European Journal of Pharmacology 733 (2014) 81–89

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Inhibitory effect of a novel naphthoquinone derivative on proliferation of vascular smooth muscle cells through suppression of platelet-derived growth factor receptor β tyrosine kinase Yohan Kim a,1, Joo-Hui Han a,1, Eunju Yun b, Sang-Hyuk Jung a, Jung-Jin Lee a,c, Gyu-Yong Song b,c, Chang-Seon Myung a,c,n a b c

Department of Pharmacology, Chungnam National University College of Pharmacy, 99 Daehakno, Yuseong-gu, Daejeon 305-764, Republic of Korea Department of Medicinal 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

art ic l e i nf o

a b s t r a c t

Article history: Received 25 January 2014 Received in revised form 10 March 2014 Accepted 18 March 2014 Available online 30 March 2014

This study was designed to investigate the antiproliferative effect of a novel naphthoquinone derivative, 2‐undecylsulfonyl‐5,8‐dimethoxy‐1,4‐naphthoquinone (2-undecylsulfonyl-DMNQ), on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) and examine the possible molecular mechanism of its antiproliferative action. 2-Undecylsulfonyl-DMNQ significantly inhibited PDGF-stimulated cell number and DNA synthesis, and arrested the PDGF-stimulated progression through G0/G1 to S phase of cell cycle supported by the suppression of pRb phosphorylation and cyclin D1/E, CDK2/4 and PCNA expressions. 2-Undecylsulfonyl-DMNQ dose-dependently inhibited the PDGF-stimulated phosphorylation of phospholipase Cγ (PLCγ), protein kinase B (Akt/PKB), signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK 1/2). In addition, 2-undecylsulfonyl-DMNQ inhibited PDGF-induced PDGF receptor β (PDGF-Rβ) dimerization and the phosphorylation of Tyr579/581, Tyr716, Tyr751 and Tyr1021 in PDGF-Rβ. However, 2-undecylsulfonyl-DMNQ has no antiproliferative effect on epidermal growth factor (EGF)- or fetal bovine serum (FBS)-stimulated VSMCs. In conclusion, these findings suggest that the antiproliferative effects of 2-undecylsulfonyl-DMNQ on PDGF-stimulated VSMCs are due to the blockade of receptor dimerization and autophosphorylation on specific tyrosine residues of PDGF-Rβ, which resulted in the subsequent suppression of signaling cascades and a cell cycle arrest. Our observation may explain an important mechanism to block the integration of multiple signals generated by growth factor receptor activation for prevention of VSMC proliferation in cardiovascular diseases. & 2014 Elsevier B.V. All rights reserved.

Keywords: Vascular smooth muscle cell Proliferation Platelet-derived growth factor-receptor 2-Undecylsulfonyl-DMNQ Cardiovascular disease

β

1. Introduction

Abbreviations: 2‐undecylsulfonyl‐DMNQ, 2‐undecylsulfonyl‐5,8‐dimethoxy‐1,4‐ naphthoquinone; VSMC, vascular smooth muscle cell; PDGF, platelet-derived growth factor; PLC, phospholipase C; Akt/PKB, protein kinase B; STAT 3, signal transducers and activators of transcription 3; ERK 1/2, extracellular signalregulated kinases 1/2; PDGF-Rβ, PDGF receptor β; PI3K, phosphatidylinositol 3kinase; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; CDK, cyclin-dependent kinase; pRb, phospho-protein retinoblastoma; PCNA, proliferating cell nuclear antigen; DMEM, Dulbecco's modified Eagle's medium n Corresponding author at: Department of Pharmacology, Chungnam National University College of Pharmacy, 99 Daehakno, Yuseong-gu, Daejeon 305-764, Republic of Korea. Tel.: þ82 42 821 5923; fax: þ 82 42 821 8925. E-mail address: [email protected] (C.-S. Myung). 1 These two authors contributed equally. http://dx.doi.org/10.1016/j.ejphar.2014.03.037 0014-2999/& 2014 Elsevier B.V. All rights reserved.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a major contributor to atherosclerosis and vascular restenosis after angioplasty. Many studies, in determining the mechanisms by which growth factors control cell proliferation, have contributed to the development of treatment strategies that target specific signal transduction pathways to control proliferative disorders. The binding of specific growth factors with their selective cell surface receptor tyrosine kinases results in its autophosphorylation and activation, leading to downstream signal transduction through chains of intercommunicating proteins culminating in cell proliferation (Seedorf, 1995). Platelet-derived growth factor (PDGF) and its receptors participate in various physiological processes such as embryonic development and wound healing (Alvarez et al., 2006). An abnormally high activity of PDGF is believed to play a central role in the etiology of certain adverse pathophysiological situations, such as

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atherosclerosis and restenosis (Ross, 1993). Thus, pharmacologic inhibition of PDGF-induced VSMC proliferation and migration during lesion development could be a novel therapeutic strategy. The signaling pathway involved in mitogenesis through PDGF receptor β (PDGF-Rβ) has been relatively well characterized. The binding of PDGF to the PDGF-Rβ leads to its phosphorylation at multiple tyrosine residues. The activated PDGF-Rβ is associated with a number of SH2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) γ1 (Muller, 1997). Several molecules have been implicated in mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular regulated kinases 1 and 2 (ERK1/2) by triggering activation of Raf-1. Moreover, ERK1/2 mediates several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest, and differentiation, in a cell type-dependent manner (Hommes et al., 2003). Thus, approaches using PDGF-Rβ antagonism or protein kinase inhibitor reduced VSMC proliferation in vitro and prevented cardiovascular problems in several animal experiments (Lipson et al., 1998). Compounds with the naphthoquinone backbone are known to have pronounced biological effects. In particular, they have been credited with antitumor, antiviral, antifungal, antimycobacterial, and antiplatelet activities (Kar and Carr, 2000). In addition, a naphthoquinone analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), has long been studied as a model quinone compound in the field of toxicology (Shi et al., 1993) as well as redox signaling (Liu et al., 1998). A recent report presented that 2-decylaminoDMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and this action is through cell cycle arrest at the G0/G1 phase (Lee et al., 2011a). The results of ongoing research to identify novel DMNQ derivatives having anti-proliferative action with the blockade of growth factor receptor levels have been reported that 2-nonylamino-DMNQ inhibited PDGF-Rβ-mediated downstream signaling pathways by blocking receptor autophosphorylation (Kim et al., 2013). Among 2-amino substituted DMNQ, 2-decylamino-DMNQ and 2-nonylamino-DMNQ exerted the strongest inhibitory action of VSMC proliferation. Thus, this study was designed to examine the inhibitory effect of a newly synthesized naphthoquinone derivate, 2-undecylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone (2-undecylsulfonyl-DMNQ) which has 2-sulfur substituted DMNQ derivative instead of 2-amino, on VSMC proliferation, and investigate the underlying mechanisms at the level of growth factor receptors.

2. Materials and methods 2.1. Materials Cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Anti-phospho-ERK1/2, anti-phospho-PLCγ1, anti-phosphoPDGF-Rβ chain (Tyr751, Tyr1021), anti-phospho-STAT3 (Tyr705), antiERK1/2, anti-Akt, anti-PLCγ1, anti-PDGF-Rβ, and apoptotic marker poly ADP-ribose polymerase (PARP) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-phosphoPDGF-Rβ chain (Tyr716, Tyr579/581) and anti-phospho-Akt antibodies were obtained from Millipore Corporation (Billerica, MA, USA). Anti-PDGF-Rβ, phosphotyrosine antibody (PY99) and protein A/G plus-agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BS3 was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Anti-phospho-pRb, anti-CDK2, anti-CDK4, antiphospho PCNA, anti-cyclin D1, anti-cyclin E, anti-Akt, and anti-β-actin antibodies were purchased from Abfrontier (Geumcheon, Seoul, Korea). PDGF-BB and EGF were obtained from Upstate Biotechnology (Lake Placid, NY, USA). Other chemicals were of analytical grade.

2.2. Cell culture Rat aortic VSMCs were isolated by enzymatic dispersion as previously described (Chamley et al., 1977). Cells were cultured in DMEM, supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 8 mM HEPES and 2 mM L-glutamine at 37 1C in a humidified atmosphere of 95% air and 5% CO2 incubator. The purity of VSMCs culture was confirmed by immunocytochemical localization of β-smooth-muscle actin. The passage of VSMCs used in this experiment was 4–8. 2.3. Cell proliferation assay The proliferation of VSMCs was measured by both direct counting and nonradioactive colorimetric WST-1 assay (premix WST-1, Takara, Japan). For direct cell counting, VSMCs were seeded into 12-well culture plates at 4  104 cells/ml, and then cultured in DMEM containing 10% FBS at 37 1C for 24 h. After reaching at  70% of confluence, the cells were incubated with serumfree medium for 24 h, treated with various concentrations of 2-undecylsulfonyl-DMNQ for another 24 h in a newly fresh serum-free medium, and stimulated by PDGF-BB (25 ng/ml). 2-Undecylsulfonyl-DMNQ was dissolved in DMSO and the final concentration of DMSO in medium was not exceeded by 0.1%. After 24 h the cells were trypsinized by trypsin–EDTA and counted by using a hemocytometer under microscopy. For nonradioactive colorimetric WST-1 assay, all experimental procedures were performed as recommended by manufacturer's instructions, and the results were expressed as a percentage of control. 2.4. Cell viability assay VSMCs were seeded into 96-well culture plates at 2  104 cells/ml, and then cultured in DMEM containing 10% FBS at 37 1C for 24 h. When cells reached at  70% of confluence, cells were incubated with serum-free medium for another 24 h, and then exposed to 1 μM 2-undecylsulfonyl-DMNQ or 100 μg/ml digitonin as a cytotoxic control for given time. After 2 h of incubating cells with WST-1 reagent, the absorbance was measured at 450 nm using a microplate reader (Packard Instrument Co., Downers Grove, IL, USA). 2.5. DNA synthesis assay DNA synthesis was determined by [3H]-thymidine incorporation assay as previously described (Lee et al., 2011b). The assay condition was the same as described in the cell proliferation assay section. After adding 25 ng/ml PDGF-BB to serum-free medium, [3H]-thymidine (2 μCi/ml) was added for 4 h before harvesting. The reaction was terminated by aspirating the medium and subjecting the cultures to sequential washes on ice with phosphate-buffered saline (PBS) containing 10% trichloroacetic acid and ethanol/ether (1:1, v/v). Acid-insoluble [3H]-thymidine was extracted into 250 ml of 0.5 M NaOH/well, and this solution was mixed with 3 ml scintillation cocktail (Ultimagold, Packard Bioscience, CT), and quantified using a liquid scintillation counter (LS3801, Beckman, Düsseldorf, Germany). 2.6. Cell cycle progression analysis The measurement of cell cycle progression was performed as previously described (Lee et al., 2011b). The assay condition was the same as described in the cell proliferation assay section. After being stimulated by PDGF-BB (25 ng/ml) for 24 h, cells were trypsinized and centrifuged at 1,500g for 7 min. The centrifuged pellets were suspended in 1 ml of 1  PBS, washed twice, and

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fixed with 70% ethanol for 48 h. The fixed cells were briefly vortexed and centrifuged at 15,000g for 5 min. The ethanol was discarded and the pellets were stained with 500 μl propidium iodide (PI) solution (50 μg/ml PI in sample buffer containing 100 μg/ml of RNase A). Before analysis by flow cytometry, each sample was incubated at room temperature for 1 h. The PI–DNA complex in each cell nucleus was measured using the FACScalibur flow cytometer (Becton & Dickinson Co., Franklin Lakes, NJ, USA). The individual nuclear DNA content was reflected by fluorescence intensity of incorporated PI. The rate of the cell cycle within the G0/G1, S and G2/M phase was determined by analysis with Modfit LT software. 2.7. Immunoblotting assay Immunoblotting assay was performed as previously described (Lee et al., 2011a). VSMCs were stimulated with 25 ng/ml PDGF-BB for phosphorylation of PDGF-Rβ (3 min), ERK 1/2 and PLCγ1 (5 min), STAT3 (10 min), and Akt (15 min). For the detection of cyclin D1, cyclin E, CDK2, CDK4 and PCNA expressions, and pRb phosphorylation, cells were stimulated by PDGF-BB (25 ng/ml) for 24 h. The detected proteins were normalized by β-actin or respective total proteins. The intensities of bands were quantified using the Quantity One program (Bio-Rad, Hercules, CA, USA). As specific inhibitors, SU6656 (2 μM) for STAT3 and AG1295 (5 μM) for PDGFRβ were used. 2.8. Immunoprecipitation and analysis of receptor dimerization Cells treated with various concentrations of 2-undecylsulfonylDMNQ for 24 h were incubated with PDGF-BB (25 ng/ml) for 1 h at 4 1C. After stimulation, cells were washed with ice-cold PBS and BS3, a non-cleavable cross-linker, was added to a final concentration of 1.5 mM for 1 h. This reaction was quenched by incubation with 10 mM Tris buffer (pH 7.5) and cells were lysed in a solution containing 20 mM Tis (pH 7.4), 137 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM sodium fluoride, 2 mM EDTA, 1 mM orthovanadate, and 1 mM phenylmethylsulfonyl fluoride for 30 min at 4 1C. Insoluble materials were removed from lysates by centrifugation at 13,000 rpm for 5 min at 4 1C. Protein concentrations were determined by the BCA assay and PDGF-Rβ was immunoprecipitated by 1 μg antibody. After this mixture was shaken for overnight at 4 1C, protein A/G plusagarose was added. The precipitates were heated for 5 min at 90 1C in loading buffer and analyzed by 4% SDS-PAGE, followed by immunoblotting with phosphotyrosine antibody PY99.

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2.9. Statistical analysis Data are expressed as the mean 7standard error of the mean (S.E.M.). A one-way ANOVA was used for multiple comparisons (GraphPad, San Diego, CA, USA). If a significant difference between treated groups was found, Dunnett's test was applied. Differences with P o0.05 were considered statistically significant.

3. Results 3.1. Influence of 2-undecylsulfonyl-DMNQ on VSMC proliferation and viability Data shown in Fig. 1A illustrate that 2-undecylsulfonylDMNQ inhibited PDGF-stimulated proliferation of VSMCs in a concentration-dependent manner. The number of VSMCs significantly increased after treatment with 25 ng/ml PDGF-BB (9.871.9  104 cells/well) compared with the non-stimulated group (4.970.5  104 cells/well). As the concentration of 2-undecylsulfonyl-DMNQ was increased to 0.1, 0.5, and 1.0 μM, the cell number was significantly reduced to 6.7 71.3, 5.3 71.1, and 4.7 71.0  104 cells/well, respectively. However, 2-undecylsulfonyl-DMNQ has no negative impact on VSMCs viability in vitro (Fig. 1B). Treatment with the highest concentration of 2undecylsulfonyl-DMNQ (1 μM) at various incubation times did not show any cytotoxicity for VSMCs in serum-free medium, indicating that the antiproliferative effect of 2-undecylsulfonylDMNQ on VSMCs was not due to cytotoxicity. Digitonin (100 μg/ ml) was used as a positive control for the cytotoxic agents.

3.2. Selective inhibition of PDGF-induced VSMC proliferation by 2-undecylsulfonyl-DMNQ To examine the selectivity of 2-undecylsulfonyl-DMNQ in growth factor-induced VSMC proliferation, PDGF (25 ng/ml), EGF (10 ng/ml), and FBS (5%) were applied, and the inhibitory action of 2-undecylsulfonyl-DMNQ was measured using non-radioactive colorimetric WST-1 assay. Data shown in Fig. 2 represent that 2undecylsulfonyl-DMNQ significantly inhibited only VSMC proliferation induced by PDGF, not by EGF or FBS. Thus, these results indicate that the inhibitory action of 2-undecylsulfonyl-DMNQ is selective for PDGF-induced VSMC proliferation.

Fig. 1. Influence of 2-undecylsulfonyl-DMNQ on vascular smooth muscle cell proliferation and viability. (A) The ability of 2-undecylsulfonyl-DMNQ to inhibit PDGF-induced VSMC proliferation. VSMCs cultured in serum-free medium were treated with the given concentration of 2-undecylsulfonyl-DMNQ for 24 h, stimulated by PDGF-BB (25 ng/ ml) for another 24 h, and counted by hemocytometer under microscopy. (B) The effect of 2-undecylsulfonyl-DMNQ on cell viability. VSMCs cultured in serum-free medium were incubated with control (0.1% DMSO) or 2-undecylsulfonyl-DMNQ (1 μM) or digitonin (100 μg/ml) for indicated periods of time. Optical density was determined at 450 nm by WST-1 assay. The values are expressed as mean 7 S.E.M., and statistical difference from PDGF control (PDGF-stimulated, but no 2-undecylsulfonyl-DMNQ) was illustrated by either nPo 0.05 or nnP o 0.01.

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Fig. 2. Selective inhibition of PDGF-induced vascular smooth muscle cell proliferation by 2-undecylsulfonyl-DMNQ. VSMCs cultured in serum-free medium were treated with the given concentration of 2-undecylsulfonyl-DMNQ for 24 h and stimulated by PDGF-BB (25 ng/ml) or EGF (10 ng/ml) or FBS (5%) for 24 h. Nonradioactive colorimetric WST-1 assay was performed as described under Section 2. The values are expressed as mean 7 S.E.M., and statistical difference from PDGF control (PDGF-stimulated, but no 2-undecylsulfonyl-DMNQ) was illustrated by either nP o0.05 or nnPo 0.01.

2-undecylsulfonyl-DMNQ concentrations of 0.1, 0.5 and 1.0 μM, respectively. This finding indicates that 2-undecylsulfonyl-DMNQ may act in the early events of the cell cycle to be effective against DNA synthesis induced by PDGF.

3.5. Effect of 2-undecylsulfonyl-DMNQ on cyclin D/E, CDK 2/4 and PCNA expression, and pRb phosphorylation

Fig. 3. Effect of 2-undecylsulfonyl-DMNQ on PDGF-induced increase in [3H]thymidine incorporation. PDGF-BB (25 ng/ml)-stimulated VSMCs cultured in serum-free medium were treated with or without the indicated concentration of 2-undecylsulfonyl-DMNQ for 24 h. [3H]-thymidine (2 μCi/ml) was added for 4 h before harvesting. Radioactivity was determined using a liquid scintillation counter. The results are representative of 3 similar experiments, each performed in quadruplicate. The values are expressed as mean7 S.E.M., and statistical difference from PDGF control (PDGF-stimulated, but no 2-undecylsulfonyl-DMNQ) was illustrated by either nP o0.05 or nnP o 0.01.

3.3. Effect of 2-undecylsulfonyl-DMNQ on DNA synthesis The effect of 2-undecylsulfonyl-DMNQ on DNA synthesis was assayed using [3H]-thymidine incorporation. Data shown in Fig. 3 represent that [3H]-thymidine incorporation significantly increased after stimulation with 25 ng/ml PDGF-BB (16996.9 7 2886.5 cpm/well) compared to the non-stimulated group (1583.37 445.6 cpm/well). PDGF-stimulated [3H]-thymidine incorporation into DNA in VSMCs was inhibited by 2-undecylsulfonyl-DMNQ in a concentration-dependent manner. The inhibition rates of 0.1, 0.5 and 1.0 μM 2-undecylsulfonyl-DMNQ were 43.13 78.79%, 61.59 79.17% and 71.447 5.23%, respectively. 3.4. Effect of 2-undecylsulfonyl-DMNQ on cell cycle progression The effect of 2-undecylsulfonyl-DMNQ on cell cycle progression was also analyzed by flow cytometry. Data shown in Fig. 4A represent that serum deprivation of VSMCs for 24 h resulted in approximately 86.4 71.6% synchronization of the cell cycle in the G0/G1 phase, and the percentage of cells in S phase increased from 3.3 71.2% to 18.6 72.7% for 24 h after PDGF was added. 2-Undecylsulfonyl-DMNQ significantly blocked cell cycle progression in a dose-dependent manner. The reduced percentage of cells in the S phase was 16.6 71.8%, 13.6 71.7% and 9.1 70.5% at

To characterize mechanism of cell cycle arrest by 2-undecylsulfonyl-DMNQ, the expressions of cyclin D1, cyclin E, CDK 2 and CDK 4 were measured. During the G1–S transition, the cyclin D1CDK 4 complex and the cyclin E-CDK 2 complex mediate hyperphosphorylation of pRb, which results in E2F release and the transcription of growth-associated gene (Black et al., 1999). Hyperphosphorylated pRb binds to the E2F family of transcription factors, and thus inhibits transcription of E2F-responsive genes necessary for cell cycle progression. Data shown in Fig. 4B illustrate that 2-undecylsulfonyl-DMNQ significantly suppressed the expression of cyclin D, cyclin E, CDK 2 and CDK 4 in a concentration-dependent manner. Moreover, 2-undecylsulfonylDMNQ induced a concentration-dependent inhibition of hyperphosphorylation of pRb and the expression of PCNA. Note that 2-undecylsulfonyl-DMNQ did not affect cell apoptosis marker PARP cleavage (Fig. 4C). These results indicate the inhibition of cell cycle in the S phase via G0/G1 arrest by 2-undecylsulfonylDMNQ without affecting cell apoptosis.

3.6. Effect of 2-undecylsulfonyl-DMNQ on the phosphorylation of PLCγ1, Akt, ERK1/2, and STAT3 in PDGF-stimulated VSMCs PDGF binding to PDGF receptor leads to the activation of several intracellular signaling cascades including PLCγ1, Akt, ERK1/2 and STAT3. Data shown in Fig. 5A present that the increased phosphorylation of PLCγ1, Akt, and ERK1/2 stimulated by PDGF-BB (25 ng/ml) was significantly inhibited with 2-undecylsulfonyl-DMNQ in a concentration-dependent manner. Total amounts of PLCγ1, Akt, and ERK1/2 were not affected by 2-undecylsulfonyl-DMNQ. The phosphorylation of STAT3 was stimulated by addition of PDGF, and this increased phosphorylation was dose-dependently inhibited by preincubating with 2undecylsulfonyl-DMNQ (Fig. 5B). Preincubation with 2 μM SU6656, a Src family kinase inhibitor, completely abolished phosphorylation of STAT3 (Tyr705), indicating a Src-dependent but JAK2-independent phosphorylation of STAT3 on PDGF treatment in VSMCs (Schwaiberger et al., 2010).

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2-Undecylsulfonyl-DMNQ Fig. 4. Effect of 2-undecylsulfonyl-DMNQ on cell cycle progression and cell cycle regulatory proteins in PDGF-stimulated vascular smooth muscle cells. (A) The ability of 2-undecylsulfonyl-DMNQ to regulate cell cycle progression. Serum-starved VSMCs were treated with or without the indicated concentration of 2-undecylsulfonyl-DMNQ for 24 h, stimulated with PDGF-BB (25 ng/ml) for 24 h, and harvested as described under Section 2. Cellular DNA was stained with propidium iodide and flow cytometric analysis of DNA contents was performed. Each item was derived from a representative experiment, where data from at least 10,000 events were obtained. The results are an average of 3 similar and independent experiments. (B) The ability of 2-undecylsulfonyl-DMNQ to regulate cell cycle-related proteins. Quiescent VSMCs cultured in serum-free medium were stimulated by PDGF-BB (25 ng/ml) to markedly express cell cycle regulatory proteins, and the effect of 2-undecylsulfonyl-DMNQ on the expression of CDK2, CDK4, cyclin D, cyclin E, PCNA and the phosphorylation of pRb was measured by SDS-PAGE followed by immunoblot as described under Section 2. (C) Analysis of PARP cleavage (cell apoptosis marker) by 2-undecylsulfonyl-DMNQ. The effect of 2-undecylsulfonyl-DMNQ on the expression of full length PARP (116 kDa) and its cleaved fragment (89 kDa) was measured as described under Section 2. Total β-actin was used for normalization. The gel pictures display representative blots of four similar, independent experiments.

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Fig. 5. Effect of 2-undecylsulfonyl-DMNQ on the phosphorylation of PLCγ1, Akt, ERK1/2, and STAT3 in PDGF-stimulated vascular smooth muscle cells. (A) The ability of 2-undecylsulfonyl-DMNQ to regulate the activation of PLCγ1, Akt, and ERK1/2. Serum-starved VSMCs were treated with or without the indicated concentrations of 2-undecylsulfonyl-DMNQ for 24 h, and then stimulated with 25 ng/ml PDGF-BB for PLCγ1 and ERK1/2 activation (for 5 min), or Akt (for 15 min), and the ability of 2-undecylsulfonyl-DMNQ to regulate the phosphorylation of PLCγ1, Akt, and ERK1/2 was measure by SDS-PAGE followed by immunoblot as described under Section 2. (B) The ability of 2-undecylsulfonyl-DMNQ and SU6656 to regulate the activation of STAT3. Quiescent VSMCs cultured in serum-free medium were treated with or without the indicated concentrations of 2-undecylsulfonyl-DMNQ or SU6656, a Src family kinase inhibitor, for 24 h, and then stimulated with PDGF-BB (25 ng/ml) for 10 min, and the ability of 2-undecylsulfonyl-DMNQ or SU6656 to regulate the phosphorylation of STAT3 was measured as described under Section 2. The gel pictures display representative blots of four similar, independent experiments.

3.7. Inhibitory action of 2-undecylsulfonyl-DMNQ on PDGF-induced VSMC proliferation by inhibiting receptor dimerization and the phosphorylation of PDGF-Rβ tyrosine kinases To investigate the effect of 2-undecylsulfonyl-DMNQ on PDGFinduced receptor dimerization, monomeric and dimeric forms of PDGF-Rβ after treatment of 2-undecylsulfonyl-DMNQ in PDGFstimulated VSMCs were measured using immunoprecipitation and immunoblotting. Gel picture shown in Fig. 6A illustrates that dimeric receptors induced by PDGF stimulation were significantly reduced by 2-undecylsulfonyl-DMNQ in concentration-dependent manner. PDGF-induced PDGF-Rβ dimerization was blocked by 1 μM 2-undecylsulfonyl-DMNQ about 40% of PDGF control (PDGF-stimulated, but no 2-undecylsulfonyl-DMNQ) (Fig. 6B). These results indicate that 2-undecylsulfonyl-DMNQ inhibits PDGF-induced receptor dimerization.

Data shown in Fig. 7A representthat PDGF-BB (25 ng/ml)-induced phosphorylation of PDGF-Rβ (Tyr751) was dose-dependently inhibited by 2-undecylsulfonyl-DMNQ. Total amount of PDGF-Rβ (Tyr751) was not affected by 2-undecylsulfonyl-DMNQ. Data shown in Fig. 7B present that the inhibition of PDGF-induced PDGF-Rβ (Tyr751) phosphorylation by 1 μM 2-undecylsulfonyl-DMNQ was similar to that by 5 μM AG1295, a selective inhibitor of PDGF receptor kinase. To further examine the time-dependent changes in the PDGF-induced activation of PDGF-Rβ kinase, VSMCs were stimulated by PDGF-BB (25 ng/ml) for a given time (0–30 min) and the phosphorylation of 5 tyrosine residues (Tyr579/581, Tyr716, Tyr751 and Tyr1021) in PDGF-Rβ was measured in the absence or presence of 2-undecylsulfonyl-DMNQ (1 μM). Data shown in Fig. 8A represent that the phosphorylation of PDGF-Rβ at five tyrosine residues tested was occurred at the highest level within 10 min after stimulation of PDGF. The highest level of PDGF-Rβ phosphorylation occurred 7 min after stimulation for

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Tyr579/581 (Fig. 8B), at 5 min for Tyr716 and Tyr1021 (Figs. 8C and E), and 10 min for Tyr751 (Fig. 8D). However, the PDGF-induced phosphorylation of PDGF-Rβ was completely blunted by 2-undecylsulfonyl-DMNQ, indicating that 2-undecylsulfonyl-DMNQ inhibited PDGF-induced VSMC proliferation by suppressing the phosphorylation of PDGF receptor kinase.

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2-Undecylsulfonyl-DMNQ Fig. 6. Effect of 2-undecylsulfonyl-DMNQ on the PDGF-Rβ dimerization in PDGFstimulated vascular smooth muscle cells. VSMCs cultured in serum-free medium were treated with given concentrations of 2-undecylsulfonyl-DMNQ for 24 h, stimulated by PDGF-BB (25 ng/ml) for another 1 h at 4 1C. All cells were then treated with 1.5 mM BS3. Cell lysates were subjected to immunoprecipitation (IP) with PDGF-Rβ antibody, and analyzed by 4% SDS-PAGE and immunoblotted (IB) with phosphotyrosine antibody PY99 (p-Tyr). (A) Immunoblot of PDGF receptor monomers and dimers. The gel pictures display representative blots of four similar independent experiments. (B) Inhibitory effect of 2-undecylsulfonyl-DMNQ on PDGFinduced receptor dimerization. The results are an average of three similar experiments, expressed as mean7S.E.M., and statistical difference from PDGF control (PDGFstimulated, but no 2-undecylsulfonyl-DMNQ) was illustrated by nnPo0.01.

4. Discussion This study has two major findings: (1) 2-undecylsulfonylDMNQ, a newly synthesized naphthoquinone derivative, has an inhibitory effect on VSMC proliferation and this effect is selectively occurred only with VSMC proliferation induced by PDGF, not by EGF or FBS, and (2) 2-undecylsulfonyl-DMNQ inhibits PDGFinduced receptor dimerization and the phosphorylation of PDGFRβ at five tyrosine residues (Tyr579/581, Tyr716, Tyr751 and Tyr1021), and subsequently suppressed PDGF-Rβ-mediated signaling pathways, including PLCγ, Akt, and ERK1/2, resulting in the inhibition of cell cycle in S phase via G0/G1 arrest. This is the first report of the antiproliferative action and underlying mechanisms of 2undecylsulfonyl-DMNQ in VSMCs. Our observation suggests that 2-undecylsulfonyl-DMNQ may be an effective inhibitor of PDGFRβ to become a potential agent for the prevention and treatment of vascular disorders such as atherosclerosis and restenosis. During atherosclerotic lesion progression, VSMC proliferation is of particular pathophysiologic importance (Gordon et al., 1990). In atherosclerotic lesions, VSMCs are exposed to mitogenic substances, such as PDGF and endothelin (Gutstein et al., 1999). Moreover, the association between PDGF and VSMCs proliferation has been demonstrated in animal experiments, in which increases and augmentations of PDGF after arterial injury were found to be correlated with neointimal cellular proliferation (Uchida et al., 1996). Therefore, the inhibition of PDGF-induced VSMC proliferation is considered as a key pharmacologic strategy during atherogenesis development. In the present study, we found that 2-undecylsulfonyl-DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation (Fig. 1A), and this effect was not due to cellular toxicity and apoptosis (Fig. 1B and 4C). Importantly, 2undecylsulfonyl-DMNQ has an effect in VSMCs stimulated only by PDGF, not by EGF or FBS (Fig. 2). EGF is a growth factor for VSMC proliferation (Beier et al., 2008) and FBS is also used as a VSMC proliferator (Peng et al., 2008). Thus, our results indicate that 2undecylsulfonyl-DMNQ has a selective antiproliferative action in VSMCs stimulated only by PDGF. The cell cycle which consists of four distinct sequential phases (G0/G1, S, and G2/M) regulates cellular proliferation. This tightly regulated temporal order is controlled by the sequential activation of certain serine/threonine protein kinases known as CDKs that phosphorylate pRb (Lundberg and Weinberg, 1998). Cell cycle

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Fig. 7. Effect of 2-undecylsulfonyl-DMNQ on the phosphorylation of PDGF-Rβ in PDGF-stimulated vascular smooth muscle cells. (A) The ability of 2-undecylsulfonyl-DMNQ to regulate the activation of PDGF-Rβ. VSMCs cultured in serum-free medium were treated with or without the indicated concentrations of 2-undecylsulfonyl-DMNQ for 24 h, and then stimulated with PDGF-BB (25 ng/ml) for 3 min, and the ability of 2-undecylsulfonyl-DMNQ to regulate the phosphorylation of PDGF-Rβ was measured by SDSPAGE followed by immunoblot as described under Section 2. The gel pictures display representative blots of four similar, independent experiments. (B) The comparison of the activity of 2-undecylsulfonyl-DMNQ and AG1295 to regulate the phosphorylation of PDGF-Rβ. Quiescent VSMCs cultured in serum-free medium were treated with or without the indicated concentrations of 2-undecylsulfonyl-DMNQ or AG1295, a selective inhibitor of PDGF receptor kinase, for 24 h, and then stimulated with PDGF-BB (25 ng/ml) for 3 min, and the ability of 2-undecylsulfonyl-DMNQ or AG1295 to regulate the phosphorylation of STAT3 was measured as described under Section 2. The results are an average of four similar experiments, expressed as mean 7 S.E.M., and statistical difference from PDGF control (PDGF-stimulated, but no 2-undecylsulfonyl-DMNQ) was illustrated by nnP o 0.01. The inset displays representative blots of four similar, independent experiments.

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Fig. 8. Effect of 2-undecylsulfonyl-DMNQ on the autophosphorylation of PDGF-Rβ tyrosine residues in PDGF-stimulated vascular smooth muscle cells. Serum-starved VSMCs were pretreated for 24 h with 1 μM 2-undecylsulfonyl-DMNQ or vehicle (1% DMSO) before adding PDGF-BB (25 ng/ml) for the indicated periods of time. Total cell lysates were then subjected to immunoblot analysis for the phosphorylation of tyrosine residues at Tyr579/581, Tyr716, Tyr751 and Tyr1021 in PDGF-Rβ kinases. Total β-actin was used for normalization. The gel pictures are representative immunoblots of three similar, independent experiments (A). The time-dependent changes of phosphorylation at each tyrosine residue (B) Tyr579/581; (C) Tyr716; (D) Tyr751; (E) Tyr1021 was illustrated by graphs depicted the compiled data of densitometrically evaluated phospho-PDGF Rβ/βactin ratio for each time point, and compared with groups between with and without treatment of 2-undecylsulfonyl-DMNQ at each time point. The results are an average of three similar, independent experiments, expressed as mean7 S.E.M., and statistical difference (two-way ANOVA followed by Bonferroni post test) from PDGF control (PDGFstimulated, but no 2-undecylsulfonyl-DMNQ) was illustrated by either nPo 0.05 or nnP o0.01.

transitions are controlled by the action of CDKs and their activating subunits, cyclins (Fingerhuth et al., 2004). The observation that 2-undecylsulfonyl-DMNQ suppressed PDGF-stimulated DNA synthesis (Fig. 3) and permitted VSMCs to arrest in G0/G1 phase (Fig. 4A) suggests that modulation of several cell cycle regulatory proteins may occur after 2-undecylsulfonyl-DMNQ treatment. The CDK 2 and CDK 4 are key mediators during the G1 to S phase progression of the cell cycle by forming complexes with cyclin E and D (Moore et al., 2005). These complexes phosphorylate a number of proteins, resulting in hyperphosphorylation of pRb, which then releases transcription factors that promote DNA synthesis (Dzau et al., 2002). Our data show that 2-undecylsulfonyl-DMNQ significantly suppressed the expression of CDK 2, CDK 4, cyclin E and cyclin D, and subsequently inhibited the phosphorylation of pRb and the expression of PCNA (Fig. 4B). Since the suppression of CDK 2 alone may be sufficient to prevent pRb hyperphosphorylation (Connell-Crowley et al., 1998), the inhibition of CDK 2, CDK 4, cyclin D and cyclin E expression as well as pRb phosphorylation may be sufficient to achieve cell cycle arrest. Moreover, the ability of 2-undecylsulfonyl-DMNQ to inhibit the expression of PCNA which is synthesized as a phospho-pRbmediated gene product in the early G0/G1 and S phase of the cell cycle (Tomita et al., 2005) confirms the arrest of cell cycle. Thus, 2-undecylsulfonyl-DMNQ inhibits cell cycle via G0/G1 phase arrest.

To investigate the signaling pathways involved in the inhibitory effect of 2-undecylsulfonyl-DMNQ on VSMC proliferation, the activations of STAT3, PLCγ1, Akt and ERK1/2 were measured. PDGFinduced activation of STAT3, PLCγ1, Akt and ERK1/2 was significantly inhibited by 2-undecylsulfonyl-DMNQ in a concentration-dependent manner (Fig. 5). These findings suggest that the antiproliferative action of 2-undecylsulfonyl-DMNQ is mediated by the inhibition of PDGF-induced activation of STAT3, PLCγ1, Akt and MAPK family member. Note that the activation of STAT3, PLCγ1, Akt and ERK1/2 is initiated by ligand-binding to the cell surface receptor, activation of the receptor, and binding of adapter molecules to phosphotyrosine residues in the activated receptor (Son et al., 2007). Thus, these observations provide a compelling reason to examine the ability of 2-undecylsulfonyl-DMNQ to act on the inhibition of PDGF receptor level. The PDGF binding to PDGF-Rβ can induce the autophosphorylation of multiple tyrosine residues, leading to the creation of binding sites for several molecules that interact with specific phosphotyrosines through their SH2 (src homology 2) domain. Currently, PLCγ, Ras GTPase-activating protein, the regulatory subunit of phosphatidylinositol 3-kinase (p85 of PI3K), growth factor receptor-bound protein 2 (GRB2), and members of the Src family have been found to bind specific phosphotyrosines in activated PDGF-Rβ (Ostman and Heldin, 2001). Tyr579 and Tyr581,

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which exist in the juxtamembrane segment of the PDGF-Rβ, are autophosphorylation sites which directly mediate the interaction with Src family kinases (Mori et al., 1993). Subsequently, Src family kinases activate STAT signaling including STAT3, and are required for PDGF-induced mitogenesis (Bowman et al., 2001). The phosphorylated Tyr716 in PDGF-Rβ kinase insert is located in such a motif and allows the direct binding of GRB2 (Arvidsson et al., 1994). The binding of GRB2 to phosphorylated PDGF-R results in the recruitment of Sos to the plasma membrane, and GRB2/Sos stimulates the Ser/Thr kinase Raf1 which stimulates MAPK ERK1/2 (Dance et al., 2008). Tyr751 in the kinase-insert region of PDGF-R is the docking site for PI3K-Akt pathway (Panayotou et al., 1992). PLC signaling is dependent on the autophosphorylation of PDGF-R at Tyr1009 and Tyr1021 (Kashishian et al., 1992). Thus, we hypothesized that PDGF-Rβ is a direct target for 2-undecylsulfonyl-DMNQ, which leads to the regulation of STAT3, ERK1/2, Akt and PLCγ, resulting in the inhibition of VSMC proliferation. Our results show that 2-undecylsulfonyl-DMNQ inhibits the PDGF-induced phosphorylation of PDGF-Rβ (Tyr751) in a dose-dependent manner (Fig. 7A), which was similar to the result with AG1295, a selective inhibitor of PDGF receptor kinase (Fig. 7B). Furthermore, 2undecylsulfonyl-DMNQ significantly inhibits the PDGF-induced phosphorylation of other 4 tyrosine residues (Tyr579/581, Tyr716, and Tyr1021) (Fig. 8). These findings suggest that the antiproliferative effects of 2-undecylsulfonyl-DMNQ on PDGF-induced VSMC proliferation are due to the specific blockade of autophosphorylation of tyrosine residues in PDGF-Rβ. 5. Conclusions In conclusion, these results provide the evidence that 2undecylsulfonyl-DMNQ selectively inhibits PDGF-stimulated VSMC proliferation and the cell cycle in S phase via G0/G1 arrest, which is mediated by selective blockade of receptor dimerization and the autophosphorylation of tyrosine residues in PDGF-Rβ, leading to the regulations of STAT3-, PLCγ-, Akt-, and ERK1/2mediated signaling pathways. Therefore, our observation suggests that 2-undecylsulfonyl-DMNQ could be a potent candidate as a preventive agent for the progression of vascular disorders such as restenosis after angioplasty and atherosclerosis.

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