Sesamin Inhibits PDGF-Mediated Proliferation of ... - ACS Publications

Aug 5, 2015 - Department of Pharmacology, Chungnam National University ... of Natural Product Chemistry, Chungnam National University College of ...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/JAFC

Sesamin Inhibits PDGF-Mediated Proliferation of Vascular Smooth Muscle Cells by Upregulating p21 and p27 Joo-Hui Han,†,‡ Sang-Gil Lee,†,‡ Sang-Hyuk Jung,† Jung-Jin Lee,§ Hyun-Soo Park,† Young Ho Kim,∥,⊥ and Chang-Seon Myung*,†,⊥ †

Department of Pharmacology, Chungnam National University College of Pharmacy, Daejeon 305-764, Republic of Korea KM Application Center, Korea Institute of Oriental Medicine, Daegu 701-300, Republic of Korea ∥ Department of Natural Product Chemistry, Chungnam National University College of Pharmacy, Daejeon 305-764, Republic of Korea ⊥ Institute of Drug Research & Development, Chungnam National University, Daejeon 305-764, Republic of Korea §

S Supporting Information *

ABSTRACT: Sesamin, an active ingredient of Asiasarum heterotropoides, is known to exhibit many bioactive functions, but the effect thereof on vascular smooth muscle cell (VSMC) proliferation remains poorly understood. Hence, we explored the antiproliferative action of sesamin on VSMCs and the underlying mechanism thereof, focusing on possible effects of sesamin on cell cycle progression. Sesamin significantly inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation (inhibition percentage at 1, 5, and 10 μM sesamin was 49.8 ± 22.0%, 74.6 ± 19.9%, and 87.8 ± 13.0%, respectively) in the absence of cytotoxicity and apoptosis, and PDGF-induced DNA synthesis; and arrested cell cycle progression in the G0/G1-to-S phase. Sesamin potently inhibited cyclin D1 and CDK4 expression, pRb phosphorylation, and expression of the proliferating cell nuclear antigen (PCNA); and upregulated p27KIP1, p21CIP1, and p53. The results thus indicate that the antiproliferative effect of sesamin on PDGF-stimulated VSMCs is attributable to arrest of the cell cycle in G0/G1 caused, in turn, by upregulation of p27KIP1, p21CIP1, and p53, and inhibition of cyclin E−CDK2 and cyclin D1−CDK4 expression. KEYWORDS: sesamin, vascular smooth muscle cell, proliferation, platelet-derived growth factor, cyclin-dependent kinase inhibitor

■

INTRODUCTION Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of various pathological conditions, including atherosclerosis and postangioplasty restenosis.1 Both of these events may be induced by cytokines and growth factors, including platelet-derived growth factor (PDGF).2 PDGF binds to receptors (PDGF-Rs) to promote VSMC proliferation by stimulating cell cycle progression. Entry of VSMCs into the cell cycle plays an important role in the development and progression of cellular proliferation. Thus, regulation of the cell cycle is a key strategy when it is sought to inhibit cellular proliferation.3−8 The mammalian cell cycle features four sequential phases; these are the resting G0 phase, and the G1, S, and G2/M phases, during the last three of which cell growth occurs. Most VSMCs in intact blood vessels are in the G0 phase, but a growth trigger such as a balloon injury or PDGF stimulation induces a phase transition regulated by the co-operative action of cyclindependent kinases (CDKs) and specific regulatory cyclin proteins.9,10 Formation and activation of CDK−cyclin complexes, principally CDK2−cyclin E and CDK4−cyclin D1, are required to trigger entry from the G1 phase into S. Activation of such complexes stimulates phosphorylation of the retinoblastoma (Rb) protein and enhances expression of the proliferating cell nuclear antigen (PCNA). The kinase activities of CDK−cyclin complexes can be negatively regulated via the binding thereof to CDK inhibitors (CKIs) such as p21CIP1 and p27KIP1. These CKIs, Cip/Kip © XXXX American Chemical Society

family, block cell-cycle progression by inhibiting activities of all CDKs.11,12 For example, cordycepin, the main compound derived from Cordyceps militaris, increases the binding of p27KIP1 to CDK2 and CDK4, and blocks the VSMC cell cycle via G0/G1 arrest.13 Obovatol, isolated from Magnolia obovata leaves, selectively upregulates p21CIP1, thus reducing the levels of CDK2−cyclin E and CDK4−cyclin D1.14 The p53 tumor suppressor acts upstream of p21CIP1 to regulate cell cycle progression; p53 acts at the G1 phase checkpoint.15 Sesamin (Figure 1) is a major lignan of sesame seed oil and is also present in several medicinal plants, including Asiasarum heterotropoides. Sesamin has been used as a traditional health food and exhibits various biological and pharmacological activities. The material exerts anti-cholesterol,16,17 antihypertensive,18 antioxidative,19,20 and antimicrobial21 effects and

Figure 1. Chemical structure of sesamin. Received: February 18, 2015 Revised: August 5, 2015 Accepted: August 5, 2015

A

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry protects liver function.22 Anticancer activities have been observed in various mammalian cancer cell lines.23−25 Sesamin retards the growth of HepG2 cells and induces apoptosis by inhibiting STAT3 signaling and upregulating p53 and p21, leading to cell cycle arrest in G2/M.26 A combination of γtocotrienol and sesamin treatment has a synergistic antiproliferative effect on +SA, MCF-7, and MDA-MB-231 mammary cancer cells and induced G1 cell cycle arrest by decreasing the levels of cyclin D1, CDK2, CDK4, CDK6, and p-Rb, and increasing p27KIP1 and p16INK4a expression, in +SA cells.27 However, the effects of sesamin on VSMCs remain poorly understood. Thus, we explored the inhibitory effects of sesamin on PDGF-induced VSMC proliferation and growth, and the molecular mechanisms of sesamin action.

■

DNA Synthesis Assay. [3H]-Thymidine incorporation was measured. The assay conditions were as described above for the cell proliferation assay, to the time of PDGF-BB treatment. After cell stimulation via addition of PDGF-BB (50 ng/mL) in serum-free medium, [3H]-thymidine (2 μCi/mL) was added 4 h prior to harvesting. Each reaction was terminated by aspiration of medium and sequential washing in PBS with 10% (v/v) trichloroacetic acid, and ethanol/ether (1:1, v/v). Acid-insoluble [3H]-thymidine was extracted into 500 μL amounts of 0.5 M NaOH/well, and these solutions mixed with 3 mL amounts of scintillation cocktail (Ultimagold; Packard Bioscience, Waltham, MA, USA). Radioactivity was measured with the aid of a liquid scintillation counter (LS3801; Beckman, Düsseldorf, Germany). Analysis of Cell Cycle Progression. Cell cycle progression was monitored as described previously.8 VSMCs were seeded into six-well culture plates and treated as described above (the cell proliferation assay) until the time of PDGF-BB treatment. After stimulation with PDGF-BB (50 ng/mL) for 24 h, cells were harvested by trypsinization and centrifuged at 1,500 rpm for 10 min. The pellets were suspended in 1 mL amounts of 1× PBS, washed twice, fixed in 70% (v/v) ethanol at −20 °C for 24 h, briefly vortexed, and centrifuged at 3,000 rpm for 10 min. The ethanol was discarded and the pellets stained with 500 μL propidium iodide (PI) solution (50 μg/mL PI in sample buffer with 100 μg/mL of DNase-free RNase A). After incubation at 4 °C for 30 min, the level of PI−DNA complex in each nucleus was measured using a FACScalibur (Becton Dickinson Co., Franklin Lakes, NJ, USA). The fluorescence intensity of incorporated PI was used as a measure of nuclear DNA content. The rate of cell cycle progression from the G0/G1 to the S and G2/M phases was determined using the Modfit LT software (Verity Software House, Topsham, ME, USA). Immunoblotting. Immunoblotting was performed as described previously with slight modifications.4,5 VSMCs were stimulated with 50 ng/mL PDGF-BB to trigger phosphorylation of JNK (3 min poststimulation [ps]); ERK 1/2 (5 min ps); p38 and PDGF-Rβ (10 min ps); PLCγ1, STAT3, and Akt (15 min ps). Cells were stimulated with 50 ng/mL PDGF-BB for 24 h prior to evaluation of cyclin D1, cyclin E, CDK2, CDK4, PCNA, p21CIP, p27KIP, and p53 expression levels; the extent of PARP cleavage; and the pRb phosphorylation level. Each reaction was terminated by washing the cells twice in icecold PBS followed by addition of Laemmli sample buffer; each lysate was next boiled for 10 min. For each blot, equal amounts of cell lysates were separated via sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE; 7.5−12.5% [w/v] acrylamide gels) and transferred to polyvinylidene fluoride (PVDF) membranes (Atto Corp., Tokyo, Japan) via application of 200 mA for 2 h. Blots were blocked with 5% (w/v) bovine serum albumin (BSA) in TBS-T and incubated with 1:500−1:1,000 dilutions of primary antibodies overnight at 4 °C, and then with 1:2,000 dilutions of secondary antibody. Bands were detected using ECL kits (Atto) and band intensities (normalized to those of β-actin) quantified using the Quantity One software (Bio-Rad, Hercules, CA, USA). RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Prior to RNA extraction, VSMCs were seeded into six-well culture plates and cultured in DMEM with 10% (v/v) FBS at 37 °C for 24 h. The assay conditions were as described above for the cell proliferation assay. RNA was isolated using Total RNA Extraction kits (Real-Biotech Co., Ping-Tung, Taiwan). All preparations were treated with DNaseI (RNase-free; Takara, Japan), and approximately 1 μg amounts of total RNA were reversetranscribed to cDNA using AccuPower CycleScript RT PreMix (dN12) (Bioneer, Daejeon, Korea). RT-PCR was performed with the aid of HiPi Plus PCR Premix (ELPIS, Daejeon, Korea) and specific primer pairs, on a MyCycler (Bio-Rad). All primers were synthesized by Bioneer, and all primer pairs spanned introns (Supplementary Table 1). The PCR conditions were as follows: 1 cycle of 5 min at 95 °C; followed by 30 cycles of 10 s at 95 °C, 10 s at 58 °C (p27) or 56 °C (p21 and p53), and 10 s at 72 °C; and 1 cycle of 5 min at 72 °C. The products were subjected to 2% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining.

MATERIALS AND METHODS

Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin/ streptomycin, and trypsin−EDTA were from Gibco BRL (Grand Island, NY). Anti-phospho-ERK 1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-JNK (Thr180/Tyr182), anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-PLCγ1 (Tyr783), anti-PLCγ1, anti-phosphoPDGF-Rβ chain (Tyr751), anti-PDGF-Rβ, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-Akt, anti-cleaved poly-ADP ribose polymerase (PARP), anti-PCNA, and anti-Alexa 555 antibodies were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-phospho-Akt (Ser473), anti-p53, and anti-p27KIP1 antibodies were obtained from Millipore Corporation (Billerica, MA, USA). Anti-phospho-pRb (Ser807), anti-CDK2, anti-CDK4, anti-cyclin D1, anti-cyclin E, and anti-β-actin were from Abfrontier (Geumcheon, Seoul, Korea). Antip21CIP1 was from Calbiochem (Darmstadt, Germany). Anti-FITC was from Sigma (St. Louis, MO, USA). PDGF-BB was obtained from Upstate Biotechnology (Lake Placid, NY, USA). All other chemicals were of the highest available analytical grade. Cell Culture. Rat aortic VSMCs were isolated via enzymatic dispersion, as previously described.28 Cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin, at 37 °C in a humidified incubator under 95% air and 5% CO2 (both v/v). VSMC purity was confirmed by immunocytochemical visualization of β-smooth-muscle actin. VSMCs were used at passages 4−8. Sesamin was dissolved in DMSO, but the final concentration of DMSO in any experimental medium did not exceed 0.1% (v/v). Cell Proliferation Assay. VSMC proliferation was measured using both an indirect colorimetric WST-1 assay (premix WST-1; Takara, Otsu, Japan) and direct cell counting. For the indirect assay, VSMCs were seeded into 96-well culture plates at 4 × 104 cells/mL and incubated in DMEM with 10% (v/v) FBS at 37 °C for 24 h. The medium was then replaced with serum-free DMEM, and incubation continued for 24 h prior to addition of various concentrations of sesamin, followed by a further 24 h of incubation in fresh serum-free medium. Cells were next stimulated by PDGF-BB (50 ng/mL) for another 24 h. After 22 h, WST-1 solution was added, and absorbances at 450 nm were determined 2 h later with the aid of a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). All results were expressed as percentages of the control value (no PDGF stimulation). To obtain direct cell counts, VSMCs were seeded into 12-well culture plates at 1 × 105 cells/mL and treated as described above, with the exception of WST-1 addition. After PDGF-BB treatment for 24 h, cells were suspended by addition of trypsin−EDTA and microscopically counted using a hemocytometer. Cell Viability Assay. VSMCs were seeded into 96-well culture plates at 4 × 104 cells/mL and incubated in DMEM with 10% (v/v) FBS at 37 °C for 24 h and in serum-free medium for another 24 h, and next they were exposed to 10 μM sesamin or 100 μg/mL digitonin (a cytotoxic control) for various times. WST-1 solution was added, and absorbances at 450 nm were measured 2 h later with the aid of a microplate reader. B

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 2. Effects of sesamin on proliferation of PDGF-stimulated VSMCs and cell viability. (A) Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB for a further 24 h. The WST-1 assay was performed, and the optical densities at 450 nm are shown (n = 3). (B) For direct cell counting, cells were harvested after treatment with 50 ng/mL PDGF-BB and microscopically counted using a hemocytometer (n = 4). (C) Cell viability levels were determined using the WST-1 assay. Serum-deprived VSMCs were incubated with control solution (0.1% [v/v] DMSO), or sesamin (10 μM) or digitonin (100 μg/mL), for various times (3−48 h). The values shown are means ± SEM, and significant differences from PDGF control (PDGF-stimulated, but no sesamin) values are shown by either * (P < 0.05) or ** (P < 0.01). (D) The extent of VSMC apoptosis was determined by assessment of PARP (a cell apoptosis marker) cleavage levels. The levels of full-length PARP (116 kDa) and the cleaved fragment thereof (89 kDa) were measured by immunoblotting as described in Materials and Methods. Band densities were normalized to those of β-actin. The images shown are representative of those obtained in three similar, independent experiments. Immunofluorescence. To detect PCNA, p21, and p27 via immunofluorescence, VSMCs were seeded onto glass coverslips in 24-well culture plates. The assay conditions were the same as described above for the cell proliferation assay. After treatment of VSMCs, the cells were washed twice with ice-cold PBS, fixed in 4% (v/v) formaldehyde in PBS for 15 min, and quenched in 100 mM glycine in PBS for 10 min. The cells were next washed twice with PBS; permeabilized with 0.1% (v/v) Triton X-100 for 3 min; blocked in 5% (v/v) goat serum in PBS for 1 h; and incubated with anti-PCNA antibody solution (1:500 dilution in 3% [w/v] BSA in PBS), anti-p21 antibody solution (1:100 dilution in 3% [w/v] BSA in PBS), or antip27 antibody solution (1:200 dilution in 3% [w/v] BSA in PBS), for 1.5 h. After washing with PBS, the cells were incubated with secondary antibody (FITC-IgG; 1:200 dilution in 3% [w/v] BSA in PBS) for 1.5 h. Cells were washed with PBS, mounted in Mounting Medium containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA), and observed under a confocal laser microscope (LSM5 live configuration Variotwo VRGB; Zeiss, Jena, Germany). siRNA Transfection. The p27 KIP1 siRNA (accession no. NM_031762.3), p21CIP1 siRNA (accession no. NM_080782.3), and negative control siRNA (si-con) oligonucleotides were synthesized by Bioneer Corp. (Daejeon, Korea). The target sequences of the siRNA for p27KIP1 (si-p27) were sense 5′-AGU ACA CUU GAU CAC UGA A(dTdT)-3′ and antisense 5′-UUC AGU GAU CAA GUG UAC U(dTdT)-3′, and for p21CIP1 (si-p21) were sense 5′-UGA CAG UGA AGC AGU CAC A(dTdT)-3′ and antisense 5′-UGU GAC UGC UUC ACU GUC A(dTdT)-3′. Transfection of siRNAs was performed using TransIT-X2 (Mirus BIO LLC, Madison, WI, USA) according to the manufacturer’s protocol. Final concentrations of 50 nM si-con, sip27, and si-p21 oligonucleotides with TransIT-X2 in OptiMEM were incubated with VSMCs for 6 h; then transfection medium was

removed and cells were changed to serum free medium. The ability of the siRNA oligonucleotide to knockdown target protein expression was analyzed by immunoblotting of whole cell extracts. Statistical Analysis. Data are expressed as means ± SEM. Oneway ANOVA was used to perform multiple comparisons (GraphPad Software, San Diego, CA, USA). Dunnett’s test was applied if a significant difference was evident between treatment groups. A p value less than 0.05 was considered to reflect significance.

■

RESULTS Effects of Sesamin on VSMC Proliferation and Viability. To evaluate the effect of sesamin on PDGF-induced VSMC proliferation, we performed both the WST-1 and direct cell counting assays. As shown in Figure 2A, sesamin significantly inhibited PDGF-BB-stimulated VSMC proliferation in a concentration-dependent manner. The levels of inhibition at 1, 5, and 10 μM sesamin were 49.8%, 74.6%, and 87.8%, respectively. Direct cell counting showed that VSMC numbers increased significantly after treatment with 50 ng/mL PDGF-BB (to 17.7 ± 1.5 × 104 cells/well) compared to the nonstimulated group (10.7 ± 1.8 × 104 cells/well) (Figure 2B). As the sesamin concentration increased to 1, 5, and 10 μM, the cell numbers fell significantly to 15.2 ± 2.4, 13.4 ± 1.8, and 11.2 ± 2.0 × 104 cells/well, respectively. We used the WST-1 assay to show that the antiproliferative effect was not attributable to cytotoxicity (Figure 2C). Exposure to the highest tested sesamin concentration (10 μM) for various times was not cytotoxic to VSMCs suspended in serum-free medium. Digitonin (100 μg/mL), which permeabilizes the cell C

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 3. Effects of sesamin on PDGF-Rβ downstream signaling in PDGF-stimulated VSMCs. Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB to activate PDGF-Rβ downstream signaling. After termination of the reactions, cells were lysed and immunoblotting was performed as described in Materials and Methods (A, B). Regulation by sesamin of the PDGF-induced phosphorylation of PLCγ1, Akt, PDGF-Rβ, STAT3, JNK, p38, and ERK1/2 was assessed. The gel images are representative of those obtained in four similar, independent experiments.

Figure 4. Effects of sesamin on DNA synthesis by and cell cycle progression of PDGF-stimulated VSMCs. (A) Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB for a further 24 h. To measure DNA synthesis, [3H]-thymidine (2 μCi/mL) was added 4 h prior to harvesting. Radioactivity levels were determined using a liquid scintillation counter (n = 3). All data are expressed as means ± SEM, and significant differences compared to the PDGF control (PDGF-stimulated, but no sesamin) are shown by ** (P < 0.01). (B) Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB for 24 h. Cells were harvested via trypsinization and the DNA contents of individual nuclei analyzed by flow cytometry. Each value shown was derived by counting at least 10,000 events. The numbers of cells in the G0/G1, S, and G2/M phases are expressed as percentages of total cells. The values shown are averages of data obtained in three similar, independent experiments.

membrane, was used as a positive cytotoxic control.29 To explore whether sesamin caused apoptosis PARP cleavage, a marker of cell apoptosis, was examined via immunoblotting. As shown in Figure 2D, PARP was not obviously cleaved in the presence of 1−10 μM sesamin, and we thus concluded that sesamin did not cause apoptotic cell death. Effects of Sesamin on PDGF-Rß Downstream Signaling in PDGF-Stimulated VSMCs. After angioplasty, or in the early stages of atherosclerosis, PDGF-R is overexpressed and PDGF-BB may bind thereto, triggering downstream phosphorylation of PDGF-R, phospholipase Cγ (PLCγ), protein kinase B (Akt/PKB), signal transducer, and activator of transcription 3 (STAT3), members of the mitogen-activated protein kinase (MAPK) family including c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinase 1/2 (ERK1/2).30 To explore the molecular basis of the antiproliferative activity of sesamin, we determined whether PDGF-R-activated downstream signaling molecules were induced when PDGF bound to its receptors. Figure 3A shows that stimulation of PLCγ1, Akt, PDGF-Rß, and STAT3 phosphorylation by PDGF-BB (50 ng/ mL) was not inhibited by sesamin; also, the total amounts of these proteins were not affected by sesamin. Phosphorylation of JNK, p38, and ERK1/2 was stimulated by addition of PDGF-

BB (50 ng/mL), and this was not affected by preincubation with sesamin (Figure 3B). These results indicate that the antiproliferative activity of sesamin did not involve an effect of sesamin on the PDGF-R-induced activation of downstream signaling pathways. Effects of Sesamin on DNA Synthesis and Cell Cycle Progression in PDGF-Stimulated VSMCs. To assess the effects of sesamin on DNA synthesis, we measured [3H]thymidine incorporation by VSMCs. As shown in Figure 4A, such incorporation increased significantly after stimulation with 50 ng/mL PDGF-BB (to 25,325.0 ± 3372.5 cpm/well) compared to the nonstimulated control (3,856.2 ± 813.2 cpm/well). [3H]-Thymidine incorporation into DNA of PDGF-stimulated VSMCs was significantly inhibited by sesamin, in a concentration-dependent manner. The counts after addition of 1, 5, and 10 μM sesamin were 21,628, 19,122, and 4,354 cpm/well, respectively. Flow cytometry showed that sesamin affected cell cycle progression in PDGF-induced VSMCs. As shown in Figure 4B, about 72.8 ± 3.0% and 6.5 ± 1.0% of VSMCs were in the G0/ G1 and S phases in the absence of PDGF, but these percentages changed to 49.5 ± 1.4% and 23.0 ± 1.6% upon stimulation with PDGF. Sesamin significantly inhibited cell cycle progression, in D

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 5. Effects of sesamin on cyclin E, CDK2, cyclin D1, CDK4, and PCNA expression levels, and pRb phosphorylation in PDGF-stimulated VSMCs. (A, B) Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB for 24 h. After termination of the reactions, immunoblotting was performed as described in Materials and Methods. The expression levels of CDK2, cyclin E, CDK4, and cyclin D1 and the phosphorylation status of pRb were measured via immunoblotting and normalized to the total levels of β-actin. The values shown were derived from three similar, independent experiments. Data are expressed as means ± SEM, and significant differences compared to the PDGF control (PDGF-stimulated, but no sesamin) are shown using either * (P < 0.05) or ** (P < 0.01). (C) PCNA expression levels were normalized to those of total β-actin. The values shown were derived from three similar, independent experiments. Data are expressed as means ± SEM, and significant differences compared to the PDGF control (PDGF-stimulated, but no sesamin) are shown using either * (P < 0.05) or ** (P < 0.01). (D) PCNA expression levels were measured by confocal microscopy. Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB for a further 24 h. Immunofluorescence evaluation was performed using anti-PCNA and anti-Alexa 555 antibodies. The images are representative of those obtained in two similar, independent experiments. Scale bar = 20 μm.

ylation was shown to reduce PCNA expression (Figure 5C), as also revealed by immunofluorescence (Figure 5D). Together, the results indicate that sesamin affects the expression of G0/G1 phase- and S phase-associated cell cycle components of VSMCs. Effect of Sesamin on Expression of CDK Inhibitors. The kinase activities of CDK−cyclin complexes are regulated by CKIs. We explored whether the inhibitory effect of sesamin on VSMC proliferation was mediated by modulation of CKI expression. First, the effect of sesamin alone on the expression of CKIs was measured. The results showed that sesamin enhanced expression of p27KIP1, p21CIP1, and p53 in a concentration-dependent manner (Figure 6). Since sesamin alone did not cleave PARP at the concentration rage of 1−10 μM, sesamin alone did not induce apoptosis. In PDGF-stimulated proliferative condition, the mRNA (Figure 7A) and protein (Figure 7B) levels of p27KIP1 fell upon addition of PDGF-BB. The level of p27KIP1 in PDGFstimulated VSMCs was about 52.2% that of the control (no PDGF) (Figure 7C). Inhibition of p27KIP1 expression was rescued to the extents of 10.8%, 19.6%, and 30.0% upon treatment with 1, 5, and 10 μM sesamin, respectively. The p21CIP1 protein level increased by about 36.6% upon addition of PDGF-BB. p21CIP1 expression was increased, in a concentration-dependent manner, by sesamin (the increases were 75.5%, 102.1%, and 193.5% upon treatment with 1, 5, and 10

a concentration-dependent manner. The reductions in S-phase cell numbers were 19.3 ± 2.0% (P < 0.01), 14.8 ± 3.5% (P < 0.01), and 13.2 ± 2.1% (P < 0.01) at sesamin concentrations of 1, 5, and 10 μM, respectively. The percentages of cells in the G0/G1 phase increased by 53.6 ± 3.0% (P < 0.01), 61.3 ± 2.7% (P < 0.01), and 63.6 ± 2.1% (P < 0.01), at the same sesamin concentrations. These results indicated that sesamin inhibited PDGF-induced VSMC proliferation by triggering cell-cycle arrest in the G0/G1 phase, and that this effect was associated with a reduction in DNA synthesis. Effect of Sesamin on Cell-Cycle-Related Protein Expression and Phosphorylation. Cell-cycle progression is controlled by CDKs and the cyclin proteins that regulate CDK activities. During the G0/G1-S transition, the cyclin E−cyclin dependent kinase (CDK)2 complex and the cyclin D1−CDK4 complex hyperphosphorylate pRb, inducing expression of PCNA, E2F release from pRb, and transcription of growthassociated genes.31 To explore how sesamin induced cell cycle arrest, we measured the expression levels of cyclin E, CDK2, cyclin D1, CDK4, and PCNA and the extent of pRb hyperphosphorylation. The data of Figures 5A and 5B show that sesamin significantly suppressed PDGF-induced cyclin D1 and CDK4 expression, and slightly suppressed that of cyclin E and CDK2, in a dose-dependent manner. Also, sesamin dosedependently inhibited pRb hyperphosphorylation. Sesamininduced inhibition of VSMC proliferation and pRb phosphorE

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

p27KIP1 and p21CIP1 are involved in inhibition of VSMC proliferation by sesamin. Effects of siRNA for p27KIP1 and p21CIP1 on SesaminInduced Inhibition of DNA Synthesis. To determine whether the inhibition of VSMC proliferation by sesamin is mediated via p27KIP1 and p21CIP1, cells were transfected with specific siRNA for p27KIP1 or p21CIP1 to knock down these proteins. Since p27KIP1 expression is higher in serum-deprived condition than PDGF-treated condition, it was examined only in serum-deprived condition, but p21CIP1 expression is increased by PDGF-BB, and it was examined in both conditions. In Figures 8A and 8B, p27KIP1 expression and p21CIP1 expression were silenced about ∼60% in VSMCs. The sesamin-induced inhibition of DNA synthesis was decreased in cells transfected with either siRNA (Figure 8C). Under si-con (negative control siRNA) treatment in VSMCs, 10 μM sesamin inhibited 91% of DNA synthesis. However, the inhibitory effect was alleviated to 50.6% by si-p27 (p27KIP1 siRNA) and 25% by si-p21 (p21CIP1 siRNA). Together, the results indicate that p27KIP1 and p21CIP1 may contribute to sesamin-induced inhibition of VSMC proliferation.

Figure 6. Effects of sesamin alone on p21CIP1, p27KIP1, and p53 expression levels and PARP cleavage in VSMCs. Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 48 h, and the cell lysates were obtained after reactions terminated. Immunoblotting was performed as described in Materials and Methods using the specific antibodies for p21CIP1, p27KIP1, p53, and PARP. β-Actin was used for normalization. The images are representative of those obtained in three similar, independent experiments.

μM sesamin, respectively), compared to the no-PDGF group. As expected, the mRNA and protein levels of p53, an upstream regulator of p21CIP1, increased in a similar manner. As the concentration of sesamin increased (from 1 to 5 and 10 μM sesamin), the p53 protein expression levels rose by 34.0%, 61.3%, and 92.8%, respectively. The images of cells shown in Figure 7D (p27KIP) and Figure 7E (p21CIP1) are consistent with the development of sesamin-induced alterations in mRNA and protein expression levels. Together, the results indicate that

■

DISCUSSION We present two major findings: (1) sesamin exerts an antiproliferative action on VSMCs by inhibiting cell cycle progression via arrest in G0/G1; and (2) sesamin enhances the

Figure 7. Sesamin upregulation of p21CIP1, p27KIP1, and p53 in PDGF-stimulated VSMCs. (A) Serum-deprived VSMCs were exposed to sesamin (1−10 μM) for 24 h and then stimulated with 50 ng/mL PDGF-BB for a further 24 h. Total RNA was extracted and RT-PCR analysis performed as described in Materials and Methods. Representative blots from three similar, independent experiments are shown; band intensities were normalized to those of total β-actin. (B) p21CIP1, p27KIP1, and p53 expression levels were quantified by immunoblotting after normalization of all band intensities to those of total β-actin (C). The values in the bar graphs (means ± SEM) were derived in three similar, independent experiments. Significant differences from the PDGF control (PDGF-stimulated, but no sesamin added) are noted as either * (P < 0.05) or ** (P < 0.01). (D, E) p27KIP1 and p21CIP1 were detected via confocal microscopy. Serum-deprived VSMCs were treated with sesamin (1−10 μM) for 24 h and next stimulated with 50 ng/mL PDGF-BB for a further 24 h. Immunofluorescence detection featured the use of anti-p27KIP1, anti-p21CIP1, and anti-FITC-conjugated IgGs. The images are representative of those obtained in two similar, independent experiments. Scale bar = 20 μm. F

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 8. Effects of siRNA for p27KIP1 or p21CIP1 on sesamin-induced inhibition of VSMC proliferation. The siRNAs for negative control (si-con, 50 nM), p27KIP1 (si-p27, 50 nM) or p21CIP1 (si-p21, 50 nM) were transfected for 54 h in VSMCs. TransIT-X2 was used as transfection reagent (T.F), and normal means no treatment with T.F and siRNA. Serum-deprived VSMCs were incubated with transfection mixture for 6 h and changed to serum-free medium. After 48 h, cells were lysed, and p27KIP1 (A) and p21CIP1 (B) expression was detected. (C) After incubated with transfection medium for 6 h, medium was removed and cells in serum-free medium were treated with sesamin (1−10 μM) for 24 h. After 24 h, VSMCs were stimulated with 50 ng/mL PDGF-BB for a further 24 h. To measure DNA synthesis, [3H]-thymidine incorporation was performed as described in Materials and Methods (n = 3). All data are expressed as means ± SEM, and significant differences compared to the PDGF control (PDGFstimulated, but no sesamin) are shown by ** (P < 0.01), and compared to si-con treated group are shown by ## (P < 0.01).

expression of CKIs including p27KIP1, p21CIP1, and p53; thus suppressing the binding and activation of CDK−cyclin complexes. The data suggest that sesamin may play a role in suppressing PDGF-induced VSMC proliferation in vitro, and explain the molecular mechanisms in play. VSMCs are the principal cells of arterial vessels; abnormal proliferation of such cells is associated with progression of atherosclerotic lesions.32 PDGF released mainly in activated platelet stimulates the growth of VSMCs.33 Associations between PDGF levels and the extent of VSMC proliferation have been noted in various models; inhibition of PDGF signaling prevented neointimal cellular proliferation.34 Thus, it seemed worthwhile to screen for compounds that might limit inappropriate VSMC growth under conditions of high-level PDGF expression. Sesamin is a major lignan constituent of sesame seed oil and has many biological activities, but no study to date has explored the effects of sesamin on signaling pathways associated with PDGF, the principal growth factor regulating VSMC proliferation after angioplasty. We found that sesamin inhibited PDGF-induced VSMC proliferation, but that this was not attributable to cytotoxicity or apoptosis (Figure 2). However, sesamin did not suppress PDGF-induced phosphorylation of PDGF-Rβ or downstream signaling molecules thereof, including PLCγ1, Akt, STAT3, and MAPK kinase family members (Figure 3). Thus, the antiproliferative action of sesamin is not mediated by PDGF-R-induced activation of early signaling pathways. We next explored whether sesamin acted at another level of the cellular machinery.

PDGF-stimulated cell cycle progression of VSMCs requires the binding and activation of CDK−cyclin complexes. Activation of cyclin D1, followed by cyclin E, CDK4, and CDK2, triggers phosphorylation of the Rb protein. Such phosphorylation plays a role in the G1−S transition by sequestering E2F, and stimulating the expression of PCNA, which promotes DNA synthesis.35 Sesamin suppressed PDGFstimulated DNA synthesis and caused VSMCs to arrest in the G0/G1 phase (Figure 4), implying that the actions of several cell cycle regulatory proteins may be affected by sesamin. We found that sesamin significantly suppressed cyclin D1 and CDK4 expression, thus inhibiting pRb hyperphosphorylation (Figures 5A and 5B). Although the sesamin-induced inhibition of cyclin E and CDK2 expression was weak, this was nonetheless adequate to prevent pRb hyperphosphorylation, as both cyclin D1 and CDK4 were potently suppressed.36 Moreover, sesamin inhibited PCNA expression, normally induced by hyperphosphorylated pRb in the early G0/G1 and S phases of the cell cycle (Figures 5C and 5D). CKIs are classified two families. One is Cip/Kip family including p21CIP1, p27KIP1, and p57KIP2, the other is INK4 family including p15INK4b, p16INK4a, p18INK4c, and p19INKd. It is known that the Cip/Kip family members bind to both cyclin and CDK subunits, cyclin D−, E−, A−, and B−CDK complexes, and regulate their activities. INK4 family members bind only to CDK4 or CDK6 not to cyclins and, thus, affect cell cycle progression.37 Since sesamin was shown to decrease cyclin E−CDK2 and cyclin D−CDK4 in Figure 5, p21CIP1 and p27KIP1, Cip/Kip family members, were focused on in this study. Interestingly, expression of p21CIP1, p27KIP1, and p53, G

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry upstream of p21CIP1, is increased by sesamin alone without influence of PARP cleavage (Figure 6). p53, tumor suppressor protein, regulates p21CIP1 activation in G1−S transition point, thus occurred cell cycle arrest.38 In another aspect, it is known that p53 leads to DNA damage, senescence, or apoptosis. However, p53-induced apoptotic effect may not occur in every cell line, and other cofactors or modifications are required for dead pathway.39 Furthermore, it is reported that some substances have antiproliferative effects on VSMCs by upregulation of p21CIP1 and p53.40−43 Thus, these phenomena might be cytostatic, not cytotoxic or apoptotic. The expression levels of CKIs were upregulated by sesamin in VSMCs treated with PDGF-BB (Figure 7). Upregulated CKIs bind to cyclin−CDK complexes and inhibit the catalytic activity of these complexes, and subsequently Rb phosphorylation was decreased. Sesamin-induced inhibition of DNA synthesis was restored in PDGF transfected with p27KIP1 or p21CIP1 siRNAs, and recovery rate in cells treated with p27KIP1 siRNA is larger than that in p21CIP1 siRNA (Figure 8). These results suggest that p27KIP1 may be the major factor compared to p21CIP1; however, further studies are needed. Thus, our results indicate that the inhibitory effect of sesamin on PDGFstimulated VSMC proliferation is attributable to upregulation of p27KIP1, p21CIP1, and p53, in turn triggering G0/G1 cell cycle arrest. In conclusion, we provide the first evidence that sesamin, the most abundant lignan of sesame seed oil, present also in several medicinal herbs including A. heterotropoides, inhibits PDGFinduced VSMC proliferation in vitro, attributable to upregulation of p27KIP1, p21CIP, and p53, in turn downregulating the expression levels of CDK−cyclin complexes. Therefore, sesamin may be valuable to prevent and treat vascular disorders such as restenosis developing after angioplasty, and atherosclerosis.

■

inhibitor; DMEM, Dulbecco’s modified Eagle’s medium; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinases; MAPK, mitogenactivated protein kinase; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PDGF, plateletderived growth factor; PLCγ, phospholipase Cγ; PVDF, polyvinylidene fluoride; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; STAT3, signal transducer and activator of transcription 3; VSMCs, vascular smooth muscle cells

■

(1) Ross, R. Cell biology of atherosclerosis. Annu. Rev. Physiol. 1995, 57, 791−804. (2) Ahn, H. Y.; Hadizadeh, K. R.; Seul, C.; Yun, Y. P.; Vetter, H.; Sachinidis, A. Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transductionpathway in vascular smooth muscle cells and inhibits transformation of sistransfected NIH 3T3 fibroblasts and human glioblastoma cells (A172). Mol. Biol. Cell 1999, 10, 1093−1104. (3) Dzau, V. J.; Braun-Dullaeus, R. C.; Sedding, D. G. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat. Med. 2002, 8, 1249−1256. (4) Kim, Y.; Han, J. H.; Yun, E.; Jung, S. H.; Lee, J. J.; Song, G. Y.; Myung, C. S. Inhibitory effect of a novel naphthoquinone derivative on proliferation of vascular smooth muscle cells through suppression of platelet-derived growth factor receptor beta tyrosine kinase. Eur. J. Pharmacol. 2014, 733, 81−89. (5) Kim, Y.; Lee, J. J.; Lee, S. G.; Jung, S. H.; Han, J. H.; Yang, S. Y.; Yun, E.; Song, G. Y.; Myung, C. S. 5,8-Dimethoxy-2-nonylaminonaphthalene-1,4-dione inhibits vascular smooth muscle cell proliferation by blocking autophosphorylation of PDGF-receptor β. Korean J. Physiol. Pharmacol. 2013, 17, 203−208. (6) Lee, J. J.; Yi, H.; Kim, I. S.; Kim, Y.; Nhiem, N. X.; Kim, Y. H.; Myung, C. S. 2S)-Naringenin from Typha angustata inhibits vascular smooth muscle cell proliferation via a G0/G1 arrest. J. Ethnopharmacol. 2012, 139, 873−878. (7) Lee, J. J.; Yu, J. Y.; Zhang, W. Y.; Kim, T. J.; Lim, Y.; Kwon, J. S.; Kim, D. W.; Myung, C. S.; Yun, Y. P. Inhibitory effect of fenofibrate on neointima hyperplasia via G0/G1 arrest of cell proliferation. Eur. J. Pharmacol. 2011, 650, 342−349. (8) Lee, J. J.; Zhang, W. Y.; Yi, H.; Kim, Y.; Kim, I. S.; Shen, G. N.; Song, G. Y.; Myung, C. S. Anti-proliferative actions of 2-decylamino5,8-dimethoxy-1,4-naphthoquinone in vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 2011, 411, 213−218. (9) Xiong, Y.; Hannon, G. J.; Zhang, H.; Casso, D.; Kobayashi, R.; Beach, D. p21 is a universal inhibitor of cyclin kinases. Nature 1993, 366, 701−704. (10) Hunter, T.; Pines, J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 1994, 79, 573−582. (11) Coqueret, O. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 2003, 13, 65−70. (12) Besson, A.; Dowdy, S. F.; Roberts, J. M. CDK inhibitors: cell cycle regulators and beyond. Dev. Cell 2008, 14, 159−169. (13) Jung, S. M.; Park, S. S.; Kim, W. J.; Moon, S. K. Ras/ERK1 pathway regulation of p27KIP1-mediated G1-phase cell-cycle arrest in cordycepin-induced inhibition of the proliferation of vascular smooth muscle cells. Eur. J. Pharmacol. 2012, 681, 15−22. (14) Lim, Y.; Kwon, J. S.; Kim, D. W.; Lee, S. H.; Park, R. K.; Lee, J. J.; Hong, J. T.; Yoo, H. S.; Kwon, B. M.; Yun, Y. P. Obovatol from Magnolia obovata inhibits vascular smooth muscle cell proliferation and intimal hyperplasia by inducing p21Cip1. Atherosclerosis 2010, 210, 372−380. (15) Macleod, K. F.; Sherry, N.; Hannon, G.; Beach, D.; Tokino, T.; Kinzler, K.; Vogelstein, B.; Jacks, T. p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 1995, 9, 935−944.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03374. Table of primers used for PCR (PDF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Department of Pharmacology, Chungnam National University College of Pharmacy, 99 Daehak-ro, Yuseong-gu, Daejeon 305764, Republic of Korea. Tel: +82-42-821-5923. Fax: +82-42821-8925. E-mail: [email protected]. Author Contributions ‡

J.-H.H. and S.-G.L. contributed equally to this work and therefore share first authorship. Funding

This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (Grant No. 2009-0093815). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED Akt/PKB, protein kinase B; BSA, bovine serum albumin; CDK, cyclin-dependent kinase; Rb, retinoblastoma; CKI, CDK H

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry (16) Rogi, T.; Tomimori, N.; Ono, Y.; Kiso, Y. The mechanism underlying the synergetic hypocholesterolemic effect of sesamin and alpha-tocopherol in rats fed a high-cholesterol diet. J. Pharmacol. Sci. 2011, 115, 408−416. (17) Liang, Y. T.; Chen, J.; Jiao, R.; Peng, C.; Zuo, Y.; Lei, L.; Liu, Y.; Wang, X.; Ma, K. Y.; Huang, Y.; Chen, Z. Y. Cholesterol-lowering activity of sesamin is associated with down-regulation on genes of sterol transporters involved in cholesterol absorption. J. Agric. Food Chem. 2015, 63, 2963−2969. (18) Lee, C. C.; Chen, P. R.; Lin, S.; Tsai, S. C.; Wang, B. W.; Chen, W. W.; Tsai, C. E.; Shyu, K. G. Sesamin induces nitric oxide and decreases endothelin-1 production in HUVECs: possible implications for its antihypertensive effect. J. Hypertens. 2004, 22, 2329−2338. (19) Nakano, D.; Kurumazuka, D.; Nagai, Y.; Nishiyama, A.; Kiso, Y.; Matsumura, Y. Dietary sesamin suppresses aortic NADPH oxidase in DOCA salt hypertensive rats. Clin. Exp. Pharmacol. Physiol. 2008, 35, 324−326. (20) Lee, W. J.; Ou, H. C.; Wu, C. M.; Lee, I. T.; Lin, S. Y.; Lin, L. Y.; Tsai, K. L.; Lee, S. D.; Sheu, W. H. Sesamin mitigates inflammation and oxidative stress in endothelial cells exposed to oxidized lowdensity lipoprotein. J. Agric. Food Chem. 2009, 57, 11406−11417. (21) Bankole, M. A.; Shittu, L. A.; Ahmed, T. A.; Bankole, M. N.; Shittu, R. K.; Kpela, T.; Ashiru, O. A. Synergistic antimicrobial activities of phytoestrogens in crude extracts of two sesame species against some common pathogenic microorganisms. Afr. J. Tradit., Complementary Altern. Med. 2007, 4, 427−433. (22) Periasamy, S.; Hsu, D. Z.; Chang, P. C.; Liu, M. Y. Sesame oil attenuates nutritional fibrosing steatohepatitis by modulating matrix metalloproteinases-2, 9 and PPAR-γ. J. Nutr. Biochem. 2014, 25, 337− 344. (23) Hibasami, H.; Fujikawa, T.; Takeda, H.; Nishibe, S.; Satoh, T.; Fujisawa, T.; Nakashima, K. Induction of apoptosis by Acanthopanax senticosus HARMS and its component, sesamin in human stomach cancer KATO III cells. Oncol. Rep. 2000, 7, 1213−1219. (24) Ju, Y.; Still, C. C.; Sacalis, J. N.; Li, J.; Ho, C. T. Cytotoxic coumarins and lignans from extracts of the northern prickly ash (Zanthoxylum americanum). Phytother. Res. 2001, 15, 441−443. (25) Miyahara, Y.; Komiya, T.; Katsuzaki, H.; Imai, K.; Nakagawa, M.; Ishi, Y.; Hibasami, H. Sesamin and episesamin induce apoptosis in human lymphoid leukemia Molt 4B cells. Int. J. Mol. Med. 2000, 6, 43− 46. (26) Deng, P.; Wang, C.; Chen, L.; Wang, C.; Du, Y.; Yan, X.; Chen, M.; Yang, G.; He, G. Sesamin induces cell cycle arrest and apoptosis through the inhibition of signal transducer and activator of transcription 3 signalling in human hepatocellular carcinoma cell line HepG2. Biol. Pharm. Bull. 2013, 36, 1540−1548. (27) Akl, M. R.; Ayoub, N. M.; Abuasal, B. S.; Kaddoumi, A.; Sylvester, P. W. Sesamin synergistically potentiates the anticancer effects of γ-tocotrienol in mammary cancer cell lines. Fitoterapia 2013, 84, 347−359. (28) Chamley, J. H.; Campbell, G. R.; McConnell, J. D.; GroschelStewart, U. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res. 1977, 177, 503−522. (29) Eid, S. Y.; El-Readi, M. Z.; Wink, M. Digitonin synergistically enhances the cytotoxicity of plant secondary metabolites in cancer cells. Phytomedicine 2012, 19, 1307−1314. (30) Claesson-Welsh, L. Platelet-derived growth factor receptor signals. J. Biol. Chem. 1994, 269, 32023−32026. (31) Black, A. R.; Jensen, D.; Lin, S. Y.; Azizkhan, J. C. Growth/cell cycle regulation of Sp1 phosphorylation. J. Biol. Chem. 1999, 274, 1207−1215. (32) Gutstein, W. H.; Teresi, J. A.; Wu, J. M.; Ramirez, M.; Salimian, F.; Cui, Y.; Paul, I.; Jabr, S. Increased serum mitogenic activity for arterial smooth muscle cells associated with relaxation and low educational level in human subjects with high but not low hostility traits: implications for atherogenesis. J. Psychosom. Res. 1999, 46, 51− 61.

(33) Uchida, K.; Sasahara, M.; Morigami, N.; Hazama, F.; Kinoshita, M. Expression of platelet-derived growth factor B-chain in neointimal smooth muscle cells of balloon injured rabbit femoral arteries. Atherosclerosis 1996, 124, 9−23. (34) Leppanen, O.; Janjic, N.; Carlsson, M. A.; Pietras, K.; Levin, M.; Vargeese, C.; Green, L. S.; Bergqvist, D.; Ostman, A.; Heldin, C. H. Intimal hyperplasia recurs after removal of PDGF-AB and -BB inhibition in the rat carotid artery injury model. Arterioscler., Thromb., Vasc. Biol. 2000, 20, E89−95. (35) Zhu, P.; Chen, J. M.; Chen, S. Z.; Zhang, C.; Zheng, S. Y.; Long, G.; Chen, J.; Zhou, Z. L.; Fan, R. X.; Fan, X. P.; Chen, Y. F.; Zhuang, J. Matrine inhibits vascular smooth muscle cell proliferation by modulating the expression of cell cycle regulatory genes. Acta Pharmacol. Sin. 2010, 31, 1329−1335. (36) Connell-Crowley, L.; Elledge, S. J.; Harper, J. W. G1 cyclindependent kinases are sufficient to initiate DNA synthesis in quiescent human fibroblasts. Curr. Biol. 1998, 8, 65−68. (37) Sherr, C. J.; Roberts, J. M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999, 13, 1501−1512. (38) el-Deiry, W. S.; Tokino, T.; Velculescu, V. E.; Levy, D. B.; Parsons, R.; Trent, J. M.; Lin, D.; Mercer, W. E.; Kinzler, K. W.; Vogelstein, B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993, 75, 817−825. (39) Aylon, Y.; Oren, M. Living with p53, dying of p53. Cell 2007, 130, 597−600. (40) Igata, M.; Motoshima, H.; Tsuruzoe, K.; Kojima, K.; Matsumura, T.; Kondo, T.; Taguchi, T.; Nakamaru, K.; Yano, M.; Kukidome, D.; Matsumoto, K.; Toyonaga, T.; Asano, T.; Nishikawa, T.; Araki, E. Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression. Circ. Res. 2005, 97, 837−844. (41) Ki, S. H.; Lee, J. W.; Lim, S. C.; Hien, T. T.; Im, J. H.; Oh, W. K.; Lee, M. Y.; Ji, Y. H.; Kim, Y. G.; Kang, K. W. Protective effect of nectandrin B, a potent AMPK activator on neointima formation: inhibition of Pin1 expression through AMPK activation. Br. J. Pharmacol. 2013, 168, 932−945. (42) Wu, W. Y.; Yan, H.; Wang, X. B.; Gui, Y. Z.; Gao, F.; Tang, X. L.; Qin, Y. L.; Su, M.; Chen, T.; Wang, Y. P. Sodium tanshinone IIA silate inhibits high glucose-induced vascular smooth muscle cell proliferation and migration through activation of AMP-activated protein kinase. PLoS One 2014, 9, e94957. (43) Chien, M. H.; Lee, T. S.; Liang, Y. C.; Lee, W. S. β-Sitosterol inhibits cell cycle progression of rat aortic smooth muscle cells through increases of p21cip1 protein. J. Agric. Food Chem. 2010, 58, 10064− 10069.

I

DOI: 10.1021/acs.jafc.5b03374 J. Agric. Food Chem. XXXX, XXX, XXX−XXX