英-细胞系GSPE

gra ay an yse C. bosch Received 16 April 2007; received in revised form 22 August 2007; accepted 31 August 2007 Keywords: PI3-K; PKB/Akt; Apoptosis; Colon cancer result of increasing genomic instability that leads tion, differentiation and metabolism [8–11]. Disrup- tion of normal PI3-K signaling has been documented as a frequent occurrence in several human cancers and appears to play an important role in their progression [12,13]. The PI3-K signal- ights reserved. * Corresponding author. Tel.: +27 21 8084573; fax: +27 21 8083145. E-mail address: ame@sun.ac.za (A.-M. Engelbrecht). Available online at www.sciencedirect.com Cancer Letters 258 (2007 0304-3835/$ - see front matter � 2007 Elsevier Ireland Ltd. All r 1. Introduction Colorectal cancer is a leading cause of cancer death in men and women, and affects more than one million people worldwide every year [1]. Colon cancer develops through several stages and is mani- fested as an increasing mass of cells, driven by pro- liferation, but potentially caused by the malignant cells failing to die. These features usually occur pro- gressively over a protracted period of time as a to up-regulation of oncogenes and down-regulation of tumour suppressor genes [2–4]. Research over the past few years has shown that major intracellular signaling pathways are altered during tumourigene- sis and that this can lead to dysregulation of pro- cesses such as proliferation and survival [5–7]. The serine/threonine protein kinase, protein kinase B (a member of the PI3-K pathway), is a crucial regu- lator of widely divergent cellular processes including apoptosis (programmed cell death), cell prolifera- Abstract The aim of this investigation was to evaluate the chemopreventative/antiproliferative potential of a grape seed proanth- ocyanidin extract (GSPE) against colon cancer cells (CaCo2 cells) and to investigate its mechanism of action. GSPE (10–100 lg/ml) significantly inhibited cell viability and increased apoptosis in CaCo2 cells, but did not alter viability in the normal colon cell line (NCM460). The increased apoptosis observed in GSPE-treated CaCo2 cells correlated with an attenuation of PI3-kinase (p110 and p85 subunits) and decreased PKB Ser473 phosphorylation. GSPE might thus exert its beneficial effects by means of increased apoptosis and suppression of the important PI3-kinase survival-related pathway. � 2007 Elsevier Ireland Ltd. All rights reserved. Proanthocyanidin from PI3-kinase/PKB pathw in a colon c A.-M. Engelbrecht *, M. Matthe R. Smith, S. Peters, Department of Physiological Sciences, Stellen doi:10.1016/j.canlet.2007.08.020 pe seeds inactivates the and induces apoptosis cer cell line , B. Ellis, B. Loos, M. Thomas, Smith, K. Myburgh University, Stellenbosch 7600, South Africa ) 144–153 www.elsevier.com/locate/canlet vinifera) seeds. Polymeric and oligomeric proanth- A.-M. Engelbrecht et al. / Cancer Letters 258 (2007) 144–153 145 ocyanidins, also called condensed tannins, are one type of polyphenol and consist of chains of fla- van-3-ol units, (+)-catechin, and (�)-epicatechin, linked through C4–C6 and C4–C8 interflavan bonds. Furthermore, oligomeric proanthocyani- dins are the only macromolecular constituent pres- ent in grape seed extract, which contains nearly equal amounts of monomeric catechin and epicat- echin chains [15]. GSPE induces apoptosis in human prostate can- cer cells without affecting the growth and viability of the normal cells [16]. The cytotoxic effects of GSPE have also been demonstrated in a variety of other cancer cell lines [17,18]. Malik and co-workers [19] demonstrated a 60% inhibition of growth of human HT-29 colon cancer cells with GSPE. The above- mentioned studies did not assess molecular mecha- nisms involved in the cytotoxic effect of GSPE on cancer cells; thus the intracellular mechanisms responsible remain to be elucidated. We hypothesize that altered phosphorylation events in the PI3-K pathway may mediate the beneficial effects reported for GSPE. 2. Material and methods 2.1. Materials CaCo2 cells, Eagle’s minimum essential medium (MEM), fetal bovine serum (FBS), trypsin and penicil- lin/streptomycin were obtained from Highveld Biological (Lyndhurst, RSA). NCM460 cells were purchased from InCell Corporation (San Antonia, TX, USA) to serve as control culture. Cell culture plastics were purchased from Greiner Bio-one (Frickenhausen, Germany). Dimethyl sulfoxide (DMSO) was purchased from Merck (Darms- tadt, Germany). Rabbit polyclonal antibodies and rabbit monoclonal antibodies were purchased from Cell Signal- ing pathway should therefore be considered as a potential target for chemotherapy. For a therapeutic agent to be effective, it should be able to kill cancer cells without harming normal cells. Grape seed proanthocyanidin extract (GSPE) may be such an agent. Proanthocyanidins are naturally occurring polyphenolic bioflavonoids diverse in chemical structure, pharmacology and characteristics and widely available in vegetables, fruits, seeds, nuts, flowers and bark [14]. Grape seed proanthocyanidins (monomers 12–16%; dimers 9–17%, and oligomers 40–45%) refer to procyanidin mixtures extracted from grape (Vitis ing Technology (Beverly, Massachusetts, USA). All elec- trophoresis equipment was from Bio-Rad (Herculus, CA, USA). Polyvinylidene fluoride (PVDF; 0.45 lm) membranes for Western blots were obtained from Milli- pore (Bedford, Massachusetts, USA). The chemilumines- cence system (ECL Plus and ECL), autoradiography film as well as anti-rabbit horseradish peroxidase (HRP)- linked secondary antibody (from donkey) were obtained from Amersham Biosciences (Arlington Heights, IL, USA). 2.2. Cell culture CaCo2 and NCM460 cells were maintained at 37 �C in a humidified 5% CO2 atmosphere in MEM supplemented with 10% FBS and 1% penicillin/streptomycin (standard medium). Cells were routinely sub-cultured before reach- ing confluency. Cell numbers were determined using a haemocytometer following trypsinization. For 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays, CaCo2 and NCM460 cells were seeded in 35 mm Petri dishes (120,000 and 350,000, respectively). For Western blotting, CaCo2 cells were seeded in 35 mm (120,000) and 60 mm Petri dishes (240,000), respectively. 2.3. Treatment of cells with GSPE Cells were incubated for 24 h with 0, 10, 50, and 100 lg/ml GSPE. Following treatment, the medium was removed and the cells were washed with phosphate buf- fered saline (PBS). For controls, the cells were incubated with medium and vehicle alone. 2.4. Cell viability assessment For the assessment of cell viability a modification of the MTT assay described by Gomez and co-workers was used [20]. The assay is based upon the principle of reduc- tion of MTT into blue formazan pigments by viable mito- chondria in healthy cells. At the end of the experiment, the medium was removed from the Petri dishes and the cells washed twice with PBS. MTT (0.01 g/ml) was dis- solved in PBS, and 500 ll was added to each Petri dish. Cells were subsequently incubated for 2 h at 37 �C in an atmosphere of 5% CO2. This time period was found to be optimal for color development associated with forma- zan product formation. After the incubation period, cells were washed twice with PBS, and one ml of HCl–isopro- panol–Triton (1% HCl in isopropanol; 0.1% Triton X- 100; 50:1) was added to each Petri and gently agitated for 5 min. This caused the cell membranes to lyse and lib- erate the formazan pigments. The suspension was then centrifuged at 131g for 2 min. The optical density (OD) was determined spectrophotometrically at a wavelength of 540 nm and the values expressed as percentages of con- trol values. 2.5. Morphological analysis of cell death For fluorescence staining, CaCo2 cells were incubated with Propidium iode (PI), (5 mg/ml, was added in a 1:200 dilution and incubated for 20 min at room temperature) and Hoechst 33342 (10 mg/ml was additionally added in a 1:200 dilution and incubated for 10 min at room temper- 2.6. Western blot analysis Tween 20 (TBST) and then incubated with the primary antibodies that recognize phospho-specific and total PTEN, PKB Ser473, PKB Thr308, CREB, FKHR, BAD as well as PI3-K p85 and p110, caspase-3 (p17 fragment pAb) and PARP (116 kDa and p85 fragment pAb). Membranes were subsequently washed with large volumes of TBST (5 · 5 min) and the immobilized anti- a particular blot. These analyses were performed under ANOVA was performed for each group of treatments, rosis. re sta of a ining xpres 146 A.-M. Engelbrecht et al. / Cancer Letters 258 (2007) 144–153 Tissue kinases as well as caspase-3 and poly (ADP- ribose) polymerase (PARP) protein were extracted with a lysis buffer containing (in mM): Tris 20, p-nitrophe- nylphosphate 20, EGTA 1, sodium fluoride (NaF) 50, sodium orthovanadate 0.1, phenylmethyl sulphonyl fluo- ride (PMSF) 1, dithiothreitol (DTT) 1, aprotinin 10 lg/ml, leupeptin 10 lg/ml. The lysate protein content was determined using the Bradford technique [21]. The tis- sue lysates were diluted in Laemmli sample buffer and boiled for 5 min; 10 lg (for kinases) or 50 lg protein (for caspase-3 and PARP) was separated by electropho- resis. The separated proteins were transferred to a PVDF membrane (ImmobilonTM P, Millipore). These membranes were routinely stained with Ponceau Red for visualization of proteins and stripped and reprobed with anti-actin antibody to ensure equal loading. Non- specific binding sites on the membranes were blocked with 5% fat-free milk in Tris-buffered saline–0.1% Fig. 1. The effect of GSPE supplementation on apoptosis and nec and supplemented with GSPE (10–100 lg/ml) for 24 h. (a) Cells we and necrosis under fluorescent microscopy. (b and c) Quantification cells were counted under fluorescence microscopy after Hoechst sta calculation of arbitrary pixel values after PI staining. Results are e ature). Images were acquired with a Nikon Eclipse E 400, equipped with the ACT-1 acquisition software, using a 40· Nikon Plan Fluor objective. The fluorescence images were visualized using a rhodamine filter (exitation at 530– 560 nm/emission at 580 nm) and a DAPI filter (exitation at 340–380 nm/emission at 430 nm), respectively. Cells were evaluated by the following grading system for apop- tosis and necrosis: normal nuclei exhibit blue chromatin with organized structure and apoptotic nuclei exhibit bright fluorescent blue chromatin which is highly con- densed or fragmented when stained with Hoechst. Necro- tic cells exhibit enlarged nuclei and stained red with PI. To evaluate necrosis, six randomly chosen cells per field, of 3 fields per experimental condition, were analyzed. Data expressed in arbitrary pixel values were exported to Microsoft excel and statistically analyzed. The apoptotic index [percentage of apoptotic cells] was calculated as number of the apoptotic cell/total cells counted · 100. Scoring was done blindly. versus control (n = 3). using Bonferroni’s post hoc test. p-values <0.05 were regarded as statistically significant. 3. Results 3.1. The effect of GSPE on cell viability in CaCo2 cells and normal colon epithelial cells (NCM 460 cells) To establish whether GSPE could induce cell death in the cancer cell model without harming normal cells, the MTT cell viability assay was used. Different concen- trations of GSPE [10–100 lg/ml] were used to evaluate the effect on the two cell lines. No significant differences in viability were detected when concentrations of 10, 50, and 100 lg/ml GSPE were compared to the control group in the normal cell line. However, concentrations of 10, 50 and 100 lg/ml significantly reduced cell Cells were cultured in standard medium until 70–80% confluency ined with Hoechst 33342 (blue) and PI (red) to evaluate apoptosis poptosis and necrosis induced by GSPE. The number of apoptotic and expressed as percent of total cells. Necrosis was evaluated by sed as means ± SEM for three independent experiments, *p < 0.05 c conditions where autoradiographic detection was in the linear response range. 2.7. Statistical analysis All results are expressed as means ± SEM. One-way body conjugated with a diluted horseradish peroxidase- labeled secondary antibody (Amersham LIFE SCI- ENCE). After thorough washing with TBST, mem- branes were covered with ECLTM detection reagents and quickly exposed to an autoradiography film (Hyperfilm ECL, RPN 2103) to detect light emission through a non-radioactive method (ECLTM Western blotting). Films were densitometrically analyzed (UN- SCAN-IT, Silkscience) and phosphorylated protein values were corrected for minor differences in protein loading, if required. All blots were scanned at a resolu- tion of 150 dpi. The exact outline of each band was demarcated in the UN-SCAN-IT programme, which takes all aspects of density and distribution into account. The full experimental range was analyzed on 0 5 10 15 20 25 30 35 co ntr ol 10 µg /ml 50 µg /ml 10 0 µ g/m l % o f t ot al c el ls 0 20 40 60 80 100 120 co ntr ol 10 µg/ ml 50 µg/ ml 100 µg /ml Fl uo re sc en ce in te ns ity (ar bit ra ry pi xe l v alu es ) * * * * * A.-M. Engelbrecht et al. / Cancer Letters 258 (2007) 144–153 147 1a–c) ory ( gulato 148 A.-M. Engelbrecht et al. / Cancer Letters 258 (2007) 144–153 The Hoechst and PI staining techniques have been employed by many laboratories for distinguishing apop- totic and necrotic cell death [22,23]. In the present study, we observed that the nuclei of untreated cells viability in the cancer cell line compared to the control group [for 10 lg/ml (44.67 ± 2.84%), 50 lg/ml (45 ± 7.93%), and for 100 lg/ml (63.67 ± 2.72%) vs con- trol (100%)]. 3.2. The effect of GSPE on CaCo2 cell morphology (Figs. 0 co ntr ol 10 ug /m l 10 0 u g/m l Fig. 2. The effect of GSPE on the catalytic (p110) and the regulat Western-blotting using antibodies recognizing the catalytic and re three independent experiments, *p < 0.05 vs control (n = 3). 20 40 60 80 100 120 % o f c on tro l * p85 subunit of PI3-K exhibit loose chromatin, stained blue with Hoechst (Fig. 1a) and did not stain with PI. Treatment of cells with 10 lg/ml GSPE significantly increased apoptosis, but did not induce PI-positive nuclei. Concentrations of 50 and 100 lg/ml caused a significant increase in the apoptotic index as well as in PI-positive stained cells Fig. 1. 3.3. The effect of GSPE on the catalytic (p110) and regulatory (p85) subunits of PI3-K in CaCo2 cells (Fig. 2) It should be noted that all the subsequent biochem- ical assays were performed at the same time point as the cytotoxicity assay (24 h). At a low concentration, GSPE (10 lg/ml) induced a significant decrease in the endogenous levels of the regulatory (p85) subunits of PI3-K, but was unable to significantly reduce the level of the catalytic (p110) subunit. However, when a higher concentration (100 lg/ml) GSPE was used, the level of the catalytic subunit was reduced by approximately 50%. 3.4. The effect of GSPE on PTEN phosphorylation in CaCo2 cells (Fig. 3) GSPE (10 and 100 lg/ml) significantly decreased PTEN phosphorylation. 3.5. The effect of GSPE on PKB/Akt phosphorylation in CaCo2 cells (Fig. 4) Both concentrations of GSPE caused a significant decrease in Ser473 phosphorylation, whereas only 100 lg/ ml significantly inhibited Thr308 phosphorylation. co ntr ol 10 ug /m l 10 0 u g/m l * p110 subunit of PI3-K p85) subunits of PI3-K in CaCo2 cells. Samples were analyzed by ry subunits of PI3-K. Results are expressed as means ± SEM for 3.6. The effect of GSPE on CREB and FKHR phosphorylation in CaCo2 cells (Fig. 5) Both concentrations of GSPE significantly decreased CREB- and FKHR-phosphorylation. 3.7. The effect of GSPE on BAD phosphorylation in CaCo2 cells (Fig. 6) GSPE significantly inhibited BAD phosphorylation. 3.8. The effect of GSPE on apoptosis in CaCo2 cells (Fig. 7) GSPE significantly reduced uncleaved caspase-3. GSPE also induced a significant increase in PARP cleav- age compared with the control group. 4. Discussion The present study was undertaken to evaluate the chemopreventive/antiproliferative potential of A.-M. Engelbrecht et al. / Cancer Letters 258 (2007) 144–153 149 Total PTEN GSPE on a colon cancer cell line and to investigate its mechanism of action. Treatment with GSPE (10– 100 lg/ml for 24 h) resulted in a significant decrease in the viability of cancer cells while the viability of normal human colon epithelial cells was unaffected. 0 20 40 60 80 100 120 co ntr ol 10 ug/ m % o f c o n tr o l P-PTEN Fig. 3. The effect of GSPE on PTEN phosphorylation in CaCo2 cel recognizing phospho- and total PTEN. Results are expressed as mean (n = 3). 0 20 40 60 80 100 120 co ntr ol 10 ug /m l 10 0 u g/m l % o f c on tro l * Thr308 Phosphorylation Total Akt/PKB Thr308 S Fig. 4. The effect of GSPE on PKB/Akt phosphorylation (Thr308 and Se recognizing phospho- and total PKB/Akt. Results are expressed as mea (n = 3). Low and high concentrations of GSPE also induced morphological and biochemical features of apoptotic cell death (i.e., nuclear condensation, formation of apoptotic bodies, and caspase-3 and PARP cleav- age). However, concentrations of 50 and 100 lg/ml l 100 ug /ml * * ls. Samples were analyzed by Western-blotting using antibodies s ± SEM for three independent experiments, *p < 0.05 vs control co ntr ol 10 ug /m l 10 0 u g/m l * Ser473 Phosphorylation * re 473 r473). Samples were analyzed by Western-blotting using antibodies ns ± SEM for three independent experiments, *p < 0.05 vs control 150 A.-M. Engelbrecht et al. / Cancer Letters 258 (2007) 144–153 20 40 60 80 100 120 % o f c o n tr o l * * P-CREB Total CREB P-CREB also induced a number of morphological features of necrosis (e.g., plasmamembrane rupture and nuclear expansion). Tumour formation can be associated with the following characteristics: evasion of apoptosis, self sufficiency in growth signals, insensitivity to anti-growth signals, sustained angiogenesis, tissue invasion and metastasis, and limitless replicative potential [24]. It is notable that PI3-K signaling appears to be involved in at least the first five of the six above-mentioned characteristics [25]. PI3-K is an essential part of the signaling net- 0 co ntr ol 10 ug /m l 10 0 u g/m l Fig. 5. The effect of GSPE on CREB and FKHR phosphorylation in antibodies recognizing phospho- and total CREB and FKHR. Results a *p < 0.05 vs control (n = 3). 0 20 40 60 80 100 120 co ntr ol 10 ug /ml 10 0 u g/m l % o f c o n tr o l * * P-BAD Total BAD Fig. 6. The effect of GSPE on BAD phosphorylation in CaCo2 cells. Samples were analyzed by Western-blotting using antibod- ies recognizing phospho- and total BAD. Results are expressed as means ± SEM for three independent experiments, *p < 0.05 vs control (n = 3). work that blocks programmed cell death (apopto- sis) and enables cells to survive when they are in a favourable environment. These pathways are activated so that a continuous survival signal from growth factors is required to block apopto- sis [26]. Upon growth factor activation of receptor tyro- sine kinases, PI3-K is recruited to the receptor in the plasma membrane and phosphorylates phospha- tidylinositol-4,5-bisphosphate (PIP2) on the 3-OH group, generating phosphatidylinositol-3,4,5-tris- co ntr ol 10 ug /m l 10 0 u g/m l * * P-FKHR Total FKHR P-FKHR CaCo2 cells. Samples were analyzed by Western-blotting using re expressed as means ± SEM for three independent experiments, phosphate (PIP3). PI3-kinases are composed of a catalytic subunit (p110) and a regulatory subunit (p85). GSPE (10 lg/ml) significantly reduced the regulatory subunit (p85), while the high concentra- tion (100 lg/ml) significantly attenuated the cata- lytic subunit of PI3-K, both of which lead to a decrease in