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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Food Chem Toxicol. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2888903
NIHMSID: NIHMS103309

Hop proanthocyanidins induce apoptosis, protein carbonylation, and cytoskeleton disorganization in human colorectal adenocarcinoma cells via reactive oxygen species

Abstract

Proanthocyanidins (PCs) have been shown to suppress the growth of diverse human cancer cells and are considered as promising additions to the arsenal of chemopreventive phytochemicals. An oligomeric mixture of PCs from hops (Humulus lupulus) significantly decreased cell viability of human colon cancer HT-29 cells in a dose-dependent manner. Hop PCs, at 50 or 100 μg/ml, exhibited apoptosis-inducing properties as shown by the increase in caspase-3 activity. Increased levels of intracellular reactive oxygen species (ROS) was accompanied by an augmented accumulation of protein carbonyls. Mass spectrometry-based proteomic analysis in combination with 2-alkenal-specific immunochemical detection identified β-actin and protein disulfide isomerase as major putative targets of acrolein adduction. Incubation of HT-29 cells with hop PCs resulted in morphological changes that indicated disruption of the actin cytoskeleton. PC-mediated hydrogen peroxide (H2O2) formation in the cell culture media was also quantified; but, the measured H2O2 levels would not explain the observed changes in the oxidative modifications of actin. These findings suggest new modes of action for proanthocyandins as antitumorgenic agents in human colon cancer cells, namely, promotion of protein oxidative modifications and cytoskeleton derangement.

Keywords: Acrolein, actin, apoptosis, proanthocyanidins, protein carbonyls, HT-29 cells

1. Introduction

Proanthocyanidins (PCs) or condensed tannins are mixtures of oligomers and polymers composed of flavan-3-ol units found in fruits, vegetables, cereals, seeds, nuts, bark, spices, cocoa, and beverages (Rasmussen et al., 2005). PCs are of increasing interest because of their potential health benefits and cancer chemopreventive properties (Dixon et al., 2005; Prior and Gu, 2005). The potential chemopreventive properties of PCs against colon cancer has been demonstrated in mice dosed with 1,2-dimethylhydrazine (Gali-Muhtasib et al., 2001) and in rats treated with azoxymethane (Singletary and Meline, 2001). PCs isolated from various fruits have been shown to suppress growth and to induce apoptosis in colon cancer cells (Engelbrecht et al., 2007; Hostanska et al., 2007; Lizarraga et al., 2007; Seeram et al., 2006).

The anticarcinogenic effects of PCs may be explained by various mechanisms such as free radical scavenging activity, regulation of signal transduction pathways by altered expression of key enzymes such as protein kinases, cell cycle arrest, suppression of oncogenes, induction of apoptosis, modulation of enzyme activity related to carcinogen detoxification, inhibition of DNA synthesis, and stimulation of DNA repair (Aron and Kennedy, 2008). The induction of apoptosis in colon cancer cells by grape seed PCs was correlated with the inactivation of the PI3-kinase/PKB pathway (Engelbrecht et al., 2007).

Hops (Humulus lupulus) may be tapped as a rich source of PCs for the chemoprevention of colon cancer. Hop PCs are mixtures of oligomers that consist of monomer units such as catechin, epicatechin, gallocatechin, epigallocatechin, afzelechin, and epiafzelechin (Li and Deinzer, 2006). These PCs also contain unique dimers such as prodelphinidins with gallocatechin units and the propelargonidin dimer, afzelechin-(4 → 8)-catechin (Li and Deinzer, 2006; Taylor et al., 2003).

One of the properties that makes PCs particularly attractive in the chemoprevention of colon cancer is that the PC concentrations may reach low mM (< 3) concentrations in the colon (Scalbert and Williamson, 2000) and retention in the colon may give these polyphenolics the potential to function as local anti-colon cancer agents. In the colon, the microbial flora may convert the PCs into products that may have improved cell membrane permeability and anti-cancer activities. We have examined the growth inhibitory effect of PCs from hops in the human colon cancer cell line, HT-29, and investigated the possible molecular mechanisms underlying these effects. The present study shows that hop PCs are cytotoxic to HT-29 colorectal adenocarcinoma cells through formation of ROS, leading to protein carbonylation and to cytoskeleton disorganization.

2. Materials and methods

2.1. Materials

5-(and -6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2 DCFDA), rhodamine phalloidin and biotinylated anti-DNPH rabbit polyclonal antibody were obtained from Molecular Probes (Eugene, OR). The fluorogenic caspase-3 substrate, Ac-DEVD-AMC [N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin)], was purchased from EMD Biosciences, Inc. (San Diego, CA, USA). 2,4-Dinitrophenylhydrazine (DNPH), epigallocatechin gallate (EGCG), and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were obtained from Sigma-Aldrich (St. Louis, MO). Coomassie Plus Protein assay kit, Laemmli sample buffer, pre-cast 10-well 12% Tris-HCl gels, prestained SDS-PAGE standards, Coomassie Blue G250 solution, and goat anti-mouse IgG-HRP conjugate were obtained from Bio-Rad Laboratories (Hercules, CA). Monoclonal anti-acrolein IgG and monoclonal anti-HNE IgG were purchased from COSMO BIO CO., LTD. (Carlsbad, CA) and Oxis International, Inc. (Foster City, CA), respectively. Monoclonal anti-β actin IgG and anti-PDI IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). SuperSignal West Pico Chemiluminescent Substrate was obtained from Pierce Biotechnology, Inc. (Rockford, IL). All other test chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

2.2. Cell culture

HT-29 cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown in 75 cm2 tissue culture flasks in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. The cell cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2.

2.3. Cytotoxicity assay

The cells were harvested from the culture flasks and plated on a 96-well microtiter plate, with each well containing 0.2 ml of complete phenol-red free RPMI 1640 medium per well. After 24 h of incubation at 37 °C in 5% CO2, the medium was replaced with fresh phenol-red free and serum-free RPMI 1640 medium containing hop PC mixture at increasing concentrations (10, 50, and 100 μg/ml). The composition of this PC mixture was described in previous work (Stevens et al., 2002). These concentrations of the PC mixture were chosen on the basis of previous cytotoxicity studies of grape seed proanthocyanidins conducted in HT-29 cells (Kaur et al., 2006). Vehicle-treated control cells were fed RPMI medium containing methanol (used for dissolving the hop PC mixture) not to exceed 0.1%. After 24 h, cell viability was measured using the MTT assay as described by Chung et al. (2007). This assay was based on the reduction of a tetrazolium dye to a blue formazan by functioning mitochondria.

2.4. Caspase-3 activity

Caspase-3 activity was used to examine the effect of PCs on apoptosis in HT-29 cells using the method described by Chung et al. (2007). HT-29 cells were seeded at a density of 5 × 105 per well onto 6-well plates. After 24 h of incubation, the cells were fed serum-free RPMI media containing 0.1% methanol or hop PC mixture at various concentrations. The cells were incubated for another 24 h incubation before harvesting in caspase-3 buffer (25 mM HEPES, pH 7.4, 10% glycerol, and 1 mM EDTA) using a cell scraper. The cells were lysed by sonication and then centrifuged at 10,000 × g for 5 min. The resulting supernatant was used to measure caspase-3 activity with the fluorogenic caspase-3 tetrapeptide-substrate, Ac-DEVD-AMC, at a final concentration of 30 μM. according to the manufacturer's instructions. Fluorescence intensity of control and treated wells was measured by a microplate fluorescence reader (SpectraMax Gemini, Molecular Devices, Sunnyvale, CA) with excitation wavelength at 380 nm and emission wavelength at 460 nm.

2.5. Detection of ROS in HT-29 cells

The formation of intracellular ROS in HT-29 cells was detected by the use of CM-H2DCFDA. This dye is cell membrane-permeable and is cleaved by non-specific esterases to remove the acetyl groups inside the cell to form CM-H2DCF which is then oxidized to a fluorescent product by ROS. Cells were cultured in serum-free RPMI medium in the presence and absence of increasing concentrations of hop PC mixture. After a 24-h incubation at 37 °C, the cells were washed twice with phenol red-free and serum-free RPMI medium and then incubated for 20 min at 37 °C in phenol red-free RPMI with 10 μM CM-H2 DCFDA. The cells were then washed of excess extracellular dye, trypsinized and washed with PBS before examination by fluorescence microscopy.

2.6. Measurement of hydrogen peroxide in cell culture media

The PC mixture (10, 50 or 100 μg/ml) was added to phenol red-free and serum-free RPMI 1640 media with or without HT-29 cells. After a 24-h incubation at 37 °C in 5% CO2, the media were analyzed for H2O2 content by a ferrous ion oxidation-xylenol orange method using the PeroXOquantTM Quantitative Peroxide Assay kit (Pierce) according to the manufacturer's directions. The samples were transferred to 96-well plates and the absorbance of the samples was read at 595 nm using a Microplate reader (SpectraMax 190).

2.7. Determination of protein carbonyls by ELISA

Protein carbonylation of control and PC-treated HT-29 cells was determined by an ELISA according to the method of Buss and Winterbourn (2002). Briefly, 96-well high-binding ELISA plates were coated overnight at 4 °C with aliquots (200 μl/well, containing 1 μg of protein) of test samples or of increasing concentrations of oxidized BSA standards (Zenith PC Test Kits). Prior to loading, samples and BSA standards were first reacted with 3 volumes of 10 mM DNPH in 6 M guanidine hydrochloride in 0.5 M phosphate buffer, pH 2.5, for 45 min at room temperature. The plates were washed with PBS and then incubated with PBS containing 0.1% Tween 20 (PBS-T). The plates were extensively washed with PBS and then incubated with 200 μl of biotinylated anti-DNPH rabbit polyclonal antibody per well. Following a 30-min incubation at room temperature, the wells were washed with PBS and then 200 μl of Streptavidin-HRP was added to each well. After 1 h of incubation, the wells were washed and 200 μl of TMB + Substrate-chromogen (Dako, Carpinteria, CA) was added to each well. After a 5-min incubation, the reaction was stopped by adding 100 μl of 2.5 M sulfuric acid. The absorbance was read at 450 nm using a Microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA).

2.8. SDS-PAGE and Western blots

HT-29 cells were treated with PC mixture (0, 10, 50, or 100 μg/ml) and after 24 h, the cells were harvested and collected by centrifugation at 200 × g for 3 min. The cell pellet was resuspended in PBS containing 0.5% Triton X-100, vortexed vigorously and stored at -80 °C for 20 min. The cells were thawed, vortexed, and then centrifuged at 10,000 × g for 10 min. The resulting supernatant was analyzed for protein content by using the Coomassie Plus Protein assay kit. Aliquots (20 μg) of the samples were mixed with Laemmli sample buffer prior to loading onto a 10-well 12% Tris-HCl gel with prestained SDS-PAGE protein standards. After electrophoresis, the proteins were transferred to a nitrocellulose membrane for Western blot analysis as described by Chung et al. (2007) using anti-acrolein IgG, anti-HNE IgG, anti-β actin IgG, or anti-PDI IgG as probes. Proteins were detected using the chemiluminescence reagent, SuperSignal West Pico Chemiluminescent Substrate, and the resulting immunoreactive protein bands were visualized on X-ray film.

To identify the proteins recognized by these probes, parallel gels were stained with IEF gel staining solution. The bands of interest were excised and the protein bands were digested with trypsin and desalted with Ziptip C18. The extracted peptides were analyzed by tandem mass spectrometry using a MALDI-TOF/TOF instrument.

2.9. Two-dimensional (2D) Electrophoresis

Proteins (100 μg) were dissolved in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.2% (v/v) biolytes, 100 mM dithiothreitol (DTT), and 0.001% bromophenol blue) and sonicated for 10 min before applying to a Ready Strip IPG (pH 3–10). The strips were then actively rehydrated at 50 V for 15 h in a protean IEF cell (Bio-Rad). Isoelectric focusing (IEF) was performed at 20 °C as follows: 250V for 15 min, 8,000 V for 2.5 h rapid gradient and 35,000 V·h. Prior to second dimension separation, gel strips were equilibrated for 10 min in 375 mM Tris– HCl (pH 8.8) containing 6 M urea, 2% (w/v) sodium dodecyl sulfate, 20% (v/v) glycerol, and 2% DTT. The strips then were equilibrated for 10 min in the same buffer containing 2.5% iodoacetamide in place of DTT. Gradient criterion Tris–HCl gels (8-16%, Bio-Rad) were used to perform second dimension electrophoresis. Prestained protein standards were run along with the sample at 130 V for 115 min. After electrophoresis, the proteins were transferred to a nitrocellulose membrane and blocked with 5% non-fat milk in TBS (Tris-buffered saline) for 2 h at room temperature. The membrane was probed with anti-acrolein IgG. The membrane was washed 6 times with TBS-T (TBS with 0.1% tween-20) and then incubated with goat anti-mouse IgG-HRP conjugate. After washing, the membrane was incubated with SuperSignal West Pico Chemiluminescent Substrate for 5 min. The acrolein-positive bands were then visualized on X-ray film.

2.10. Identification of immunoreactive proteins by tandem mass spectrometry

To identify the proteins recognized by anti-acrolein antibody, a parallel gel was stained with Coomassie Blue G250 solution for 2 h at room temperature. The gel was destained with water. The bands or protein spots of interest were excised. The protein bands or spots were digested with trypsin and desalted with Ziptip C18 (Millipore, ZTC18S096). The peptides were analyzed by MALDI tandem mass spectrometry as described by Chavez et al. (2006).

2.11. Visualization of actin cytoskeleton by fluorescence microscopy

HT-29 cells were grown in a 35 mm petri dish with a 14 mm microwell coverglass (Mat Tek Corporation, Ashland, MA) and treated with 0, 10, 50 or 100 μg/ml PC mixture for 24 h. After treatment, the cells were washed twice with PBS and then fixed with 3.7% formaldehyde solution in PBS for 10 min. After washing with PBS, the cells were incubated with 0.1% Triton X-100 for 5 min. The cells were again washed with PBS and then stained with rhodamine phalloidin (5 units/ml) in PBS. The cells were washed with PBS and then examined by using a fluorescence microscope with a 40× objective and a video camera for recording the images.

2.12. Statistics

Unpaired Student's t-test was performed to assess the differences between groups. Differences in means between treatment groups were considered statistically significant if P < 0.05. All values are given as mean ± SE.

3. Results

3.1. Effect of hop PCs on HT-29 cell viability

Treatment of HT-29 cells with the mixture of PCs isolated from hops produced a significant dose-dependent decrease in cell viability as measured by the MTT assay (Fig. 1). Cell viability of HT-29 cells treated with 10, 50, and 100 μg/ml PC mixture were 104, 75.2, and 47.2 of control value. The cytotoxicity of the PC mixture was not significantly affected by the addition of catalase (100 units/ml) to the culture medium (Fig. 1), indicating that the observed effect was not due to H2O2 generated by autooxidation of the polyphenols in the cell culture medium.

Fig. 1
Percent viability of HT-29 cells treated with increasing concentrations of hop PCs in the presence and absence of catalase (100 units/ml) in the culture medium

3.2. H2O2 content of RPMI media

PCs as many other antioxidants are prone to oxidation in diverse cell culture media resulting in the formation of H2O2 and semiquinones/quinones (Long et al., 2000). The chemical composition of PCs varies from source to source. Hop PCs differ from grapeseed or tea PCs by the absence of gallate esters and the presence of PC units with one OH group at the B-rings. The effect of hop PCs on H2O2 formation and acrolein adduction to cellular proteins has not previously been studied. Because H2O2 formation was found to be associated with higher levels of acrolein adduction to proteins, we determined the levels of H2O2 in the culture media in the presence and absence of HT-29 cells as well as the intracellular levels of H2O2.

Both fresh and incubated phenol red-free RPMI 1640 media contained 1.1 μM H2O2 (Fig. 2A). Incubation of RPMI 1640 with the PC mixture in the absence of cells for 24 h at 37 °C caused a dose-dependent increase in the levels of H2O2, reaching up to 50 μM in the culture media treated with 100 μg/ml PCs. When the RPMI 1640 medium was added to cells and incubated for 24 h at 37 °C in the absence of PCs, the amount of H2O2 in the culture medium was 0.56 μM. In the presence of cells, the H2O2 concentration determined after 24 h was 2.94 μM for the media containing 100 μg/ml PCs. These findings suggest that the hop PCs induced the formation of H2O2 in the medium and that the cells' antioxidant enzymes (i.e. catalase, glutathione peroxidase and peroxiredoxins) are capable of efficiently removing the PC-generated H2O2 in concordance with a study of Antunes and Cadenas (2000). The addition of H2O2 (3 -100 μM) to the culture medium with cells did not cause a significant increase in H2O2 content of the culture medium after 24 h of incubation (Fig. 2B).

Fig. 2
(A) H2O2 production in fresh RPMI 1640 media (no cells, not incubated) and in media with or without cells incubated with 0 (control), 10, 50 or 100 μg/ml PCs for 24 h at 37 °C. Values are mean ± SE, n = 4. *P < 0.05 vs. ...

3.3. Effect of hop PCs on caspase-3 activity

Caspase-3-like activity of the cytosol fraction of HT-29 cells pretreated with 50 or 100 μg/ml PCs was significantly increased by 3- and 4-fold, respectively, compared to control cells (Fig.3). No significant increase in caspase-3 activity was observed at 10 μg/ml PCs.

Fig. 3
Effect of hop PCs on caspase-3 activity of HT-29 cells. The cells were exposed to PC for 24 h before measuring caspase-3 activity of the cytosol fraction of HT-29 cells using Ac-DEVD-AMC as a substrate

3.4. Effect of hop PC mixture on intracellular ROS levels

Intracellular ROS was detected in HT-29 cells using CM-H2 DCFDA which is oxidized to the highly fluorescent DCF. Exposure to 50 or 100 μg/ml PCs caused an increase of ROS levels in treated cells as reflected by increased fluorescence intensity of the treated as compared to untreated control cells (Fig. 4). However, this assay does not allow deciphering if the ROS detected in cells were generated intracellularly or extracellularly. The results reported in Figure 2 show that there is a PC dose-dependent increase of H2O2 levels in the medium and that the extracellular H2O2 levels are lower in the presence of cells. These data clearly indicate that the medium is a prominent source of H2O2 formation and that extracellular H2O2 may migrate into cells across the cell membrane (Antunes and Cadenas, 2000). However, intracellular H2O2 formation due to PCs taken up by the cells cannot be excluded.

Fig. 4
Effect of treatment of HT-29 cells with hop PCs on intracellular ROS levels as measured by fluorescence microscopy after staining the cells with 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate

3.5. Effect of hop PCs on protein carbonyl content

Whole cell homogenates of control and PC-treated HT-29 cells were examined for protein oxidative damage using anti-DNP antibody in ELISA. Fig. 5A shows a significant increase in protein carbonyls of HT-29 cells with increasing concentrations of hop PC mixture over control levels. Treatment of HT-29 cells with 10, 50 or 100 μg/ml PCs resulted in 14-, 20- and 22-times higher protein carbonyl levels with respect to the control cells. Exposing the HT-29 cells to bolus additions of 15 and 50 μM H2O2 resulted in no significant elevation of protein carbonyl levels; whereas exposure to 100 μM H2O2 resulted in marked increase in protein carbonyl levels (Fig. 5B).

Fig. 5
(A) Protein carbonyl content of HT-29 cells incubated with 10, 50 or 100 μg/ml PCs for 24 h at 37 °C. Values are mean ± SE, n = 4. *P < 0.05 vs. control group. (B) Protein carbonyl content of HT-29 cells incubated with ...

3.6. Identification of β-actin and PDI as the major targets of PC-induced oxidative stress in HT-29 cells

To determine if PCs treatments of HT-29 cells affect protein levels, we separated whole protein cell lysates on SDS-PAGE (Fig. 6A), excised protein bands and subjected the gel pieces to trypsin digestion. Resulting peptide extracts were subjected to MALDI-TOF/TOF tandem mass spectrometry for obtaining peptide sequencing data and the proteins were identified using the MASCOT search engine. The levels of the following proteins appeared to be lower in the PC-treated cells (lanes 3, 50 μg/ml PC mixture; and lane 4, 100 μg/ml PC mixture, Fig. 6A) than in the controls (lane 1): myosin-9, heat shock protein 90-α and β, α-enolase, 14-3-3 protein gamma, and triosephosphate isomerase. In contrast, the levels of α-actin 1 (band no. 2) were higher in cells treated with 50 (lane 3) or 100 μg/ml (lane 4) PC mixture than in the controls (lane 1). Other proteins showed no obvious change in their cellular concentration in the SDS-PAGE and they were identified as follows: 78 kDa glucose regulatory protein; heat shock protein 71; protein disulfide isomerase (PDIA3); cytoplasmic 1 actin (β-actin; Fig. 6A, band no.8,); keratin type I; keratin type II; glyceraldehyde 3-phosphate dehydrogenase; 14-3-3 protein gamma; and glutathione S-transferase. The mass spectrometry data of the identified proteins are summarized in Table 1.

Fig. 6
SDS-PAGE (A) and Western blot using anti-acrolein antibody (B), anti-HNE antibody (C), anti-β-actin antibody + anti-PDI antibody (D) of cell lysates from HT-29 cells treated with 10, 50, and 100 μg/ml PC for 24 h, and of cell lysates from ...
Table 1
Mass spectrometry data of proteins identified in the gel regions 1 to 14 (Figure 6) and 1 to 4 (Figure 7).

To further examine if the elevated protein carbonyls levels are, at least in part, related to oxidative modifications caused by reactive lipid peroxidation products, we used 2-alkenal specific antibodies in Western blot analyses. Proteins that were recognized by anti-acrolein antibody in Western blots were protein disulfide isomerase, β-actin, and keratin type I (Fig. 6B). The levels of immunoreactive β-actin detected by anti-acrolein antibody were markedly increased by treatment of the cells with 50 or 100 μg/ml PC mixture. PDI, with a monomeric molecular weight of 55 kDa, was also detected by anti-acrolein IgG in these PC-treated cells but not in control cells (Fig. 6B). The levels of immunoreactive keratin type I were not altered by treatment of the cells with the PC mixture.

In Western blots probed with anti-HNE antibody, the levels of an immunoreative band corresponding β-actin were highly increased in HT-29 cells treated with 50 or 100 μg/ml PC mixture (Fig. 6C). No immunoreactive band was detected by anti-HNE in the region of the PDI (Fig. 6C).

The nitrocellulose membrane used in Figure 6B was re-probed with anti-β-actin monoclonal antibody, followed by anti-PDI IgG and the resulting blot is shown in Fig. 6D. Only one protein band corresponding to β-actin was detected by the anti-β-actin antibody in control and PC-treated cells. When the same blot was re-probed with anti-PDI, a second band appeared corresponding to PDI. The levels of β-actin and PDI proteins appeared to be unchanged after treating the cells with the PC mixture (Fig. 6D). HT-29 cells treated with 50 or 100 μM H2O2 for 24 h did not show an increase in immunoreactive β-actin detected by anti-acrolein antibody (Fig. 6E).

To unequivocally confirm the formation of oxidatively modified β-actin, protein samples used in the 1D electrophoresis were separated by 2D electrophoresis (Fig. 7). Tandem mass spectrometry of the tryptic peptides extracted from the excised spots indicated that β-actin was present in multiple spots and that treatment of HT-29 cells with 100 μg/ml hop PCs resulted in increased diversity and levels of post-translationally modified β-actin proteins.

Fig. 7
Coomassie Blue G250-stained gels (A, control HT-29 cells and B, HT-29 cells treated with 100 μg/mL PCs and the respective Western blots (right top and right bottom panels) probed with anti-acrolein IgG

Western blot analysis of the 2D gel with anti-acrolein antibody indicated that several of the modified β-actin proteins stained highly positive in HT-29 cells treated with 100 μg/ml PCs (Fig. 7B) but were only appearing as faint spots in the control sample (Fig. 7A).

In addition, the 2D Western blot analysis confirmed unequivocally PDI as an additional target of acrolein adduction. In the center panel, two spots labeled as 4 were seen in control (Fig. 7A) and PC-treated cells (Fig. 7B) and these spots were identified as PDIA3. The two PDIA3 spots were recognized by anti-acrolein IgG in the PC-treated cells (right lower panel, Fig. 7B) but not in the control cells (right upper panel, Fig. 7A). The mass spectrometry data used for the identification of β-actin and PDIA3 in the 2D-gel are included in Table 1.

3.7. PC-induced disruption of the actin cytoskeleton

Chemical modification of β-actin has been previously linked to filament misfolding and cytoskeleton disorganization (Allingham et al., 2006). We therefore examined the organization of the HT-29 cytoskeleton before and after treatment with hop PCs. The actin filaments were stained with phalloidin covalently linked to rhodamine and visualized using a fluorescence microscope. In control cells, the actin filament showed a ring-like organization at the cell periphery whereas in PC-treated cells, actin appeared in clusters as shown by the increased fluorescence intensity in the affected cells (100 μg/ml PC vs. control cells, Fig. 8). Cluster appearance of actin has also been reported by Feick et al. (2006) after treating HT-29 cells with formaldehyde. The cells also appear to be aggregated but this is normal for HT-29 cells if these cells are grown on a petri dish or in a culture flask before becoming confluent. The aggregation of HT-29 cells has been described previously (Feick et al., 2006).

Fig. 8
Effect of hop PCs on actin cytoskeleton organization in HT-29 cells. Control cells and cells treated with 10, 50 or 100 μg/ml PCs for 24 h were stained with rhodamine phalloidin and examined by fluorescence microscopy (with 40× power objective) ...

4. Discussion

The present study revealed that hop PCs are cytotoxic to HT-29 colon cancer cells and their toxicity was associated with elevated levels of intracellular ROS, increased formation of protein carbonyls and cytoskeletal disorganization.

Many earlier studies have shown that PCs from diverse polyphenols can act either as antioxidants or prooxidants depending on the cell type and concentrations used (Hadi et al., 2007; Oikawa et al., 2003; Sakano et al., 2005). For example, (epi)catechin caused oxidative DNA damage, as indicated by the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H2O2)-resistant cell line derived from HL-60 (Oikawa et al., 2003). Similarly, procyanidin B2 (epicatechin-(4beta-8)-epicatechin) inhibited the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in HL-60 cells treated with an H2O2-generating system, but high concentration of procyanidin B2 (> 500 μg/ml) increased the formation of 8-oxodG in these cells (Sakano et al., 2005). Several studies indicate that at least some of the tumoricidal activities of EGCG can be attributed to the cytotoxicity of H2O2 generated during the oxidation of polyphenols to the semiquinone and quinone derivatives, a reaction promoted by the presence of superoxide radical anion (Chan et al., 2006; Yamamoto et al., 2004). For HT-29-18-C1 cells, the median lethal dose for one h exposure to H2O2 was approximately 0.1 mM (Watson et al., 1994). Whereas, a bolus addition of 30 μM H2O2 directly to the culture media did not affect cell viability of HT-29 cells (Klenow et al., 2008).

Polyphenolic compounds get readily oxidized in cell culture media to semiquinones/quinones, whereby H2O2 is released into the cell medium (Halliwell et al., 2000; Halliwell, 2003; Lambert et al., 2007). It is therefore conceivable that H2O2 generated in the cell culture medium may at least partly contribute to the observed cytotoxicity and intracellular effects attributed to polyphenols exposure. In our study, the addition of the PC mixture (100 μg/ml) and 24-h incubation at 37 °C resulted in H2O2 concentrations in RPMI 1640 media of 50 μM and 2.9 μM in the absence and presence of HT-29 cells, respectively. A previous study by Cadenas and colleagues (Antunes and Cadenas, 2000) demonstrates that the rapid enzymatic consumption of H2O2 inside the cell results in a steep H2O2 gradient across the plasma membrane after a bolus addition of H2O2 and low intracellular concentration of H2O2. Therefore, the cells' arsenal of H2O2-consuming enzymes and capability of regulating membrane permeability for H2O2 will affect the intracellular effects of polyphenolics. In our hands, the presence of catalase (100 units/ml) in the culture medium did not affect the cytotoxicity of the PC mixture on the HT-29 cells (Fig. 1). We also tested the impact of different concentrations of H2O2 on protein carbonyl levels. These experiments revealed that protein carbonyl levels (Fig. 5) as well as acrolein adduction to β-actin (Fig. 6E) were unaffected by bolus additions of H2O2 up to 50 μM. The H2O2 content of culture media with cells was not significantly increased after a 24-h incubation with 100 μM H2O2. Therefore, we concluded that low amounts of H2O2 generated in the media in the presence of cells were unlikely responsible for the cytotoxic effects observed for the hop proanthocyanidin mixture tested.

Measurement of intracellular DCF fluorescence showed that treatment of HT-29 cells with hop PCs led to increased intracellular levels of ROS (Fig. 4). During condition of oxidative stress reduced glutathione (GSH) is converted into glutathione disulfide (GSSG) and, consequently, GSH may get depleted. Glutathione peroxidase converts H2O2 and lipid hydroperoxides to water and lipid alcohols, respectively, whereby GSH provides the reducing equivalents (Doroshow et al., 1990). In addition, GSH readily forms adducts with a great variety of both endogenous and exogenous electrophiles. In our system, it is conceivable that reactive lipid peroxidation products, such as acrolein and HNE, as well as phenolic semiquinone/quinones contribute to GSH consumption. Maintenance of GSH/GSSG homeostasis is critical for cell survival. Depletion of GSH triggers apoptotic pathways and we demonstrate a dose-dependence of caspase-3 like activity. Disturbance of the cellular redox balance results in increased protein oxidative insults.

We measured total carbonyl levels using a DNPH-based ELISA and found that hop PCs induce the formation of protein carbonyls in HT-29 cells. We performed proteomic analysis (1D or 2D gel electrophoresis followed by mass spectrometry) to gain an insight into the nature of the proteins that were targets of oxidative stress-related modifications. Protein carbonylation reactions are chemically diverse; beside the direct oxidation of amino acid side chains to semialdehydes (Requena et al., 2001), the adduction of nucleophilic sites to α,β-unsaturated lipid peroxidation products contributes to elevated carbonyl levels (Aldini et al., 2007b). With the use of anti-acrolein IgG as a probe, we found that β-actin and PDI were the major targets of protein adduction by acrolein in HT-29 cells exposed to hop PCs (Figs. 6 and and7).7). The increase in β-actin-acrolein adduct formation is not the result of increased levels of β-actin protein because β-actin levels were not altered by PC treatment as shown in the Western blot probed with anti-β-actin antibody (Fig. 6D). HNE also contributed to the carbonylation of β-actin as shown by the Western blot probed with anti-HNE IgG (Fig. 6C). Our finding is in agreement with previous studies which showed that β-actin is a major target of protein carbonylation as in gerbil synaptosomes treated with acrolein (Mello et al., 2007), human neuroblastoma SH-SY5Y cells treated with 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (Aldini et al., 2007c), and Alzheimer's disease brain (Butterfield et al., 2002).

Adduction of acrolein to β-actin may result in a disruption of the actin cytoskeleton in cells treated with the PC mixture. To test this hypothesis, we exposed cells to various concentrations of the PC mixture for 24 h and then examined the actin cytoskeleton organization by fluorescence microscopy using a rhodamine-phalloidin stain. Treatment of the cells with PC caused a disorganization of the ring-like cytoskeleton structure which was most evident in cells treated with 50 and 100 μg/ml PC (Fig. 8). Disruption of the actin cytoskeleton structure has been observed in human gingival fibroblasts treated with acrolein (Poggi et al., 2002) and in HT-29 cells exposed to 1 mM formaldehyde for 1 h (Feick et al., 2006). Disorganization of the actin cytoskeleton was also seen in bovine lung microvascular endothelial cells treated with HNE (25 μM) for 30 min (Usatyuk et al., 2006). Thus, alterations in the cytoskeleton as a result of increased carbonylation of β-actin by acrolein and HNE may be one possible mechanism of cell death induced by oxidative damage in HT-29 cells treated with hop PCs.

Acrolein has a propensity to undergo Michael addition with cysteine, histidine, and lysine and is considered the most reactive of the α,β-unsaturated aldehydes produced by lipid peroxidation (Aldini et al., 2007a; Aldini et al., 2007b; Esterbauer et al., 1991; Uchida, 1999). In in vitro studies employing G-actin, Cys-374 was identified as the site of carbonylation by acrolein (Aldini et al., 2007b; Aldini et al., 2007c; Dalle-Donne et al., 2007). 2-Alkenals are bifunctional electrophiles and Michael type-addition would allow for secondary reactions, such as Schiff's base formation, resulting in protein crosslinks. Noteworthy, the 2D gel electrophoretic maps indicated that in PC-treated HT-29 cells β-actin was found to be extensively modified as evident in the large number of spots visible in the 2D gel. Clearly, other modifications occurred beside the adduction to the two identified lipid peroxidation products, acrolein and HNE. Possible oxidative modifications may include hydroxyl radical-triggered side chain oxidations, direct oxidation of cysteine thiols, S-glutathionylation, S-alkylation involving polyphenolic semiquinones/quinones and adduction to other reactive lipid peroxidation products, e.g. 15d-PGJ2, a reactive α,β-unsaturated cyclopentenone derived from arachidonic acid via the cyclooxygenase pathway with particular relevance to colorectal adenoma carcinomas (Gayarre et al., 2006). Thus, further work is needed to evaluate to what extent the increased formation of β-actin-acrolein and β-actin-HNE adducts contribute towards the disruption of the actin cytoskeleton and the cytotoxic activity of hop PCs.

PDI-acrolein adduct formation in HT-29 cells treated with the PC mixture was evident in the Western blots probed with anti-acrolein antibody (Fig. 6B and Fig. 7B, right bottom panel). PDI is an endoplasmic reticulum chaperone which promotes correct disulfide formation, thereby participating in the maturation of newly synthesized proteins. An active site cysteine residue of PDI has been shown to be modified by both HNE and acrolein resulting in the inhibition of its enzyme activity (Carbone et al., 2005). The reduced form of PDI can be inactivated by acrolein (IC50 = 10 mM) at pH 6.3, and to a lesser extent, at pH 7 (Liu and Sok, 2004). However, to which extent PDI inactivation contributes to possible mechanisms by which hop PCs exert their cytotoxic effects in HT-29 cells would need further investigation.

ROS generated from other polyphenolics such as epicatechin and EGCG have been shown to induce apoptosis and inhibit the growth of cancer cells (Azam et al., 2004; Chan et al., 2006) which may explain the anticancer activities of these compounds. In HT-29 cells, the antiproliferative and pro-apoptotic effects of EGCG have been attributed to increased oxidative stress in the treated cells (Chen et al., 2003). Other compounds, such as alpha-lipoic acid, have been shown to induce apoptosis in HT-29 cancer cells by a prooxidant mechanism (Wenzel et al., 2005a). There is a large body of evidence that a variety of cancer cells essentially operate under oxidative stress. Colorectal adenoma carcinoma cells express high levels of Nox1, a superoxide generation NADPH oxidase (Laurent et al., 2008), and cyclooxygenases (Antonakopoulos and Karamanolis, 2007). In rapidly growing cancer cells, metabolism is characterized by limited oxidative phosphorylation in order to provide protection from reactive oxygen species (ROS)-mediated cellular damage during phases of DNA replication and high biosynthetic activities (Wenzel et al., 2005b). In contrast in normal cells, the cellular redox status is tightly controlled and levels of ROS are counterbalanced by enzymatic and non-enzymatic antioxidant defense systems. Accordingly, the use of phytochemicals that increase ROS production and overburden the antioxidative capacity may drive cancer cells ultimately into apoptosis. The differential sensitivity of cancer cells to oxidative stress has been considered to be useful in alternative chemoprevention and chemotherapy strategies (McEligot et al., 2005). The low systemic bioavailability of PCs is an advantage when used in the prevention of intestinal/colon cancers in which the cancerous tissues could be exposed to high levels of PCs after ingestion. Unabsorbed PCs will go through the colon where the PCs may come in direct contact with the cancer cells and reach.high micromolar concentrations at the cancer site.

Acknowledgments

We thank Ms. Deborah J. Hobbs, Linus Pauling Institute, Oregon State University, for the analysis of protein carbonyls, and Ms. Tamara Fraley, Environmental Health Sciences Center and Department of Biochemistry and Biophysics, Oregon State University, for conducting the fluorescence microscopy studies. This study was supported by grants from the Agricultural Research Foundation. Parts of the proteomic methods were developed with the help of a grant from the NIH/NIA (R01 AG025372). The Mass Spectrometry core and services facility and the Cell Culture facility of the Environmental Health Sciences Center at Oregon State University are supported in part by NIH/NIEHS P30 ES00210.

Abbreviations

Ac-DEVD-AMC
N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin)
CM-H2 DCFDA
5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate
DCF
dichlorofluorescein
DNPH
2,4-dinitrophenylhydrazine
DTT
dithiothreitol
EGCG
epigallocatechin gallate
GSH
glutathione
HNE
4-hydroxy-2-nonenal
HRP
horseradish peroxidases
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PBS
phosphate buffered saline
PI3-kinase
phosphatidylinositol 3-kinase
PCs
proanthocyanidins
PDI
protein disulfide isomerase
PKB
protein kinase B
ROS
reactive oxygen species
TMB
3,3′-5,5′ tetramethylbenzidine

Footnotes

Conflict of interest statement: There are no conflicts of interest.

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