Cyanidin-3-Glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2
© Xu et al; licensee BioMed Central Ltd. 2010
Received: 20 July 2010
Accepted: 29 October 2010
Published: 29 October 2010
Ethanol is a tumor promoter. Both epidemiological and experimental studies suggest that ethanol may enhance the metastasis of breast cancer cells. We have previously demonstrated that ethanol increased the migration/invasion of breast cancer cells expressing high levels of ErbB2. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis. We sought to identify agents that can prevent or ameliorate ethanol-induced invasion of breast cancer cells. Cyanidin-3-glucoside (C3G), an anthocyanin present in many vegetables and fruits, is a potent natural antioxidant. Ethanol exposure causes the accumulation of intracellular reactive oxygen species (ROS). This study evaluated the effect of C3G on ethanol-induced breast cancer cell migration/invasion.
C3G attenuated ethanol-induced migration/invasion of breast cancer cells expressing high levels of ErbB2 (BT474, MDA-MB231 and MCF7ErbB2) in a concentration dependent manner. C3G decreased ethanol-mediated cell adhesion to the extracellular matrix (ECM) as well as the amount of focal adhesions and the formation of lamellipodial protrusion. It inhibited ethanol-stimulated phosphorylation of ErbB2, cSrc, FAK and p130Cas, as well as interactions among these proteins. C3G abolished ethanol-mediated p130Cas/JNK interaction.
C3G blocks ethanol-induced activation of the ErbB2/cSrc/FAK pathway which is necessary for cell migration/invasion. C3G may be beneficial in preventing/reducing ethanol-induced breast cancer metastasis.
Excessive ethanol consumption is associated with an increased risk for breast cancer [1–5]. Epidemiological studies indicate that alcohol consumption is associated with advanced and invasive breast tumors [6, 7]. We have previously demonstrated that breast cancer cells or mammary epithelial cells expressing high levels of ErbB2 are sensitive to ethanol-mediated migration/invasion; ethanol stimulates migration/invasion of breast cancers with high ErbB2 levels more robustly than cells expressing lower levels of ErbB2 [8–10]. ErbB2 belongs to the ErbB family of receptor kinases which consists of EGFR, ErbB2, ErbB3 and ErbB4. Among the ErbB family, ErbB2 is most directly related to breast cancer and is implicated in breast cancer metastasis. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis and relapse [11, 12].
We sought to identify agents that may ameliorate ethanol's promoting effect on breast cancer cell migration/invasion. Cyanidin-3-glucoside (C3G) is a member of the anthocyanin family which is present in various vegetables and fruits, especially edible berries. C3G is a potent antioxidant and displays anti-cancer properties in vitro and in vivo[13–18]. Since ethanol exposure causes the accumulation of intracellular oxygen species (ROS) and many biological effects of ethanol are believed to be mediated by ROS, we hypothesize that C3G may inhibit ethanol-induced migration/invasion of breast cancer cells. We examined the effect of C3G on ethanol-mediated migration/invasion of breast cancer cells expressing high levels of ErbB2. We demonstrate here that C3G effectively blocks ethanol-induced cell migration/invasion. We further investigate the effect of C3G on the cell/extracellular matrix (ECM) interaction and the associated ErbB2/cSrc/FAK pathway.
Materials and methods
Human plasma fibronectin was obtained from Chemicon International (Temecula, CA). Anti-paxillin antibody was purchased from Invitrogen Corporation (Carlsbad, CA). Anti-phospho-ErbB2 (Tyr1248) (polyclonal), phospho-p130Cas and ErbB2 (polyclonal) antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA). Anti-Neu/Her2/ErbB2 (monoclonal), FAK, cSrc, JNK and phospho-Src (Tyr216) antibodies and Protein A/G beads were purchased from Santa Cruz Biotechnology (San Diego, CA). Anti-phospho-Her2/ErbB2 (Tyr1248) (monoclonal) and phospho-FAK (Tyr861) antibodies were purchased from Biosource (Camarillo, CA). Anti-p130Cas antibody was obtained from BD Transduction Laboratory (San Jose, CA). Anti-active JNK antibody was obtained from Promega Corporation (Madison, WI). Phalloidin 488, Alex Fluor-labeled secondary antibodies, Prolong Gold anti-fade reagent and reactive oxygen species detection reagents were obtained from Invitrogen Molecular Probes (Eugene, OR). MTT assay kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Matrigel Invasion Chambers were purchased from BD Biosciences (Bedford, MA). Transwell was obtained from Costar Corp. (Acton, MA). C3G was purified from blackberry fruit tissue as previously described . The purity of C3G is greater than 95%. Alcohol (200 Proof) was obtained from Fisher Scientific (Pittsburgh, PA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Cell culture and ethanol exposure
MCF7ErbB2 (MCF7 cells overexpressing ErbB2) and MDA-MB231 breast cancer cells were grown in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100 U/ml)/streptomycin (100 U/ml), 1 μg/ml hydrocortisone and 10 μg/ml insulin at 37°C with 5% CO2. BT474 cells were grown in RPMI 1640 medium containing 10% FBS, penicillin (100 U/ml)/streptomycin (100 U/ml) and 10 μg/ml insulin. A method utilizing sealed containers was used to maintain ethanol concentrations in the culture medium. The containers were placed in a humidified environment and maintained at 37°C with 5% CO2.
Cell invasion and migration
Cell invasion was assayed using Matrigel Invasion Chambers (BD Biosciences). Briefly, cells were placed on the upper compartment of invasion chambers and treated with ethanol in the presence or absence of C3G. Culture medium containing 10% FBS was added into the lower compartment of invasion chambers and served as chemoattractants for the cells. Cells were maintained in the invasion chambers for 48 hours. The invaded cells were fixed in 3.7% paraformaldehyde and stained with 0.5% crystal violet in 2% ethanol. Membranes were washed and the dye was eluted with 10% acetic acid. Absorbance was measured at 595 nm using a microtiter platereader (Beckman coulter).
Cell migration was analyzed using a Transwell Migration System (Costar). Briefly, cells were plated into upper chambers (Transwells with 8.0 μm pore size) in serum free medium. The lower compartment of the chamber contained regular medium containing 10% FBS. The chambers were cultured at 37°C in 5% CO2 for 12 hours. Migrated cells were fixed and stained with 0.5% crystal violet, followed by dye elution and absorbance measurement as described above.
Wound healing migration assay
The wound healing migration assay was performed as described previously . MDA-MB231 cells were grown on 35 mm dishes to 100% confluence and then scratched to form a wound using sterile pipette tips. The cells were then treated with ethanol (0 or 400 mg/dl) in the presence or absence of C3G (10 μM) for 24 hours. The images were recorded using a Zeiss Axiovert 40C photomicroscope.
Analysis of cell adhesion
Cell adhesion to fibronectin was analyzed as described previously [19–21]. Briefly, 96-well cell culture plates were precoated with fibronectin (10 μg/ml) for 60 min at 37°C. Plates were then incubated with 3% BSA in PBS for 30 min to block non-specific binding sites, followed by several washes with PBS. Cells were exposed to ethanol with/without C3G for 48 hours. After exposure, cells (5 × 104/well) were seeded on fibronectin precoated plates, allowing attachment for 1 hour at 37°C with 5% CO2. Non-adherent cells were removed by washing with PBS. The attached cells were fixed with 3.7% paraformaldehyde for 10 min, washed 3 times in PBS, and stained with 0.1% crystal violet in 2% ethanol for 10 min. Cells were rinsed with water and dried. Crystal violet was eluted in 10% acetic acid and the absorbance (attached cells) was measured at 595 nm using a microtiter platereader.
The MTT assay was employed to determine the number of viable cells in culture. Briefly, the cells were plated into 96-well plates and exposed to ethanol with/without C3G for indicated times. After the treatment, 10 μl of MTT reagent was added into each well and the plates were incubated at 37°C for 4 hours. The cultures were solubilized and spectrophotometric absorbance was measured at 595 nm using a microtiter platereader.
The procedure for immunofluorescence microscopy has been previously described . Briefly, after treatments, cells were seeded on fibronectin (10 μg/ml) precoated coverslips. Cells were fixed with 3.7% paraformaldehyde for 10 min, washed 3 times in PBS and permeabilized with 0.5% Triton X-100 for 5 min. Cells were blocked with 5% BSA and incubated with primary antibodies for 1 hour. The concentrations of primary antibodies were: phospho-FAK (Tyr861), 1:50; paxillin, 1:800; and phalloidin, 1:200. Following incubation with primary antibodies, cells were washed and treated with Alexa Fluor-labeled secondary antibodies and rinsed several times with PBS. Coverslips were mounted with Prolong Gold anti-fade reagent and immunofluorescence images were examined with a LEICA SP1 inverted confocal microscope. The fluorescent signals were measured with the same pinhole, detector gain and amplifier offset. The focal adhesions were detected by immunostaining for phosphorylated FAK and quantified randomly on 10 or more cells for each treatment condition.
Immunoprecipitation and immunoblotting
After the treatment of ethanol and/or C3G, cells were trypsinized and seeded on fibronectin (10 μg/ml) precoated dishes allowing attachment for indicated times. Cells were then rinsed twice in cold PBS to remove non-adherent cells. Attached cells were lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris, 1% NP-40, 0.25% sodium deoxycholate, 1 mM sodium vanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF), 5 μg/ml of aprotinin, and 2 μg/ml of leupeptin). The procedure for immunoprecipitation and immunoblotting has been previously described [10, 19]. Briefly, equal amounts of proteins (about 500-800 μg) were incubated with anti-ErbB2, FAK, p130Cas or cSrc antibodies overnight at 4°C, followed by treatment with TrueBlot anti-mouse Ig or anti-rabbit Ig beads (eBioscience, San Diego, CA) for 2 hours at 4°C. Immunoprecipitates were collected by centrifugation at 10,000 g for 5 min at 4°C. Samples were washed five times with RIPA buffer, one time with cold PBS and boiled in sample buffer (187.5 mM Tri-HCl, pH 6.8, 6% SDS, 30% glycerol, 150 mM DTT and 0.03% bromophenol blue). Proteins were resolved in SDS-PAGE and the separated proteins were transferred to nitrocellulose membranes. The membranes were probed with indicated primary antibodies, followed by the appropriate TrueBlot horseradish peroxidase-conjugated secondary antibodies and developed by enhanced chemiluminescence.
Detection of intracellular reactive oxygen species
Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent dye CM-H2DCFDA (Invitrogen Corporation, Carlsbad, CA) as previously described . CM-H2DCFDA is converted to a non-fluorescent derivative inside the cells and when oxidized forms a highly fluorescent product by intracellular ROS. Briefly, cells were treated with ethanol with/without C3G or other antioxidants for 48 hours. After the treatment, cells were washed with cold PBS and incubated with 5 μM CM-H2DCFDA for 30 min, followed by several additional washes with cold PBS. Cells were trypsinized and transferred into polystyrene round-bottom tubes; intracellular ROS levels were measured with a flow cytometer (FACScalibur, BD Biosciences, San Jose, CA) at an emission wavelength of 525 nm.
Differences among treatment groups were analyzed using analysis of variance (ANOVA). Differences in which p was less than 0.05 were considered statistically significant. In cases where significant differences were detected, specific post-hoc comparisons between treatment groups were examined with Student-Newman-Keuls tests.
C3G inhibits ethanol-enhanced migration/invasion and attachment of breast cancer cells
C3G attenuates ethanol-stimulated ErbB2 signaling
C3G inhibits ethanol-induced formation of lamellipodia and focal adhesions
C3G scavenges ethanol-induced accumulation of reactive oxygen species (ROS)
C3G as a potent agent to alleviate ethanol-induced cell migration/invasion
In search for better chemopreventive or chemotherapeutic agents, we isolated a natural antioxidant cyanidin-3-glucoside (C3G) from blackberries [14, 32]. C3G is a member of the anthocyanin family which is present in various vegetables and fruits, especially edible berries. We have confirmed C3G's antioxidant property [14, 23]. C3G has been implicated in some beneficial health actions including reducing age-associated oxidative stress, improving cognitive brain function, as well as anti-diabetic, anti-inflammation, anti-atherogenic and anti-obesity activity . C3G exhibits anti-cancer properties in various in vitro and animal models of carcinogenesis and tumor development; the effects of C3G include the inhibition of tumor cell proliferation and the attenuation of cell migration/invasion as well as metastasis in vivo[13–18].
We demonstrate here that C3G inhibits ethanol-mediated migration/invasion in cells expressing high levels of ErbB2. C3G has a greater inhibitory effect on the invasion of cells treated with 200 mg/dl ethanol compared to 400 mg/dl (Figure 1B); the underlying mechanism is unclear. C3G is effective at 10 μM, a concentration that is lower than previously reported for its anti-cancer effects. C3G inhibits the migration/invasion of A549 lung cancer cells at 40-100 μM [14, 34]. It is unlikely that C3G-mediated inhibition of tumor cell migration/invasion in this study results from decreased cell viability. C3G up to 40 μM does not affect the viability of breast cancer cells (Figure 1D). It is reported that C3G at 100 μM fails to reduce the viability of A549 lung cancer cells . However, at 20 μM, C3G significantly decreases the viability of HS578T human breast cancer cells . Thus, cells apparently display differential sensitivity to C3G. For BT474 cells, C3G does not have a dose-dependent inhibitory effect. It is likely BT474 cells are more sensitive to C3G and the concentration may need to be lower in order to see the dose-dependent inhibition.
Consistent with its effects on migration/invasion, C3G affects early events associated with cell motility. C3G inhibits ethanol-mediated cell adhesion to the ECM, formation of focal adhesions and development of lamellipodia. These events are prerequisites for cell migration/invasion. These results suggest that ethanol-induced migration/invasion is initiated by tumor cell/ECM interaction and C3G blocks this interaction.
C3G and ethanol-stimulated cell signaling
Ethanol-stimulated tumor cell/ECM interaction may be initiated by its effect on ErbB2 activity. Ethanol increases the phosphorylation of ErbB2 and enhances the adhesion of breast cancer cells with high levels of ErbB2 to the ECM as well as the assembly of focal adhesions in these cells . These effects were not observed in breast cancer cells expressing low levels of ErbB2.
FAK is a critical regulator of cell/ECM interaction and is strongly implicated in tumor aggressiveness [35, 36]. It has been shown that FAK is essential for ErbB2/ErbB3-induced oncogenesis and breast cancer invasion . The phosphorylation of FAK at Tyr 861 plays an important role in the invasion of breast cancer cells . FAK is a substrate of cSrc. The activation of ErbB2 recruits cSrc and FAK, resulting in the phosphorylation of cSrc at Tyr 216 and FAK at Tyr 861 in breast cancer cells [39, 40]. An ErbB2 inhibitor Tyrphostin (AG825) abolishes the ethanol-stimulated interaction between ErbB2 and FAK, as well as the adhesion of breast cancer cells to the ECM . It appears C3G targets ErbB2 since C3G is able to inhibit ethanol-mediated phosphorylation of ErbB2, cSrc and FAK. Additionally, it inhibits the ethanol-mediated interaction among these proteins.
Previously we have shown that JNK activation is required in ethanol-induced migration/invasion of breast cancer cells . It was reported that JNK activation during cell migration is mediated by p130Cas/Crk coupling or JSAP1 (JNK/stress-activated protein kinase-associated protein 1)[42, 43]. p130Cas (Crk-associated substrate) is an adaptor protein and it binds to and is phosphorylated by FAK in a FAK/cSrc dependent manner . Activation of p130Cas leads to recruitment of Crk to form an adaptor complex which results in activation of Rac1 and JNKs. We show here that ethanol increases the association of p130Cas with FAK/cSrc and the phosphorylation of p130Cas. Ethanol also promotes the association between p130Cas and JNKs. C3G inhibits ethanol-mediated p130Cas/JNK interaction (Figure 5). Together, our results indicate that ethanol activates p130Cas and JNKs through the ErbB2/cSrc/FAK pathway. Blocking ErbB2/cSrc/FAK signaling by C3G inhibits ethanol-mediated activation of JNKs which is necessary for cell migration/invasion. At times the effect of C3G on cell signaling components, such as FAK, cSrc and JNK, is not dose-dependent, which is not entirely consistent with its effect on ethanol-induced cell migration/invasion. This suggests that the mechanism of ethanol-induced cell migration/invasion is complex and probably multiple signaling pathways are involved.
Antioxidant property of C3G
Ethanol exposure causes the accumulation of intracellular ROS [10, 23]. The antioxidant property of C3G is confirmed in this study and we show that C3G effectively blocks ethanol-induced ROS production in breast cancer cells. ROS is reported to be involved in the activation of EGFR . To evaluate the involvement of ROS, we compared the effect of C3G with other antioxidants. We have titrated antioxidants and identified concentrations for these antioxidants to produce a ROS scavenging capacity similar to C3G at 10 μM. Although these antioxidants have a similar capacity of scavenging ROS, they are less effective in alleviating ethanol-induced cell invasion and ErbB2 phosphorylation. Broad antioxidants, such as NAC and vitamin C, display modest inhibition on ethanol-induced cell invasion and ErbB2 phosphorylation, but to a lesser extent compared to C3G. More specific antioxidants, SOD (for superoxide) plus catalase (for hydrogen peroxide) fail to inhibit ethanol-stimulated invasion and ErbB2 phosphorylation. These data suggest that although the antioxidant property may be involved, C3G may also act through other mechanisms to regulate ErbB2 signaling and subsequent migration/invasion. We have previously shown that C3G is able to reverse ethanol-induced inhibition of neurite outgrowth in neuronal cells; however, its antioxidant property is minimally involved .
C3G has drawn increasing attention because of its potential anti-cancer properties. A recent animal study investigates the pharmacokinetics of C3G and demonstrates that pharmacologically relevant concentrations of C3G are achievable in vivo through oral administration or intravenous injection in mice without apparent adverse effects . Further analysis suggests that concentrations required for in vivo action of C3G could be much lower than that of in vitro[14, 45]. In future studies, we will evaluate the effect of C3G on ethanol-induced tumor promotion in animal models. C3G may offer a novel avenue for treating alcohol-related disorders.
focal adhesion kinase
c-Jun N-terminal kinases
reactive oxygen species
This research was supported by grants from the National Institutes of Health (AA01540 and AA017226).
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