JOURNAL OF MEDICINAL FOOD J Med Food 00 (00) 1–7 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.0159
FULL COMMUNICATION
Anticancer Activity of Protocatechualdehyde in Human Breast Cancer Cells Jieun Choi, Xiaojing Jiang, Jin Boo Jeong, and Seong-Ho Lee Department of Nutrition and Food Science, College of Agriculture and Natural Resources, University of Maryland, College Park, Maryland, USA. ABSTRACT Protocatechualdehyde (PCA) is a natural polyphenol compound isolated from the root of the herb S. miltiorrhiza and barley tea plants. PCA possesses antiproliferative and pro-apoptotic properties in human colorectal cancer cells. However, the cellular mechanism has not been fully understood. b-catenin and cyclin D1 are proto-oncogene that is overexpressed in many types of cancers and leads to cancer development. The present study was performed to elucidate the molecular mechanism by which PCA stimulates cell growth arrest and apoptosis in human breast cancer cells. PCA repressed cell proliferation and induced apoptosis in dose-dependent manner. PCA suppressed the expression of b-catenin and cyclin D1 with no changes in mRNA levels. Inhibition of proteosomal degradation using MG-132 and Ada-(Ahx)3-(Leu)3-vinyl sulfone ameliorates PCA-induced downregulation of b-catenin and cyclin D1. PCA treatment decreased the half-life of b-catenin and cyclin D1. PCA-mediated b-catenin downregulation depends on GSK3b. We further provide the evidence that PCA increased nuclear translocation of nuclear factor kappa-B (NF-jB) and the blockage of NF-jB using Bay11-7082 inhibited PCAmediated b-catenin downregulation. The current study demonstrates that PCA suppress b-catenin expression through GSK3band NF-jB-mediated proteosomal degradation. In addition, PCA decreased cyclin D1 expression independent to b-catenin through proteosomal degradation. KEY WORDS: b-catenin breast cancer cyclin D1 protocatechualdehyde
particular, GSK3b is inactivated by the PI3K/Akt-mediated phosphorylation at serine 9 residue, and leads to b-catenin accumulation in the cytosol.10 Free b-catenin translocate into nucleus where b-catenin protein binds to the members of the LEF/TCF family and activates the expression of several target genes, including cyclin D1.11,12 Thus, activation of b-catenin signaling leads to the induction of cyclin D1 at the transcriptional level and furthers the progression of the G1 to S phase and the increase of proliferation. In this study, we propose anticancer activity of PCA in breast cancer. PCA inhibits cell growth and increases apoptosis of breast cancer cells by downregulating b-catenin and cyclin D1 via multiple mechanisms.
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n the united states, breast cancer is most prevalent in cancer incidence and the second leading cause of death in women.1 In 2012, there were an estimated 226,870 new cases of and 39,510 cancer-related deaths from breast cancer.1 Chemoprevention using phytochemicals has received considerable attention as an effective and promising approach for breast cancer prevention. Protocatechualdehyde (PCA) is a (3,4-dihydroxybenzaldehyde)-polyphenolic compound derived from the root of the herb S. miltiorrhiza2 and barley tea.3 PCA has been reported to possess antioxidant, antitumor, and antiinflammatory properties.4,5 In human colorectal cancer, PCA suppressed cell proliferation and downregulated cyclin D1 and histone deacetylase.2,6 Many signaling pathways controls breast tumorigenesis. Specifically, the Wnt and PI3K signaling pathways play important roles in tumorigenesis and cancer progression.7,8 The b-catenin protein interacts with several cellular proteins including glycogen synthase kinase (GSK) 3b, adenomatous polyposis coli (APC), casein kinase 1a (CK1a), or axin.9 In
MATERIALS AND METHODS Chemicals PCA was purchased from Sigma Aldrich (St. Louis, MO, USA), and cell culture media (Dulbecco’s modified Eagle medium [DMEM]/F-12) was purchased from Lonza (Walkersville, MD, USA). Antibodies for b-catenin (#9562) and PARP (#9542), phospho GSK3b (Ser9, #9336), GSK3b (#9315), p65 (#8242), TBP (#8515), and b-actin (#5125) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody for cyclin-D1 (sc-718) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,
Manuscript received 24 October 2013. Revision accepted 12 February 2014. Address correspondence to: Seong-Ho Lee, PhD, Department of Nutrition and Food Science, 0112 Skinner Building, University of Maryland, College Park, Maryland, 20742, USA, E-mail: [email protected]
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USA). Ada-(Ahx)3-(Leu)3-vinyl sulfone and BAY 11-7082 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). MG-132 and cyclohexamide were purchased from Calbiochem (San Diego, CA, USA). Unless otherwise designated, all chemicals were purchased from Fisher Scientific (Hampton, NH, USA). Cell culture and treatment Human breast cancer cell (MCF-7 and MDA-MB-231) was purchased from American Type Culture Collection (Manassas, VA, USA) and was grown in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and a mixture of antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin). The cells were maintained in a 37C incubator at 5% CO2. PCA was dissolved in dimethyl sulfoxide (DMSO) and treated to the cells. DMSO was used for a vehicle and the final concentration of DMSO did not exceed 0.1% (v/v). The cells were pre-treated with various inhibitors (MG-132, Ada-(Ahx)3-(Leu)3-vinyl sulfone, SB216763, Bay11-7082) and then co-treated with 100 lM of PCA for 24 hours as indicated in legends of Figures 3B, 3C, 4A and 5B. For cycloheximide experiment, the cells were pretreated with DMSO or PCA for 6 hours and then co-treated with 10 lg/mL of cycloheximide for different times as indicated in legends of Figures 3D and 3E. Measurement of cell proliferation Cell Proliferation Assay system (Promega, Madison, WI, USA) was used for measuring proliferative activity of the cells. MCF-7 cells (2000 cells/well) were plated onto 96well plates. On the next day, the cells were treated with 0, 5, 10, 25, 50, or 100 lM of PCA in media containing 10% FBS for 0, 24, or 48 hours. Twenty microliters of CellTiter96 Aqueous One solution was added to each well and further incubated for 1 hour at 37C. The absorbance was measured at 490 nm using enzyme-linked immunosorbent assay plated reader (Bio-Tek Instrument Inc., Winooski, VT, USA). Measurement of apoptosis Flow cytometric detection of apoptotic cells was performed according to the manufacturer’s instructions of Annexin V kits (Trevigen, Gaithersburg, MD, USA). Briefly, the cells were plated in 6-well tissue culture dishes and treated with 0, 50, and 100 lM of PCA for 24 hours. The attached and floating cells were harvested together with trypsin-EDTA. After washing with phosphate-buffered saline (PBS), the whole cells were resuspended and collected by centrifugation at 1000 rpm for 5 minutes. Cell pellets were stained with Annexin V-FITC and early apoptotic cells were quantified in FACS co-facility at University of Maryland using FACSCanto II (Becton Dickinson Biosciences, San Jose, CA, USA).
were harvested with 1X hypotonic buffer and incubated for 15 minutes on ice. After centrifugation for 30 seconds at 14,000 g, the supernatants (cytoplasmic fraction) were isolated. The nuclear pellet was incubated with lysis buffer for 30 minutes at 4C under agitation. The nuclear suspension was centrifuged for 10 minutes at 14,000 g and the supernatant (nuclear fraction) was collected. Both nuclear and cytoplasmic fractions were used for Western blot analysis. Isolation of RNA and reverse-transcription polymerase chain reaction (RT-PCR) Total RNA was extracted by RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol. Briefly, the cells were lysed in RNeasy lysis buffer containing b-mercaptoethanol and then added 70% ethanol to the homogenized lysate. The total RNA bound to membrane of RNeasy spin column was eluted in RNase-free water. The 1 lg of total RNA was used for reverse-transcription (RT) reaction according to manufacturer’s protocol (Verso cDNA kit, Thermo Scientific, Pittsburg, PA, USA). Polymerase chain reaction (PCR) was conducted as follows: total 30 cycles at 94C for 30 seconds, 55C for 30 seconds, and 72C for 1 minute using PCR Master Mix kit (Promega). The primer sequences are as follows: b-catenin (CTNNB1): forward 50 CCC ACT AAT GTC CAG CGT TT-30 and reverse 50 -AAT CCA CTG GTG AAC CAA GC-30 ; cyclin D1: forward 50 AAC TAC CTG GAC CGC TTC CT-30 and reverse 50 -CCA CTT GAG CTT GTT CAC CA-30 ; GAPDH: forward 50 -ACC CAG AAG ACT GTG GAT GG-30 and reverse 50 -TTC TAG ACG GCA GGT CAG GT-30 . SDS-PAGE and Western blot To prepare samples for electrophoresis, cells were lysed in radio immuno-precipitation assay (RIPA) buffer (Boston Bioproduct Inc., Ashland, MA, USA) with mixture of protease inhibitors and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). The protein concentration of cell lysates was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) using a bovine serum albumin (BSA) as a standard. The samples were denatured with 1X loading buffer at 95–100C for 5 minutes. The proteins were separated on the sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and transferred to the nitrocellulose transfer membranes (Whatman GmbH, Dassel, Germany). The membrane was blocked using 5% non-fat milk to prevent non-specific background and incubated with the primary antibodies at 4C overnight. The next day, the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. Using Pierce ECL Western blotting substrate (Thermo Scientific, Rockford, IL, USA), chemiluminescence was detected and imaged by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
Isolation of cytosol and nucleus fraction Cytosol and nucleus fractions were extracted from cells according to manufacturer’s protocols of a nuclear extract kit (Active Motif, Carlsbad, CA, USA). Briefly, the cells
Statistical analysis Statistical analysis was measured with the Student’s unpaired t-test, with statistical significance set at P < .05.
PROTOCATECHUALDEHYDE DOWNREGULATES b-CATENIN
RESULTS PCA inhibits proliferation and stimulates apoptosis of MCF-7 cells To investigate whether or not the treatment of PCA affects the growth of breast cancer cells, we tested estrogen receptor (ER)-positive (MCF-7) and negative (MDA-MB231) breast cancer cells. Both cells were exposed to 0, 5, 10, 25, 50, and 100 lM of PCA for 0, 24, and 48 hours into media containing 10% serum. Thereafter, cell proliferation was measured. As shown in Figure 1A (top), MCF-7 cells treated with 50 lM and 100 lM of PCA significantly decreased cell growth by 11% and 20% in 24 hours and by 22% and 27% in 48 hours, respectively, when compared to the cells treated with 0 lM of PCA. However, PCA did not change the cell proliferation in MDA-MB-231 cells (Fig. 1A, bottom). Next, we explored whether PCA affect apoptosis of MCF-7 cells. The cells were exposed to 0, 50, and 100 lM of PCA for 24 hours and early apoptotic cells were detected with FACS analysis (Fig. 1B). As a result, the percentage of early apoptotic cells were increased by 1.9fold and 2.6-fold in the cells treated with 50 lM and 100 lM of PCA, respectively, when compared to the cells treated with 0 lM of PCA. These results indicate that PCA may inhibit cell proliferation and increases apoptosis in human
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breast cancer cells through ER-dependent manner because PCA suppresses proliferation of estrogen receptor (ER)positive (MCF-7) breast cancer cells, but not ER-negative (MDA-MB-231) breast cancer cells. Inhibitory effect of PCA on b-catenin and cyclin D1 expression Next, we measured PARP cleavage, a marker of apoptosis. As shown in Figure 2A and 2B, the amount of cleaved fractions increased in the cells treated with PCA in dose- and time-dependent manners. It is generally accepted that an induction of apoptosis is associated with Wnt signaling pathway. Thus, in order to test whether PCA treatment affects b-catenin protein level, Western blot was performed. As shown in Figure 2A and B, treatment of 50 lM and 100 lM PCA to MCF-7 cells resulted in a decrease of b-catenin protein level in a dose- and a time-dependent manner. Increased b-catenin translocates to the nucleus, and binds TCF/LEF transcription factor and regulates transcription of downstream target gene such as cyclin D1.12 Therefore, we reblotted the same membrane with antibody for cyclin D1. The protein level of cyclin D1 is also decreased at the cells treated with 50 lM and 100 lM of PCA and decreased 24 hours after treatment of 100 lM of PCA. These data propose the potential that antiproliferative and
FIG. 1. Inhibition of cell proliferation and induction of apoptosis by protocatechualdehyde (PCA). (A) MCF-7 (top) and MDA-MB-231 (bottom) cells were treated with 0, 5, 10, 25, 50, and 100 lM of PCA for 0, 24, or 48 hours. Cell proliferation was measured using CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit and expressed as absorbance (A490). *P < .05 compared to cells treated with 0 lM of PCA. (B) MCF-7 cells were treated with 0, 50, and 100 lM of PCA for 24 hours. The cells were stained with Annexin V-FITC and propidium iodide according to apoptosis assay kit (Trevigen, Gaithersburg, MD, USA). Apoptosis was quantified by flow cytometry as described in Materials and Methods. Early apoptotic cells were expressed in graph and values are mean – SD of 3 replicates. *P < .05 compared to cells treated with 0 mM of PCA; #P < .05 compared to cells treated with 50 lM of PCA. Color images available online at www.liebertpub.com/jmf
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CHOI ET AL. FIG. 2. Downregulation of b-catenin and cyclin D1 by PCA. (A) MCF-7 cells were treated with indicated concentrations of PCA for 24 hours. (B) MCF-7 cells were treated with 100 lM of PCA for indicated time points and total cell lysates were harvested and, subsequently, Western blot analysis was performed as described in Materials and Methods for PARP, b-catenin, cyclin D1, and actin. Data represent one experiment.
pro-apoptotic activity of PCA might be mediated via decreased expression of b-catenin and cyclin D1. Proteosomal degradation of b-catenin and cyclin D1 by PCA To investigate whether or not an inhibition of b-catenin protein level may be associated with transcriptional downregulation of b-catenin gene, MCF-7 cells were incubated with 0, 50, and 100 lM of PCA for 24 hours and RT-PCR was performed to measure b-catenin mRNA. As shown in Figure 3A, mRNA level was not different among treatments. Interestingly, cyclin D1 mRNA was not affected by PCA treatment, indicating that PCA may decrease protein level of b-catenin and cyclin D1 through enhancing proteosomal degradation. Thus, we performed experiments using the proteasome inhibitor, MG-132 and Ada-(Ahx)3-(Leu)3-vinyl sulfone (ADA). As shown in Figure 3B and 3C, pre-treatment of MG-132 and ADA blocked PCA-stimulated downregulation of b-catenin and cyclin D1. To confirm this data, we pre-
treated the cells with DMSO or PCA and then co-treated with cycloheximide (CHX), a protein synthesis inhibitor, for indicated times. We observed that b-catenin protein was degraded more rapidly in the cells treated with PCA than vehicle-treated control (Fig. 3D). Because half-life of cyclin D1 was very short after CHX treatment, we performed another experiment exposing the cells to CHX for short times. As shown in Figure 3E, PCA treatment decreased cyclin D1 more rapidly than DMSO-treated control. Overall, the data indicate that proteosomal degradation might be responsible mechanism for downregulation of b-catenin and cyclin D1 by PCA. b-catenin downregulation by PCA is depending on GSK3b Cellular b-catenin level is regulated by PI3K/AKT/ GSK3b pathway. Activation of glycogen synthase kinase3b (GSK3b) induces destabilization of b-catenin and subsequent proteosomal degradation.10 In order to investigate whether GSK3b mediates PCA-induced downregulation of
FIG. 3. Proteosomal degradation of b-catenin and cyclin D1 by PCA. (A) MCF-7 cells were treated with the indicated concentrations of PCA for 24 hours and then RT-PCR was performed as described in Materials and Methods. The GAPDH represent a loading control. (B) The cells were pretreated with 0, 5, and 10 lM of MG-132 for 2 hours and then co-treated with 0 or 100 lM of PCA for 24 hours. (C) The cells were pretreated with 1 lM of ADA (Ada-(Ahx)3-(Leu)3-vinyl sulfone) for 2 hours and then co-treated with 0 or 100 lM of PCA for 24 hours. (D, E) The cells were pre-treated with DMSO or PCA for 6 hours and then co-treated with cycloheximide (CHX; 10 lg/mL) for indicated times. Total cell lysates were harvested and subsequently Western blot analysis was performed for b-catenin, cyclin D1, and actin. Data represent one experiment.
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FIG. 4. GSK3b dependency of b-catenin downregulation by PCA. (A) The cells were pre-treated with DMSO or SB216763 (50 lM) for 2 hours and then co-treated with PCA for 24 hours. Total cell lysates were harvested and subsequently Western blot analysis was performed for b-catenin and actin. (B) MCF-7 cells were treated with 100 lM of PCA for indicated time points and total cell lysates were harvested and subsequently Western blot analysis was performed for GSK3b (Ser9) and GSK3b. Data represent one experiment.
b-catenin, we pretreated the MCF-7 cells with specific inhibitor for GSK3b (SB216763) and then co-treated with DMSO or PCA. As shown in Figure 4A, pretreatment with SB216763 suppressed PCA-mediated downregulation of b-catenin. Since phosphorylation of GSK3b (p-GSK3b) at residue Ser9 decrease enzymatic activity of GSK3b, we measured phosphorylation of Ser9. As you can see in Figure 4B, inactive GSK3b decreased by treatment with PCA for 24 hours (Fig. 4B) without a change of total GSK3b. These results suggest that GSK3b, as a downstream target of PK3/ AKT pathway, might play a pivotal role for PCA-mediated downregulation of b-catenin.
PCA induces activation of nuclear factor kappa-B pathway There is evidence to suggest that the nuclear factor kappa-B (NF-jB) activation is associated with b-catenin and affect development and progression of cancer. To test whether PCA treatment influences NF-jB pathway, we observed the expression of p65 in cytosolic and nuclear fraction of MCF7 cells after treatment of PCA. As a result, we found that PCA increased an amount of p65 in nucleus of MCF-7 cells (Fig. 5A). According to recent literatures, NF-jB interacts with b-catenin in several proposed mechanisms.13,14 Thus,
FIG. 5. NF-jB dependency of b-catenin downregulation by PCA. (A) The cells were treated with DMSO or PCA for 24 hours. Cytosol (CE) and nucleus extracts (NE) were prepared and subsequently Western blot analysis was performed for p65, TBP, and actin. Data represent one of two independent experiments with similar results. (B) The cells were pre-treated with DMSO or Bay11-7082 at indicated doses for 2 hours and then co-treated with PCA for 24 hours. Total cell lysates were harvested and subsequently Western blot analysis was performed for b-catenin and actin (top). Data represent one of three independent experiments with similar results (bottom). *P < .05 compared to control group treated with 0 lM PCA; NS, not significant. (C) The proposed anticancer mechanism of PCA in human breast cancer cells is depicted.
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to test if NF-jB mediates PCA-stimulated downregulation of b-catenin, MCF-7 cells were pre-treated with BAY 117082, selective inhibitor of NF-jB translocation and then co-treated with DMSO or PCA. As shown in Figure 5B, downregulation of b-catenin by PCA was ameliorated by pretreatment of BAY 11-7082, indicating that activation of NF-jB by PCA at least in part contributes PCA-mediated downregulation of b-catenin in human breast cancer cells. DISCUSSION PCA is a naturally occurring polyphenol in plants. There were several reports that PCA possesses anticancer activity.15,16 In previous study, we reported that PCA inhibited proliferation of human colorectal cancer cells.6 The anticarcinogenic effects of PCA have raised an important question regarding the underlying molecular mechanisms. Here, we demonstrate direct evidence that PCA downregulates b-catenin and cyclin D1 through activating proteosomal degradation. The proteosomal degradation of b-catenin is associated with GSK3b and NF-jB pathway in human breast cancer cells. b-catenin is regulated by several pathways and one is that b-catenin revitalize as a transcriptional activator via a Wnt signaling pathway.17 GSK3 is a downstream target of PI3K/ AKT and Wnt signaling pathways and has recently been recognized as one of the most important signals ensuring apoptosis in the cancer cells.10 Deregulation of PI3K/AKT/ GSK3b pathways by specific inhibitor triggers a cellular cascade involved in apoptosis in various cancer model.18,19 The present data indicate that activation of GSK3b may be a major mechanism by which PCA downregulates b-catenin. The effect of PCA on b-catenin/TCF-dependent gene regulation can be important for the PCA-induced antitumorigenesis. The importance of this effect is clearly indicated by the reduced expression of cyclin D1, a protein that plays an important role in cell-cycle transitions. In fact, cyclin D1 overexpression has been found in approximately 50% of breast cancer.20 Our data indicate that PCA-induced cyclin D1 downregulation is independent on b-catenin downregulation and completely related to its proteosomal degradation. There are multiple mechanisms by which dietary compounds represses cyclin D1 expression. One is through transcriptional regulation. In the previous study, we found that PCA decrease cyclin D1 in transcriptional downregulation.6 In fact, TCF/LEF site is located in this region of the promoter and plays an important role in bcatenin-dependent transcriptional regulation of cyclin D1.12 Another mechanism to suppress cyclin D1 expression is the activation of proteasome degradation. Our results clearly demonstrate that PCA increase proteosomal degradation. Proteolysis of cyclin D1 is one of prominent anticancer mechanism, previously reported with curcumin,21 retinoic acid,22 and troglitazone.23 NF-jB has been known as a potential molecular target for inflammation and cancer.24,25 Transcriptional activity of NF-jB is determined by cellular localization of two nuclear transcription factors, p50 and p65. Without stimulus,
p65 and p50 is sequestered in IjB-a, but activation of NF-jB signaling induce proteosomal degradation of IjB-a by activating IjB-a kinase (IKK), and increase translocation of p65 and p50 to the nucleus where coordinate transcription of several target genes associated with inflammation, cell proliferation, and apoptosis.26 In our study, we observed that PCA treatment increase nuclear translocation of p65 (Fig. 5A). The role of NF-jB in cancer development is complex. According to several recent studies, NF-jB activation mediates apoptosis of anticancer compounds.27–29 It is proposed that NF-jB activation is a cancer preventive and therapeutic target. In addition, NF-jB interacts with b-catenin and may control cancer progression. b-catenin forms a complex with NF-jB and reduces NF-jB transactivation in human breast cancer.14 Therefore, it is likely that NF-jB activation by PCA might be at least a partly responsible mechanism for b-catenin downregulation. In fact, NSAIDs such as diclofenac inhibited b-catenin signaling through activating NF-jB.30 Many studies indicated that estrogen via estrogen receptor (ER)-mediated signaling pathways participate in cancer development and progression by modulating expressions of genes involved in cell cycles and apoptosis. Our data demonstrate that PCA suppressed proliferation of breast cancer cells via estrogen-dependent manner (Fig. 1A). In fact, bcatenin is highly associated with estrogen and inhibition of ER activation could markedly suppress growth of breast cancer cells through modulation of several signaling pathways.31,32 Thus, it is likely that link of b-catenin and estrogen signaling is crucial for PCA-stimulated suppression of breast cancer cell growth. In conclusion, PCA activates GSK3b by dephosphorylating serine 9 and activates NF-jB pathway by increasing nuclear translocation of p65. Activation of GSK3b and NF-jB subsequently stimulate proteosomal degradation of b-catenin and inhibit proliferation and induces apoptosis in human breast cancer cells (Fig. 5C). The current study provides information on molecular events of anticancer activity of PCA.
ACKNOWLEDGMENTS This work was supported by start-up funds (S.-H.L.) from University of Maryland and in part by #RSG-11-133-01CCE (S.-H. L.) from the American Cancer Society. DISCLOSURE STATEMENT There are no competing financial interests. REFERENCES 1. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29. 2. Zhou Z, Liu Y, Miao AD, Wang SQ: Protocatechuic aldehyde suppresses TNF-alpha-induced ICAM-1 and VCAM-1 expression in human umbilical vein endothelial cells. Eur J Pharmacol 2005;513:1–8. 3. Etoh H, Murakami K, Yogoh T, Ishikawa H, Fukuyama Y, Tanaka H: Anti-oxidative compounds in barley tea. Biosci Biotechnol Biochem 2004;68:2616–2618.
PROTOCATECHUALDEHYDE DOWNREGULATES b-CATENIN 4. Chang ZQ, Gebru E, Lee SP, et al.: In vitro antioxidant and anti-inflammatory activities of protocatechualdehyde isolated from Phellinus gilvus. J Nutr Sci Vitaminol (Tokyo) 2011;57: 118–122. 5. Wei G, Guan Y, Yin Y, et al.: Anti-inflammatory effect of protocatechuic aldehyde on myocardial ischemia/reperfusion injury in vivo and in vitro. Inflammation 2013;36:592–602. 6. Jeong JB, Lee SH: Protocatechualdehyde possesses anti-cancer activity through downregulating cyclin D1 and HDAC2 in human colorectal cancer cells. Biochem Biophys Res Commun 2013;430: 381–386. 7. Widelitz RB, Jiang TX, Lu J, Chuong CM: Beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin. Dev Biol 2000;219:98–114. 8. Polakis P: Wnt signaling and cancer. Genes Dev 2000;14:1837– 1851. 9. Bienz M: APC: the plot thickens. Curr Opin Genet Dev 1999;9:595–603. 10. Cohen P, Frame S: The renaissance of GSK3. Nat Rev Mol Cell Biol 2001;2:769–776. 11. Shtutman M, Zhurinsky J, Simcha I, et al.: The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 1999;96:5522–5527. 12. Tetsu O, McCormick F: Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 1999:398:422–426. 13. Cho HH, Song JS, Yu JM, et al.: Differential effect of NFkappaB activity on beta-catenin/Tcf pathway in various cancer cells. FEBS Lett 2008;582:616–622. 14. Deng J, Miller SA, Wang HY, et al.: beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer. Cancer Cell 2002;2:323–334. 15. Kim KJ, Kim MA, Jung JH: Antitumor and antioxidant activity of protocatechualdehyde produced from Streptomyces lincolnensis M-20. Arch Pharm Res 2008;31:1572–1577. 16. Lee BH, Yoon SH, Kim YS, Kim SK, Moon BJ, Bae YS: Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium. Natural Product Research 2008;22:1441–1450. 17. Behrens J, Jerchow BA, Wu¨rtele M, et al.: Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 1998;280:596–599. 18. Parsons R: Human cancer, PTEN and the PI-3 kinase pathway. Semin Cell Dev Biol 2004;15:171–176. 19. Yamaguchi K, Lee SH, Eling TE, Baek SJ: Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a
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novel downstream target of phosphatidylinositol 3-kinase/AKT/ GSK-3beta pathway. J Biol Chem 2004;279:49617–49623. Gillett C, Fantl V, Smith R, et al.: Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res 1994;54:1812–1817. Mukhopadhyay A, Banerjee S, Stafford LJ, Xia C, Liu M, Aggarwal BB: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation. Oncogene 2002;21:8852–8861. Spinella MJ, Freemantle SJ, Sekula D, Chang JH, Christie AJ, Dmitrovsky E: Retinoic acid promotes ubiquitination and proteolysis of cyclin D1 during induced tumor cell differentiation. J Biol Chem 1999;274:22013–22018. Huang JW, Shiau CW, Yang YT, et al.: Peroxisome proliferatoractivated receptor gamma-independent ablation of cyclin D1 by thiazolidinediones and their derivatives in breast cancer cells. Mol Pharmacol 2005;67:1342–1348. Perkins ND, Gilmore TD: Good cop, bad cop: the different faces of NF-kappaB. Cell Death Differ 2006;13:759–772. Stark LA, Dunlop MG: Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis. Mol Cell Biol 2005;25:5985–6004. Pahl HL: Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999;18:6853–6866. Stark LA, Din FV, Zwacka RM, Dunlop MG: Aspirin-induced activation of the NF-kappaB signaling pathway: a novel mechanism for aspirin-mediated apoptosis in colon cancer cells. FASEB J 2001;15:1273–1275. Kasperczyk H, La Ferla-Bru¨hl K, Westhoff MA, et al.: Betulinic acid as new activator of NF-kappa B: molecular mechanisms and implications for cancer therapy. Oncogene 2005;24:6945– 6956. Jeong JB, Yang X, Clark R, Choi J, Baek SJ, Lee SH: A mechanistic study of the proapoptotic effect of tolfenamic acid; involvement of NF-kappaB activation. Carcinogenesis 2013;34: 2350–2360. Cho M, Gwak J, Park S, et al.: Diclofenac attenuates Wnt/betacatenin signaling in colon cancer cells by activation of NF-kappaB. FEBS Lett 2005;579:4213–4218. Kouzmenko AP, Takeyama K, Ito S, et al.: Wnt/b-catenin and estrogen signaling converge in vivo. J Biol Chem 2004;279: 40255–40258. Gupta N, Schmitt F, Grebhardt S, Mayer D: b-catenin is a positive regulator of estrogen receptor-a function in breast cancer cells. Cancers (Basel) 2011;3:2990–3001.
FULL COMMUNICATION
Anticancer Activity of Protocatechualdehyde in Human Breast Cancer Cells Jieun Choi, Xiaojing Jiang, Jin Boo Jeong, and Seong-Ho Lee Department of Nutrition and Food Science, College of Agriculture and Natural Resources, University of Maryland, College Park, Maryland, USA. ABSTRACT Protocatechualdehyde (PCA) is a natural polyphenol compound isolated from the root of the herb S. miltiorrhiza and barley tea plants. PCA possesses antiproliferative and pro-apoptotic properties in human colorectal cancer cells. However, the cellular mechanism has not been fully understood. b-catenin and cyclin D1 are proto-oncogene that is overexpressed in many types of cancers and leads to cancer development. The present study was performed to elucidate the molecular mechanism by which PCA stimulates cell growth arrest and apoptosis in human breast cancer cells. PCA repressed cell proliferation and induced apoptosis in dose-dependent manner. PCA suppressed the expression of b-catenin and cyclin D1 with no changes in mRNA levels. Inhibition of proteosomal degradation using MG-132 and Ada-(Ahx)3-(Leu)3-vinyl sulfone ameliorates PCA-induced downregulation of b-catenin and cyclin D1. PCA treatment decreased the half-life of b-catenin and cyclin D1. PCA-mediated b-catenin downregulation depends on GSK3b. We further provide the evidence that PCA increased nuclear translocation of nuclear factor kappa-B (NF-jB) and the blockage of NF-jB using Bay11-7082 inhibited PCAmediated b-catenin downregulation. The current study demonstrates that PCA suppress b-catenin expression through GSK3band NF-jB-mediated proteosomal degradation. In addition, PCA decreased cyclin D1 expression independent to b-catenin through proteosomal degradation. KEY WORDS: b-catenin breast cancer cyclin D1 protocatechualdehyde
particular, GSK3b is inactivated by the PI3K/Akt-mediated phosphorylation at serine 9 residue, and leads to b-catenin accumulation in the cytosol.10 Free b-catenin translocate into nucleus where b-catenin protein binds to the members of the LEF/TCF family and activates the expression of several target genes, including cyclin D1.11,12 Thus, activation of b-catenin signaling leads to the induction of cyclin D1 at the transcriptional level and furthers the progression of the G1 to S phase and the increase of proliferation. In this study, we propose anticancer activity of PCA in breast cancer. PCA inhibits cell growth and increases apoptosis of breast cancer cells by downregulating b-catenin and cyclin D1 via multiple mechanisms.
INTRODUCTION
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n the united states, breast cancer is most prevalent in cancer incidence and the second leading cause of death in women.1 In 2012, there were an estimated 226,870 new cases of and 39,510 cancer-related deaths from breast cancer.1 Chemoprevention using phytochemicals has received considerable attention as an effective and promising approach for breast cancer prevention. Protocatechualdehyde (PCA) is a (3,4-dihydroxybenzaldehyde)-polyphenolic compound derived from the root of the herb S. miltiorrhiza2 and barley tea.3 PCA has been reported to possess antioxidant, antitumor, and antiinflammatory properties.4,5 In human colorectal cancer, PCA suppressed cell proliferation and downregulated cyclin D1 and histone deacetylase.2,6 Many signaling pathways controls breast tumorigenesis. Specifically, the Wnt and PI3K signaling pathways play important roles in tumorigenesis and cancer progression.7,8 The b-catenin protein interacts with several cellular proteins including glycogen synthase kinase (GSK) 3b, adenomatous polyposis coli (APC), casein kinase 1a (CK1a), or axin.9 In
MATERIALS AND METHODS Chemicals PCA was purchased from Sigma Aldrich (St. Louis, MO, USA), and cell culture media (Dulbecco’s modified Eagle medium [DMEM]/F-12) was purchased from Lonza (Walkersville, MD, USA). Antibodies for b-catenin (#9562) and PARP (#9542), phospho GSK3b (Ser9, #9336), GSK3b (#9315), p65 (#8242), TBP (#8515), and b-actin (#5125) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody for cyclin-D1 (sc-718) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,
Manuscript received 24 October 2013. Revision accepted 12 February 2014. Address correspondence to: Seong-Ho Lee, PhD, Department of Nutrition and Food Science, 0112 Skinner Building, University of Maryland, College Park, Maryland, 20742, USA, E-mail: [email protected]
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USA). Ada-(Ahx)3-(Leu)3-vinyl sulfone and BAY 11-7082 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). MG-132 and cyclohexamide were purchased from Calbiochem (San Diego, CA, USA). Unless otherwise designated, all chemicals were purchased from Fisher Scientific (Hampton, NH, USA). Cell culture and treatment Human breast cancer cell (MCF-7 and MDA-MB-231) was purchased from American Type Culture Collection (Manassas, VA, USA) and was grown in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and a mixture of antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin). The cells were maintained in a 37C incubator at 5% CO2. PCA was dissolved in dimethyl sulfoxide (DMSO) and treated to the cells. DMSO was used for a vehicle and the final concentration of DMSO did not exceed 0.1% (v/v). The cells were pre-treated with various inhibitors (MG-132, Ada-(Ahx)3-(Leu)3-vinyl sulfone, SB216763, Bay11-7082) and then co-treated with 100 lM of PCA for 24 hours as indicated in legends of Figures 3B, 3C, 4A and 5B. For cycloheximide experiment, the cells were pretreated with DMSO or PCA for 6 hours and then co-treated with 10 lg/mL of cycloheximide for different times as indicated in legends of Figures 3D and 3E. Measurement of cell proliferation Cell Proliferation Assay system (Promega, Madison, WI, USA) was used for measuring proliferative activity of the cells. MCF-7 cells (2000 cells/well) were plated onto 96well plates. On the next day, the cells were treated with 0, 5, 10, 25, 50, or 100 lM of PCA in media containing 10% FBS for 0, 24, or 48 hours. Twenty microliters of CellTiter96 Aqueous One solution was added to each well and further incubated for 1 hour at 37C. The absorbance was measured at 490 nm using enzyme-linked immunosorbent assay plated reader (Bio-Tek Instrument Inc., Winooski, VT, USA). Measurement of apoptosis Flow cytometric detection of apoptotic cells was performed according to the manufacturer’s instructions of Annexin V kits (Trevigen, Gaithersburg, MD, USA). Briefly, the cells were plated in 6-well tissue culture dishes and treated with 0, 50, and 100 lM of PCA for 24 hours. The attached and floating cells were harvested together with trypsin-EDTA. After washing with phosphate-buffered saline (PBS), the whole cells were resuspended and collected by centrifugation at 1000 rpm for 5 minutes. Cell pellets were stained with Annexin V-FITC and early apoptotic cells were quantified in FACS co-facility at University of Maryland using FACSCanto II (Becton Dickinson Biosciences, San Jose, CA, USA).
were harvested with 1X hypotonic buffer and incubated for 15 minutes on ice. After centrifugation for 30 seconds at 14,000 g, the supernatants (cytoplasmic fraction) were isolated. The nuclear pellet was incubated with lysis buffer for 30 minutes at 4C under agitation. The nuclear suspension was centrifuged for 10 minutes at 14,000 g and the supernatant (nuclear fraction) was collected. Both nuclear and cytoplasmic fractions were used for Western blot analysis. Isolation of RNA and reverse-transcription polymerase chain reaction (RT-PCR) Total RNA was extracted by RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol. Briefly, the cells were lysed in RNeasy lysis buffer containing b-mercaptoethanol and then added 70% ethanol to the homogenized lysate. The total RNA bound to membrane of RNeasy spin column was eluted in RNase-free water. The 1 lg of total RNA was used for reverse-transcription (RT) reaction according to manufacturer’s protocol (Verso cDNA kit, Thermo Scientific, Pittsburg, PA, USA). Polymerase chain reaction (PCR) was conducted as follows: total 30 cycles at 94C for 30 seconds, 55C for 30 seconds, and 72C for 1 minute using PCR Master Mix kit (Promega). The primer sequences are as follows: b-catenin (CTNNB1): forward 50 CCC ACT AAT GTC CAG CGT TT-30 and reverse 50 -AAT CCA CTG GTG AAC CAA GC-30 ; cyclin D1: forward 50 AAC TAC CTG GAC CGC TTC CT-30 and reverse 50 -CCA CTT GAG CTT GTT CAC CA-30 ; GAPDH: forward 50 -ACC CAG AAG ACT GTG GAT GG-30 and reverse 50 -TTC TAG ACG GCA GGT CAG GT-30 . SDS-PAGE and Western blot To prepare samples for electrophoresis, cells were lysed in radio immuno-precipitation assay (RIPA) buffer (Boston Bioproduct Inc., Ashland, MA, USA) with mixture of protease inhibitors and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). The protein concentration of cell lysates was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) using a bovine serum albumin (BSA) as a standard. The samples were denatured with 1X loading buffer at 95–100C for 5 minutes. The proteins were separated on the sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and transferred to the nitrocellulose transfer membranes (Whatman GmbH, Dassel, Germany). The membrane was blocked using 5% non-fat milk to prevent non-specific background and incubated with the primary antibodies at 4C overnight. The next day, the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. Using Pierce ECL Western blotting substrate (Thermo Scientific, Rockford, IL, USA), chemiluminescence was detected and imaged by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
Isolation of cytosol and nucleus fraction Cytosol and nucleus fractions were extracted from cells according to manufacturer’s protocols of a nuclear extract kit (Active Motif, Carlsbad, CA, USA). Briefly, the cells
Statistical analysis Statistical analysis was measured with the Student’s unpaired t-test, with statistical significance set at P < .05.
PROTOCATECHUALDEHYDE DOWNREGULATES b-CATENIN
RESULTS PCA inhibits proliferation and stimulates apoptosis of MCF-7 cells To investigate whether or not the treatment of PCA affects the growth of breast cancer cells, we tested estrogen receptor (ER)-positive (MCF-7) and negative (MDA-MB231) breast cancer cells. Both cells were exposed to 0, 5, 10, 25, 50, and 100 lM of PCA for 0, 24, and 48 hours into media containing 10% serum. Thereafter, cell proliferation was measured. As shown in Figure 1A (top), MCF-7 cells treated with 50 lM and 100 lM of PCA significantly decreased cell growth by 11% and 20% in 24 hours and by 22% and 27% in 48 hours, respectively, when compared to the cells treated with 0 lM of PCA. However, PCA did not change the cell proliferation in MDA-MB-231 cells (Fig. 1A, bottom). Next, we explored whether PCA affect apoptosis of MCF-7 cells. The cells were exposed to 0, 50, and 100 lM of PCA for 24 hours and early apoptotic cells were detected with FACS analysis (Fig. 1B). As a result, the percentage of early apoptotic cells were increased by 1.9fold and 2.6-fold in the cells treated with 50 lM and 100 lM of PCA, respectively, when compared to the cells treated with 0 lM of PCA. These results indicate that PCA may inhibit cell proliferation and increases apoptosis in human
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breast cancer cells through ER-dependent manner because PCA suppresses proliferation of estrogen receptor (ER)positive (MCF-7) breast cancer cells, but not ER-negative (MDA-MB-231) breast cancer cells. Inhibitory effect of PCA on b-catenin and cyclin D1 expression Next, we measured PARP cleavage, a marker of apoptosis. As shown in Figure 2A and 2B, the amount of cleaved fractions increased in the cells treated with PCA in dose- and time-dependent manners. It is generally accepted that an induction of apoptosis is associated with Wnt signaling pathway. Thus, in order to test whether PCA treatment affects b-catenin protein level, Western blot was performed. As shown in Figure 2A and B, treatment of 50 lM and 100 lM PCA to MCF-7 cells resulted in a decrease of b-catenin protein level in a dose- and a time-dependent manner. Increased b-catenin translocates to the nucleus, and binds TCF/LEF transcription factor and regulates transcription of downstream target gene such as cyclin D1.12 Therefore, we reblotted the same membrane with antibody for cyclin D1. The protein level of cyclin D1 is also decreased at the cells treated with 50 lM and 100 lM of PCA and decreased 24 hours after treatment of 100 lM of PCA. These data propose the potential that antiproliferative and
FIG. 1. Inhibition of cell proliferation and induction of apoptosis by protocatechualdehyde (PCA). (A) MCF-7 (top) and MDA-MB-231 (bottom) cells were treated with 0, 5, 10, 25, 50, and 100 lM of PCA for 0, 24, or 48 hours. Cell proliferation was measured using CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit and expressed as absorbance (A490). *P < .05 compared to cells treated with 0 lM of PCA. (B) MCF-7 cells were treated with 0, 50, and 100 lM of PCA for 24 hours. The cells were stained with Annexin V-FITC and propidium iodide according to apoptosis assay kit (Trevigen, Gaithersburg, MD, USA). Apoptosis was quantified by flow cytometry as described in Materials and Methods. Early apoptotic cells were expressed in graph and values are mean – SD of 3 replicates. *P < .05 compared to cells treated with 0 mM of PCA; #P < .05 compared to cells treated with 50 lM of PCA. Color images available online at www.liebertpub.com/jmf
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CHOI ET AL. FIG. 2. Downregulation of b-catenin and cyclin D1 by PCA. (A) MCF-7 cells were treated with indicated concentrations of PCA for 24 hours. (B) MCF-7 cells were treated with 100 lM of PCA for indicated time points and total cell lysates were harvested and, subsequently, Western blot analysis was performed as described in Materials and Methods for PARP, b-catenin, cyclin D1, and actin. Data represent one experiment.
pro-apoptotic activity of PCA might be mediated via decreased expression of b-catenin and cyclin D1. Proteosomal degradation of b-catenin and cyclin D1 by PCA To investigate whether or not an inhibition of b-catenin protein level may be associated with transcriptional downregulation of b-catenin gene, MCF-7 cells were incubated with 0, 50, and 100 lM of PCA for 24 hours and RT-PCR was performed to measure b-catenin mRNA. As shown in Figure 3A, mRNA level was not different among treatments. Interestingly, cyclin D1 mRNA was not affected by PCA treatment, indicating that PCA may decrease protein level of b-catenin and cyclin D1 through enhancing proteosomal degradation. Thus, we performed experiments using the proteasome inhibitor, MG-132 and Ada-(Ahx)3-(Leu)3-vinyl sulfone (ADA). As shown in Figure 3B and 3C, pre-treatment of MG-132 and ADA blocked PCA-stimulated downregulation of b-catenin and cyclin D1. To confirm this data, we pre-
treated the cells with DMSO or PCA and then co-treated with cycloheximide (CHX), a protein synthesis inhibitor, for indicated times. We observed that b-catenin protein was degraded more rapidly in the cells treated with PCA than vehicle-treated control (Fig. 3D). Because half-life of cyclin D1 was very short after CHX treatment, we performed another experiment exposing the cells to CHX for short times. As shown in Figure 3E, PCA treatment decreased cyclin D1 more rapidly than DMSO-treated control. Overall, the data indicate that proteosomal degradation might be responsible mechanism for downregulation of b-catenin and cyclin D1 by PCA. b-catenin downregulation by PCA is depending on GSK3b Cellular b-catenin level is regulated by PI3K/AKT/ GSK3b pathway. Activation of glycogen synthase kinase3b (GSK3b) induces destabilization of b-catenin and subsequent proteosomal degradation.10 In order to investigate whether GSK3b mediates PCA-induced downregulation of
FIG. 3. Proteosomal degradation of b-catenin and cyclin D1 by PCA. (A) MCF-7 cells were treated with the indicated concentrations of PCA for 24 hours and then RT-PCR was performed as described in Materials and Methods. The GAPDH represent a loading control. (B) The cells were pretreated with 0, 5, and 10 lM of MG-132 for 2 hours and then co-treated with 0 or 100 lM of PCA for 24 hours. (C) The cells were pretreated with 1 lM of ADA (Ada-(Ahx)3-(Leu)3-vinyl sulfone) for 2 hours and then co-treated with 0 or 100 lM of PCA for 24 hours. (D, E) The cells were pre-treated with DMSO or PCA for 6 hours and then co-treated with cycloheximide (CHX; 10 lg/mL) for indicated times. Total cell lysates were harvested and subsequently Western blot analysis was performed for b-catenin, cyclin D1, and actin. Data represent one experiment.
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FIG. 4. GSK3b dependency of b-catenin downregulation by PCA. (A) The cells were pre-treated with DMSO or SB216763 (50 lM) for 2 hours and then co-treated with PCA for 24 hours. Total cell lysates were harvested and subsequently Western blot analysis was performed for b-catenin and actin. (B) MCF-7 cells were treated with 100 lM of PCA for indicated time points and total cell lysates were harvested and subsequently Western blot analysis was performed for GSK3b (Ser9) and GSK3b. Data represent one experiment.
b-catenin, we pretreated the MCF-7 cells with specific inhibitor for GSK3b (SB216763) and then co-treated with DMSO or PCA. As shown in Figure 4A, pretreatment with SB216763 suppressed PCA-mediated downregulation of b-catenin. Since phosphorylation of GSK3b (p-GSK3b) at residue Ser9 decrease enzymatic activity of GSK3b, we measured phosphorylation of Ser9. As you can see in Figure 4B, inactive GSK3b decreased by treatment with PCA for 24 hours (Fig. 4B) without a change of total GSK3b. These results suggest that GSK3b, as a downstream target of PK3/ AKT pathway, might play a pivotal role for PCA-mediated downregulation of b-catenin.
PCA induces activation of nuclear factor kappa-B pathway There is evidence to suggest that the nuclear factor kappa-B (NF-jB) activation is associated with b-catenin and affect development and progression of cancer. To test whether PCA treatment influences NF-jB pathway, we observed the expression of p65 in cytosolic and nuclear fraction of MCF7 cells after treatment of PCA. As a result, we found that PCA increased an amount of p65 in nucleus of MCF-7 cells (Fig. 5A). According to recent literatures, NF-jB interacts with b-catenin in several proposed mechanisms.13,14 Thus,
FIG. 5. NF-jB dependency of b-catenin downregulation by PCA. (A) The cells were treated with DMSO or PCA for 24 hours. Cytosol (CE) and nucleus extracts (NE) were prepared and subsequently Western blot analysis was performed for p65, TBP, and actin. Data represent one of two independent experiments with similar results. (B) The cells were pre-treated with DMSO or Bay11-7082 at indicated doses for 2 hours and then co-treated with PCA for 24 hours. Total cell lysates were harvested and subsequently Western blot analysis was performed for b-catenin and actin (top). Data represent one of three independent experiments with similar results (bottom). *P < .05 compared to control group treated with 0 lM PCA; NS, not significant. (C) The proposed anticancer mechanism of PCA in human breast cancer cells is depicted.
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to test if NF-jB mediates PCA-stimulated downregulation of b-catenin, MCF-7 cells were pre-treated with BAY 117082, selective inhibitor of NF-jB translocation and then co-treated with DMSO or PCA. As shown in Figure 5B, downregulation of b-catenin by PCA was ameliorated by pretreatment of BAY 11-7082, indicating that activation of NF-jB by PCA at least in part contributes PCA-mediated downregulation of b-catenin in human breast cancer cells. DISCUSSION PCA is a naturally occurring polyphenol in plants. There were several reports that PCA possesses anticancer activity.15,16 In previous study, we reported that PCA inhibited proliferation of human colorectal cancer cells.6 The anticarcinogenic effects of PCA have raised an important question regarding the underlying molecular mechanisms. Here, we demonstrate direct evidence that PCA downregulates b-catenin and cyclin D1 through activating proteosomal degradation. The proteosomal degradation of b-catenin is associated with GSK3b and NF-jB pathway in human breast cancer cells. b-catenin is regulated by several pathways and one is that b-catenin revitalize as a transcriptional activator via a Wnt signaling pathway.17 GSK3 is a downstream target of PI3K/ AKT and Wnt signaling pathways and has recently been recognized as one of the most important signals ensuring apoptosis in the cancer cells.10 Deregulation of PI3K/AKT/ GSK3b pathways by specific inhibitor triggers a cellular cascade involved in apoptosis in various cancer model.18,19 The present data indicate that activation of GSK3b may be a major mechanism by which PCA downregulates b-catenin. The effect of PCA on b-catenin/TCF-dependent gene regulation can be important for the PCA-induced antitumorigenesis. The importance of this effect is clearly indicated by the reduced expression of cyclin D1, a protein that plays an important role in cell-cycle transitions. In fact, cyclin D1 overexpression has been found in approximately 50% of breast cancer.20 Our data indicate that PCA-induced cyclin D1 downregulation is independent on b-catenin downregulation and completely related to its proteosomal degradation. There are multiple mechanisms by which dietary compounds represses cyclin D1 expression. One is through transcriptional regulation. In the previous study, we found that PCA decrease cyclin D1 in transcriptional downregulation.6 In fact, TCF/LEF site is located in this region of the promoter and plays an important role in bcatenin-dependent transcriptional regulation of cyclin D1.12 Another mechanism to suppress cyclin D1 expression is the activation of proteasome degradation. Our results clearly demonstrate that PCA increase proteosomal degradation. Proteolysis of cyclin D1 is one of prominent anticancer mechanism, previously reported with curcumin,21 retinoic acid,22 and troglitazone.23 NF-jB has been known as a potential molecular target for inflammation and cancer.24,25 Transcriptional activity of NF-jB is determined by cellular localization of two nuclear transcription factors, p50 and p65. Without stimulus,
p65 and p50 is sequestered in IjB-a, but activation of NF-jB signaling induce proteosomal degradation of IjB-a by activating IjB-a kinase (IKK), and increase translocation of p65 and p50 to the nucleus where coordinate transcription of several target genes associated with inflammation, cell proliferation, and apoptosis.26 In our study, we observed that PCA treatment increase nuclear translocation of p65 (Fig. 5A). The role of NF-jB in cancer development is complex. According to several recent studies, NF-jB activation mediates apoptosis of anticancer compounds.27–29 It is proposed that NF-jB activation is a cancer preventive and therapeutic target. In addition, NF-jB interacts with b-catenin and may control cancer progression. b-catenin forms a complex with NF-jB and reduces NF-jB transactivation in human breast cancer.14 Therefore, it is likely that NF-jB activation by PCA might be at least a partly responsible mechanism for b-catenin downregulation. In fact, NSAIDs such as diclofenac inhibited b-catenin signaling through activating NF-jB.30 Many studies indicated that estrogen via estrogen receptor (ER)-mediated signaling pathways participate in cancer development and progression by modulating expressions of genes involved in cell cycles and apoptosis. Our data demonstrate that PCA suppressed proliferation of breast cancer cells via estrogen-dependent manner (Fig. 1A). In fact, bcatenin is highly associated with estrogen and inhibition of ER activation could markedly suppress growth of breast cancer cells through modulation of several signaling pathways.31,32 Thus, it is likely that link of b-catenin and estrogen signaling is crucial for PCA-stimulated suppression of breast cancer cell growth. In conclusion, PCA activates GSK3b by dephosphorylating serine 9 and activates NF-jB pathway by increasing nuclear translocation of p65. Activation of GSK3b and NF-jB subsequently stimulate proteosomal degradation of b-catenin and inhibit proliferation and induces apoptosis in human breast cancer cells (Fig. 5C). The current study provides information on molecular events of anticancer activity of PCA.
ACKNOWLEDGMENTS This work was supported by start-up funds (S.-H.L.) from University of Maryland and in part by #RSG-11-133-01CCE (S.-H. L.) from the American Cancer Society. DISCLOSURE STATEMENT There are no competing financial interests. REFERENCES 1. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29. 2. Zhou Z, Liu Y, Miao AD, Wang SQ: Protocatechuic aldehyde suppresses TNF-alpha-induced ICAM-1 and VCAM-1 expression in human umbilical vein endothelial cells. Eur J Pharmacol 2005;513:1–8. 3. Etoh H, Murakami K, Yogoh T, Ishikawa H, Fukuyama Y, Tanaka H: Anti-oxidative compounds in barley tea. Biosci Biotechnol Biochem 2004;68:2616–2618.
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