from 0 to 60 µg/ml. As shown in Figure 2A, cell growth inhibition was observed in a dose-dependent manner. At 10 µg/ml, lupulones induced a 40% inhibition of cell growth after 48 h of exposure and 50% inhibition after 72 h. At 40 µg/ml, cell growth inhibition reached 70% at 48 h and 80% at 72 h. As shown in Figure 2B, lupulones at 40 and 60 µg/ml, respectively, increased the number of total dead cells with values varying from 18–20% at 48 h to 30–45% at 72 h. All further experiments with lupulones were performed at a concentration of 40 µg/ml.


Figure 2
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  		Fig. 2. Effect of lupulones on SW620 cell growth. Cells were exposed to different concentrations of lupulones varying from 0 to 60 µg/ml. Lupulones were diluted in DMSO and the final concentration of DMSO did not exceed 0.1% in the culture medium. At 0 µg/ml, cells were cultured in the presence of DMSO 0.1%. (A) Total number of cells (x106) after 48 and 72 h exposure to lupulones. (B) Percent of total dead cells obtained after 48 and 72 h of treatment. Data are the mean ± SE of at least three separate experiments. Statistical differences: a != b != c != d, P < 0.05.

 
Lupulones induced apoptosis in SW620 cells
We measured phosphatidylserines externalization by annexin-V–FITC staining. Figure 3 shows that phosphatidylserines relocalization in SW620 cells started after 48 h of treatment with lupulones. The population of apoptotic cells was significantly (P < 0.05) enhanced in lupulone-treated cells (15% at 48 h and 34% at 72 h) versus non-treated cells (4% at 48 h and 7% at 72 h).


Figure 3
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  		Fig. 3. Lupulones induces apoptosis in SW620 cells. Cells were treated with DMSO 0.1% (control) or lupulones 40 µg/ml for 48 and 72 h. At each time point, cells were stained with annexin-V–FITC and analyzed by flow cytometry as described in Materials and Methods. Apoptosis is expressed as the percent of cells positive to staining (R1) corresponding to phosphatidylserine externalization. Results are presented as cytometer dot plots. Data in the table are the mean ± SE of at least three separate experiments. Comparisons concern data for the same time point. Statistical differences: *P < 0.05.

 
Mitochondrial membrane permeabilization and Bcl-2/Bax expressions
The intrinsic apoptotic pathway involves mitochondria dysfunction caused by mitochondrial membrane permeabilization due to a loss of membrane potential (14). Changes in mitochondrial membrane permeabilization were assessed by flow cytometry after staining the cells with DiOC2(3) reagent. This cationic cyanine dye penetrates cytosol and accumulates into the mitochondria. The intensity of DiOC2(3) stain decreases in cells treated with agents disrupting the inner mitochondrial membrane potential ({Delta}{Psi}m). As illustrated in Figure 4A, a significant (P < 0.05) reduction of mitochondrial membrane potential was observed after 48 h of treatment with lupulones (40 µg/ml).


Figure 4
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  		Fig. 4. Effects of lupulones on mitochondrial membrane potential and Bcl-2/Bax expression. SW620 cells were exposed to DMSO 0.1% (control) or to lupulones 40 µg/ml for 24, 48 and 72 h. (A) Percent of cells with reduced mitochondrial membrane potential ({Delta}{Psi}m): cells were stained with DiOC2(3) probe and analyzed by flow cytometry as described in Materials and Methods. Columns represent the percent of labeled cells with reduced red fluorescence corresponding to the percent of cells with reduced {Delta}{Psi}m. (B) Percent of cells expressing Bcl-2 and Bax proteins: cells were analyzed by flow cytometry after permeabilization and staining with anti-Bcl-2–FITC antibody or with anti-Bax polyclonal and anti-F(ab’)2–FITC antibodies. Changes on green fluorescence were measured. Data are the mean ± SE of at least three separate experiments, and for each time point, columns not sharing the same superscript letter are statistically different (a != b != c, P < 0.05).

 
In the intrinsic-apoptotic pathway mediated by mitochondria, two members of the Bcl-2 family proteins, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax, are dominant regulators of apoptosis. Bcl-2 protein prevents mitochondrial integrity and blocks the release of apoptotic factors, whereas Bax induces disruption of mitochondrial integrity and favors the release of pro-apoptotic factors by forming a pore in association with other Bcl-2 family members (15,16). The ratio of Bcl-2 to Bax may determine cell death or cell survival (17). Changes in Bcl-2 and Bax expressions were evaluated by flow cytometry. Cells treated with lupulones (40 µg/ml) showed significant (P < 0.05) changes in Bcl-2 and Bax protein expression when compared with non-treated cells (Figure 4B). Bcl-2:Bax ratio remained, however, constant in cells exposed to lupulones with a mean value of RBcl-2/Bax = 2 ± 0.1, indicating that the expression of the anti-apoptotic Bcl-2 protein is 2-fold higher than the expression of Bax protein in the lupulones-treated cells.

Fas and FasL proteins expression
The extrinsic apoptotic pathway involves the activation of cell membrane death receptors, including Fas and TRAIL receptors (18). As shown in Figure 5, SW620 cells treated with the vehicle (DMSO 0.1%) did not express Fas, whereas in the presence of lupulones 40 µg/ml, Fas expression was increased in a time-dependent manner. To determine whether FasL was involved in the process, the FasL protein expression was determined. As for its receptor, FasL expression was only increased in lupulones-treated cells and the effect was also time dependent (Figure 5).


Figure 5
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  		Fig. 5. Lupulones treatment enhanced Fas and FasL expression. SW620 cells were treated with DMSO 0.1% (control) or lupulones 40 µg/ml for 24, 48 and 72 h. At different time points, cells were harvested, and Fas and FasL protein expressions were analyzed by flow cytometry after staining with FITC–anti-Fas or with phycoerythrin–anti-FasL antibodies. Data are presented as cytometer histogram plots and the shift on the right corresponds to an increase of Fas/FasL expression. Isotype control and control cells had the same profile (data not shown). Dot plots are representative of at least three different experiments.

 
Lupulones enhance TRAIL-R1 and -R2 receptors expression
Four major members of TRAIL receptors (R1–R4) have been identified. TRAIL-R1 and -R2 are also called DR4 and DR5, respectively, and by binding their specific ligands induce an apoptotic signal (19). As illustrated in Figure 6, treatment of SW620 cells with lupulones stimulated mainly TRAIL-R1 (DR4) and -R2 (DR5) proteins expression with a prominent effect on DR5. The expression of DR5 reached its maximum after 24 h of treatment and remained at the same level throughout the treatment period.


Figure 6
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  		Fig. 6. Analysis of TRAIL receptor expression. Cells were treated with DMSO 0.1% (control) or lupulones 40 µg/ml for 24, 48 and 72 h. TRAIL receptor expressions were analyzed by flow cytometry. At each time point, cells were harvested and stained with FITC-conjugated monoclonal antibodies against the four types of TRAIL receptors (from R1 to R4). Increased green fluorescence was measured and data are represented by histograms as the percent of cells expressing TRAIL receptors. Data are the mean value ± SE of at least three separate experiments. For each time period, columns not sharing a common superscript letter are statistically different, a != b != c != d, P < 0.05.

 
Animal experiments
Colon carcinogenesis was induced in rats by intra-peritoneal injections of the chemical carcinogen AOM once a week for 2 weeks. One week after the last injection rats received lupulones (0.001 or 0.005%) in their drinking fluid. After 7 months, the mucosal surface of the colon of rats receiving lupulones showed a significant reduction (P < 0.05) in the number of preneoplastic lesions. The number of ACF was reduced by 50% in rats receiving lupulones at 0.005% and by 30% in rats administered with 0.001% lupulones (Figure 7A) when compared with the AOM control group. In addition, we found that treatment with lupulones caused a significant reduction (P < 0.05) in the number of ACF with a large size. Indeed, the colon of AOM controls exhibited 60% of ACF containing four and more individual aberrant crypts whereas 45% of the ACF contained four and more aberrant crypts in lupulone-treated rats. However, no significant differences were observed with the two doses of lupulones.


Figure 7
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  		Fig. 7. Chemopreventive effects of lupulones in an experimental model of colon carcinogenesis. (A) Number of ACF in the distal colon (6 cm in length) in AOM control group and in rats receiving 0.001 or 0.005% lupulones (LP) in the drinking fluid. Values are the mean ± SE. Columns not sharing a common superscript letter differ significantly (P < 0.05). (B) Number and size (in mm3) of tumors found at autopsy 7 months after AOM injection in each control or LP-treated rat.

 
Figure 7B shows the number and size of tumors present in the colon of each animal. Striking differences were observed in the number of tumors present in the colon of AOM control and lupulones-treated rats. In the AOM control group, a total number of seven tumors were recorded, whereas only two tumors were present in the group receiving 0.001% lupulones and one tumor was detected for the group treated with 0.005% lupulones. In the AOM control group, two of six rats were found tumor free, whereas in the experimental groups receiving 0.001 and 0.005% lupulones, respectively, four of six and five of six rats were found free of tumors.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we report that lupulones induced apoptosis in SW620 cells. In order to get more insight into the mechanism of action, we studied the effects of lupulones on several effectors directly involved in the intrinsic and extrinsic apoptotic pathways. We demonstrate that lupulones up-regulated the expression of Fas and FasL as well as TRAIL-R1 (DR4) and -R2 (DR5) receptor proteins, suggesting the involvement of Fas and TRAIL receptors-mediated death pathways in lupulones-induced SW620 cell death. In addition, we showed that lupulones increased mitochondrial membrane permeability in association with an enhanced expression of two members of the Bcl-2 family proteins (Bcl-2 and Bax).

One previous study, realized with a mixture of hop bitter acids containing {alpha}- and ß-acids, has also reported that apoptotic cell death of HL60 and U937 cells was caused by mitochondrial perturbation and caspases cascade activation with implication of the Fas/FasL system (5). In our study, we used a purified fraction containing only lupulones (without humulones and oxidation-derived products) and showed that lupulones induced programmed cell death in SW620 metastatic cells through the activation of both intrinsic and extrinsic apoptotic pathways.

Concerning the intrinsic pathway, we measured the expression of two members of the Bcl-2 family. The Bcl-2 protein is an anti-apoptotic member that controls mitochondrial permeabilization and inhibits apoptosis (20). The Bax protein is a pro-apoptotic factor associated to mitochondria, and its enhanced expression may counterbalance Bcl-2 protein effect and promote apoptosis (21). In our study, even if apoptosis was induced by lupulones, both Bcl-2 and Bax proteins were enhanced but the Bcl-2:Bax protein ratio remained constant with a 2-fold increase in Bcl-2 versus Bax proteins. This suggests that Bcl-2 may not be a determinant factor in regulating lupulones-induced apoptosis (22,23). The observed mitochondrial permeabilization may in fact be a downstream consequence of the activation of the extrinsic apoptotic pathway. This is in agreement with the observed enhanced expression of both Fas and FasL after treatment with lupulones. Engagement of the Fas/FasL system results in the clustering of intracellular death domains of Fas leading to caspase-8 activation. Caspase-8 may activate not only the effector caspase-3 but also a pro-apoptotic member of Bcl-2 family proteins, the Bid protein which in turn induces mitochondrial membrane permeabilization (24,25). Thus, the observed loss of mitochondrial membrane potential resulting in organite damage after lupulones treatment may therefore be triggered by Fas/FasL system mediated by caspase-8 and Bid protein.

Lupulones enhanced also the expression of the TRAIL pro-apoptotic receptors R1 and R2. These receptors, also called DR4 and DR5, are implicated in the activation of apoptosis (26). The metastatic SW620 cells are resistant to TRAIL-induced apoptosis (27). Therefore, our observation that lupulones activated the expression of TRAIL receptors DR4 and DR5 has potentially a very high interest. Indeed, more and more studies are focused on the discovery of new drugs that are able to activate DR4 and DR5 expression. DR4 and DR5 upon linking their ligands, selectively induce apoptosis of variety of tumor and transformed cells independently of their p53 status, but not in most normal cells, and their implication has gained intense interest as a promising pathway for cancer therapy (18,19,26,28). Indeed, the SW620 cells are mutated for p53 (codon 273) and we show that lupulones are able to overcome resistance to apotosis in these cells through a p53-independent pathway. This could be of clinical relevance because most of the colorectal tumors bear a p53 mutation. This strategy seems very accurate since it was reported that TRAIL-resistant breast cancer cells can be efficiently killed by over-expression of DR4 even in the absence of exogenous TRAIL (29). Furthermore, TRAIL is expressed on different cells of the immune system and plays a role in both T-cell and natural killer cell-mediated tumor suppression (26). Here we present evidence that lupulones may represent a new drug able to activate TRAIL death receptors and improve therapeutic design.

The carcinogen-induced model used in the present study is well established. Most published studies were done in rats injected with AOM. In this model, the induced tumors share many histopathological and genetical characteristics with sporadic colon tumors in humans (30). AOM initiates a multistep carcinogenic process that transforms the normal colonic epithelia into a carcinoma with an adenomatous polyp as an intermediate step in the process (31). The distribution of carcinogen-induced colorectal tumors resembles human colon carcinoma where the great majority of tumors (80%) are recorded in the distal half of the colon (32–34). In humans, colon carcinogenesis is a long, chronic process that is thought to take up 10–20 years (35). In the AOM-induced rat model, preneoplastic lesions (aberrant crypts) appear 3–4 weeks after carcinogen injection, and the first tumors can be detected by 6 months (36), making this model very attractive for the screening of potential chemopreventive agents. We induced colon carcinogenesis by intra-peritoneal injection of AOM (once a week during 2 weeks). One week after the second injection, two groups of rats received daily, respectively, 0.001 and 0.005% lupulones in their drinking fluid. The amount of lupulones administered corresponded, respectively, to 0.8 and 4 mg/kg/day. No toxic effects in rats were observed with these two doses of lupulones, and rats treated with lupulones showed a similar body weight gain and food consumption during the whole experimental period than AOM controls. Furthermore, even a dose of 150 mg/kg was reported in animals to be the no-observed-adverse-effect level for hop acids, indicating that these substances have a wide safety margin (37). We observed 7 months after initiation of colon carcinogenesis a 30% reduction in the number of ACF present on the mucosal surface of the colon in rats receiving 0.001% lupulones in their drinking fluid as compared with the AOM control group. The number of ACF was reduced by 50% in the group receiving 0.005% lupulones. The observed reduction of the number and size of preneoplastic lesions was accompanied by a drastic reduction (70–80%) of the total number of tumors in rats consuming lupulones when compared with the AOM control group.

Our present data highlight for the first time the potent anticarcinogenic properties of lupulones. These hop ß-acids induce apoptosis in SW620 colon-derived metastatic cells by activating both Fas and TRAIL death receptor signaling, and exhibit at a low dose (4 mg/kg/day) potent antitumor effects. These observations suggest the use of lupulones for colon cancer chemoprevention trials.


   Acknowledgments
 
V.L. is supported by a doctoral studentship provided by the Conseil Régional d'Alsace. The authors thank J.-Y.Malpote and J.-F.Doriat (Brasseries Kronenbourg, Strasbourg, France) for supplying the industrial by-product containing high amounts of ß-acids.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 12, 2007; revised March 29, 2007; accepted April 2, 2007.


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