Article pubs.acs.org/JAFC
Protocatechuic Acid Inhibits Oleic Acid-Induced Vascular Smooth Muscle Cell Proliferation through Activation of AMP-Activated Protein Kinase and Cell Cycle Arrest in G0/G1 Phase Ming-Cheng Lin,†,‡ Ting-Tsz Ou,# Chun-Hua Chang,# Kuei-Chuan Chan,*,†,‡ and Chau-Jong Wang*,#,⊥ †
Department of Internal Medicine, Chung-Shan Medical University Hospital, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan ‡ School of Medicine, Institute of Medicine, Chung-Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan # Institute of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan ⊥ Department of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan ABSTRACT: Protocatechuic acid (PCA) has been implicated in the progression of atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) may play a crucial role in the pathogenesis of atherosclerosis. Adenosine 5′-monophosphateactivated protein kinase (AMPK) additionally exerts several beneficial effects on vascular function and improves vascular abnormalities. The current study sought to determine whether PCA has an inhibitory effect on VSMC proliferation under oleic acid (OA) treatment. A7r5 cells were treated with OA (150 μM) or cotreated with OA and PCA (150 μg/mL) for 24 and 48 h. PCA-treated cells were found to cause an increase in G0/G1 cell cycle arrest. Western blotting showed that PCA increased the expressions of p53 and p21Cip1, subsequently decreasing the expression of cyclin E1 and Cdk2. In addition, PCA induced phosphorylation of AMPK and inhibited the expression of fatty acid synthase, Akt-p, and Skp2 after stimulation with OA. After treatment with AMPK inhibitor, the effects of PCA mentioned above were reversed. Taken together, PCA inhibited OA-induced VSMC proliferation through AMPK activation and down-regulation of FAS and AKT signals, which then blocks G0/G1 phase cell cycle progression. These findings provide a new insight into the protective properties of PCA on VSMC, which may constitute a novel effective antiatherosclerosis agent. KEYWORDS: Akt, AMP-activated protein kinase, cell cycle, protocatechuic acid, vascular smooth muscle cell proliferation
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INTRODUCTION Atherosclerosis is a chronic inflammation and proliferative process of the arteries. The abnormal proliferation of vascular smooth muscle cells (VSMCs) is the key pathological factor in the initiation and progression of vascular disorders, including arteriosclerosis.1 VSMC activation promotes a synthetic phenotype that underlies vessel growth disorders causing instent restenosis, transplant vasculopathy, and vein bypass graft failure.2 The best way to control the proliferation of VSMCs has been a major focus in the development of new therapeutic strategies. Recently, adenosine 5′-monophosphate-activated protein kinase (AMPK), a cellular fuel sensor, has emerged as a therapeutic target for atherosclerosis.3 AMPK, a physiological sensor of cellular energy, has been reported to play a key role in energy metabolism and to exert a cardiovascular protective function under physiological conditions and diseases of the vascular system.4 It has also been reported that AMPK activation by 5-aminoimidazole-4carboxamide ribonucleoside inhibits PDGF-BB-induced human VSMC proliferation.5 In addition, AMPK activation has been reported to lead to improved endothelial function,6 attenuation of myocardial ischemia injury in mice,7 and suppression of neointimal formation in a balloon injury rat model.8 In particular, the deletion of AMPK has been reported to reduce the level of p21, probably through S-phase kinase© 2014 American Chemical Society
associated protein 2 (Skp2)-mediated degradation, thereby causing neointimal hyperplasia.9 Recently, it has been reported that AMPK plays a significant role in cell growth arrest,10,11 making it an ideal candidate for studies targeted at reducing VSMC growth. Protocatechuic acid (PCA) is chemically known as 3,4dihydroxybenzoic acid and is widely distributed and present in most edible plants.12 It is also commonly found in the human diet including in bran, brown rice, and onions.13 PCA is also found in many fruits such as plums, gooseberries, and grapes14 and plants including Sudan mallow (Hibiscus sabdariffa L.)15 and Japanese ginkgo.16 Its pharmacological effects have been reported including antioxidant,17 antibacterial,18 anticancer,19 antidiabetic,20 antiaging,21 and anti-inflammation22 activities. Natural and synthetic polyphenols that stimulate AMPK have shown protective and antiatherogenic properties in aortic atherosclerotic lesions.23 In the present study, we hypothesized that PCA would attenuate oleic acid (OA)-induced VSMC proliferation, and we attempted to explore the molecular mechanisms underlying the protective effect of PCA. Received: Revised: Accepted: Published: 235
November 3, 2014 December 15, 2014 December 16, 2014 December 16, 2014 DOI: 10.1021/jf505303s J. Agric. Food Chem. 2015, 63, 235−241
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effect of OA, PCA, and compound C (AMPK inhibitor) on growth of VSMCs. (A) A7r5 cells were cotreated with OA (150 μM) and various concentrations (50, 100, and 150 μg/mL) of PCA for 48 h. (B) A7r5 cells were cotreated with OA (150 μM) and PCA (150 μg/mL) for the indicated times (0, 24, and 48 h). Cell viability was assayed using MTT. (C) A7r5 cells were pretreated with compound C (10 μM) for 30 min and then treated with OA (150 μM), PCA (150 μg/mL), and compound C for 48 h. C, control; com. C, compound C; G0/G1, the diploid population in the G0/G1 phase of the cell cycle; S, the cell population in the S phase; G2/M, the tetraploid population in the M phase. Data are represented as the mean ± SD (n = 3). (#) p < 0.05 versus the control group; (◊) p < 0.05 versus the OA group; (∗) p < 0.05 versus the OA+PCA group.
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μM) alone, or cotreated with OA and PCA (150 μg/mL) for 48 h. The OA-treated cells were harvested with trypsin−EDTA, washed with cold PBS, and fixed with cold 70% alcohol for 4 h at −20 °C. The fixed cells were then removed from the alcohol and stained with 50 μg/mL propidium iodide (Sigma) solution containing 10 μg/mL RNase A in darkness for at least 30 min on ice. The distribution of cells in different cell cycle phases characterized by specific amounts of DNA (G0/G1 = diploid, S = DNA synthesis between diploid and tetraploid, and G2/M = tetraploid, preceding cell division) was determined using flow cytometry (FACS Calibur; BD Biosciences). A minimum of 10000 cells per sample was counted, and DNA histograms were analyzed using CellQuest software (BD Biosciences) for cell cycle analysis.25 Western Blot. Following appropriate treatment, A7r5 cells were lysed and equal amounts of cell lysates (50 μg of protein) were separated by electrophoresis on 8−12% SDS polyacrylamide gel and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were incubated with Tris-buffered saline containing 1% (w/v) nonfat milk and 0.1% (v/v) Tween-20 for 1 h to block nonspecific binding, washed with Tween-20 for 30 min, and then incubated with the appropriate primary antibody at 4 °C overnight. Specific antibodies were used to evaluate the expressions of p-AMPK, fatty acid synthase (FAS), Cip1/p21, cyclin E1, cyclindependent kinase 2 (Cdk2), p-Akt,p53,p-p53, and skp2 (all from Santa
MATERIALS AND METHODS
Cell Culture. The cell line A7r5, a smooth muscle cell line derived from rat thoracic aorta, was obtained from the American Type Culture Collection (ATCC no. CRL-1444; Manassas, VA, USA). The A7r5 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 1% penicillin−streptomycin, and 1.5 g/L sodium bicarbonate (all from Gibco/BRL, Gaithersburg, MD, USA). All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cell Growth and MTT Assay. The effect of OA (Sigma, St. Louis, MO, USA) on cell growth was determined in the A7r5 cells. The A7r5 cells were seeded in 24-well culture plates at a density of 4 × 104 cells/ well, incubated for 48 h, treated with OA (150 μM) bound to BSA to stimulate cell growth as the control, or cotreated with OA and PCA (50−150 μg/mL) (Alfa Aesar, Ward Hill, MA, USA) for 48 h and 150 μg/mL of PCA for the indicated time. To measure cell viability, 0.2 mg/mL MTT (Sigma) was added to each well and incubated at 37 °C in a humidified atmosphere of 5% CO2 for another 4 h and solubilized with isopropanol. The viable number of cells was directly proportional to the production of formazan measured spectrophotometrically at 563 nm. Cell viability was determined using an MTT assay.24 Cell Cycle Analysis Using Flow Cytometry. A7r5 cells were seeded in 6-well plates, treated with compound C (10 μM), OA (150 236
DOI: 10.1021/jf505303s J. Agric. Food Chem. 2015, 63, 235−241
Article
Journal of Agricultural and Food Chemistry
Figure 2. Inhibition of cell cycle related protein in VSMCs by PCA. A7r5 cells were cotreated with OA (150 μM) and PCA (100 and 150 μg/mL) or vehicle-treated control. (A) After treatment, the cell lysates for protein extraction were prepared and adjusted for Western blot analysis. Proteins were detected by specific antibodies (cyclin E1, CdK2, Cdc25C-p, p21), and β-actin was used as a loading control. (B) After treatment of cells with PCA 150 μg/mL, the formation of the cyclin E1−p21 complex was assayed by immunoprecipitation analysis. Each mean value is the average of three independent experiments ± SD. (∗) p < 0.05 versus the OA group. Cruz Biotechnology, Santa Cruz, CA, USA), Cdc25 (Cell Signaling, Beverly, MA, USA), and β-actin (Sigma). The membranes were then incubated with horseradish peroxidase-conjugated second antibody (Sigma) for 1 h and developed using ECL chemiluminescence (Millipore), and the densitometry was analyzed using AlphaImager Series 2200 software.26 The results were representative of at least three independent experiments. Immunoprecipitation Analysis. A7r5 (4 × 104/mL) cells were seeded into 10 cm plates and treated with OA (150 μM) alone or cotreated with OA and PCA (150 μg/mL) for 48 h. The A7r5 cells were then lysed and transferred to a new tube, followed by the addition of 25 μL of protein A/G plus agarose, incubation for 30 min on ice, and centrifugation. The supernatant was removed, and the protein quantity was determined. Eight micrograms of Cip1/p21 antibody was added per 0.5 mg of protein, and the samples were incubated for 2 h before the addition of 20 μL of Ultralink immobilized A/G bead slurry (Pierce, Rockford, IL, USA) for another 2 h. The samples were centrifuged, and the precipitate was washed more than five times. The remaining steps were the same as for the Western blot analysis. AMPK Inhibitor. Compound C, an AMPK inhibitor, was purchased from Sigma. The cells were treated with 10 μmol of compound C for 30 min, followed by treatment with PCA. Specific antibodies were used to evaluate the expressions of AMPK-p, FAS, Akt-p, p53-p, p21, and skp2 Statistical Analysis. All data are expressed as the mean ± SD of three independent experiments, and statistical comparisons were evaluated using one-way analysis of variance (ANOVA). A p value
