Methyl Protodioscin, a Steroidal Saponin, Inhibits Neointima

Article pubs.acs.org/jnp

Methyl Protodioscin, a Steroidal Saponin, Inhibits Neointima Formation in Vitro and in Vivo Yun-Lung Chung,† Chun-Hsu Pan,‡ Charles C.-N. Wang,§ Kai-Cheng Hsu,⊥ Ming-Jyh Sheu,† Hai-Feng Chen,*,∥ and Chieh-Hsi Wu*,†,‡ †

School of Pharmacy, China Medical University, Taichung 40402, Taiwan Department of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan § Department of Biomedical Informatics, Asia University, Taichung 41354, Taiwan ⊥ Cancer Biology and Drug Dsicovery, Taipei Medical University, Taipei 11031, Taiwan ∥ School of Pharmaceutical Sciences, Xiamen University, Xiamen 361005, China ‡

ABSTRACT: Restenosis (or neointimal hyperplasia) remains a clinical limitation of percutaneous coronary angioplasty. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are known to be involved in the development of restenosis. The present study aimed to investigate the ability and molecular mechanisms of methyl protodioscin (1), a steroidal saponin isolated from the root of Dioscorea nipponica, to inhibit neointimal formation. Our study demonstrated that 1 markedly inhibited the growth and migration of VSMCs (A7r5 cells). A cytometric analysis suggested that 1 induced growth inhibition by arresting VSMCs at the G1 phase of the cell cycle. A rat carotid artery balloon injury model indicated that neointima formation of the balloon-injured vessel was markedly reduced after extravascular administration of 1. Compound 1 decreased the expression levels of ADAM15 (a disintegrin and metalloprotease 15) and its downstream signaling pathways in the VSMCs. Moreover, the expressions and activities of matrix metalloproteinases (MMP-2 and MMP-9) were also suppressed by 1 in a concentration-dependent manner. Additionally, the molecular mechanisms appear to be mediated, in part, through the downregulation of ADAM15, FAK, ERK, and PI3K/Akt.

Methyl protodioscin (1), a bioactive natural compound isolated from Dioscorea collettii Makino (Dioscoreaceae), has been investigated for its numerous pharmacological activities, including those related to anti-inflammation, lipid-lowering, and anticancer activities.12−16 The antiproliferative effects of 1 have been shown to involve arresting the cell cycle and inducing apoptosis in several cancer cell lines.12−14 Accordingly, the

Balloon angioplasty is a routine surgical procedure for revascularization of acute arterial obstruction. However, this clinical intervention induces neointimal hyperplasia (also known as restenosis), a serious side effect that predominantly involves abnormal growth and migration of vascular smooth muscle cells (VSMCs) and ultimately results in recurrence of arterial lumen narrowing.1 Accordingly, the candidate drugs that potentially inhibit VSMC migration and proliferation will be investigated further for their applicability in the clinical prevention of neointima formation.2,3 The ADAMs (a disintegrin and metalloproteases) family of proteins are membrane-anchored surface glycoproteins involved in ectodomain shedding of various regulatory molecules, such as growth factors, receptors, and adhesion molecules.4,5 It has been demonstrated that ADAMs proteins are involved in critical regulation of cell migration, survival, and apoptosis.6−8 ADAM15 expression has been confirmed in VSMCs,9 and experimental data demonstrated that it plays a critical role in pathological neovascularization and atherosclerosis.10 In cultured human VSMCs and atherosclerotic lesions, upregulation of ADAM-15 showed a corresponding increase in α5β1 and αvβ3 integrin proteins, which promotes the migration and proliferation of VSMCs.11 These studies suggest that ADAM15 can be a potential target for prevention of neointima formation (or restenosis) by inhibiting abnormal VSMC proliferation and migration. © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Cell viability of A7r5 cells was analyzed by MTT assay after treatment of methyl protodioscin (1). *p < 0.05 and **p < 0.01 compared with the control group (10% FBS alone), respectively. Received: March 18, 2016

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DOI: 10.1021/acs.jnatprod.6b00217 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 2. Regulation of methyl protodioscin (1) on cell cycle stages of A7r5 cells. Data are presented as cell cycle profile (A) and histograms (B). Expression levels of Bax and Bcl-2 proteins as well as ratio of Bax/Bcl-2 were examined (C) and are shown as histograms (D). *p < 0.05 and **p < 0.01 compared with the control group (10% FBS alone).

of candidate drugs that potentially prevent restenosis. Previous reports have demonstrated that 1 has the potential to cause growth inhibition by inducing cell cycle arrest or apoptosis in numerous cancer cells.12−14 In the present study, we evaluated the inhibitory effect of 1 on the prevention of neointima formation and analyzed its bioactivities on VSMCs migration and proliferation. However, fetal bovine serum (FBS) is one of the major regulators involved in promoting the proliferation and migration of VSMCs.17 The results provide evidence that 1 can indeed suppress serum-stimulated cell proliferation and

present study attempted to examine the potential application of 1 in inhibiting neointima formation as well as to investigate whether its inhibitory effects are associated with regulation of the ADAM15-mediated signaling pathways.

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RESULTS AND DISCUSSION Effect of Methyl Protodioscin (1) on the Growth, Cell Cycle, and Apoptosis of A7r5 Cells. Since the outgrowth of VSMCs plays a critical pathological role in the progression of neointima formation, they have become a therapeutic target B



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Figure 3. Inhibitory effect of methyl protodioscin (1) was evaluated in a rat model of balloon injury-induced neointimal hyperplasia. Data are presented as original images of arterial sections (A) and histograms (B). L, lumen; N, neointimal; E, elastic fiber; M, medium. Black arrowhead indicates elastic fiber; bidirection arrowhead (white) indicates range of neointimal layer. The severity of the neointima formation was positively associated with the ratio of the neointima-to-media area. The data were treated with 1 to analyze the expression levels of ADAM15 of protein and related histograms (C); *p < 0.05 and **p < 0.01 compared with the control group (BI + PBS), respectively.

migration in A7r5 cells (Figures 1 and 2A), which explains the preventive mechanisms of 1 on neointima formation in the rat carotid artery balloon injury model (Figure 3) at a cellular level. The results showed that 1 exhibited an inhibitory effect on the A7r5 cell growth in a dose-dependent manner (Figure 1). The value of the half-maximal inhibitory concentration (IC50) of 1 growth of A7r5 cells is approximately 9 μM. Antiproliferative effects of dioscin have been proved by inducing cell apoptosis and cell cycle arrest at the G2/M phase in both HepG2 and A549 cancer cells.18−20 Also, 1 could induce hyperpolarization of mitochondria in K562 cells.21 Experimental data indicated that the cell cycle distribution of A7r5 cells was markedly changed and arrested at the G0/G1 stage after 18 h of incubation with 1 (Figure 2A,B). Furthermore, the expression levels of Bax and Bcl-2 proteins were also evaluated

Figure 4. Computational simulations of the degradation rate of ADAM15 due to methyl protodioscin (1) (6 μm). C



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Figure 5. Binding conformation and interactions of methyl protodioscin (1) and ADAM15. (A) Docking pose of 1 and ADAM15. (B) Interactions between 1 and ADAM15. (C) Diagram of the interactions.

hydroxyl groups of part A yield various hydrogen bonds with residues A317, M316, and H352 and the metal ion (Figure 5B and C). In addition, one of the ring groups has van der Waals interactions with residues G311, V314, and G315 (Figure 5B). Part B is bulky and hydrophobic and anchors the region adjacent to the zinc-binding site (Figure 5B). Part B forms extensive van der Waals interactions with residues H335, S336, G341, K374, T382, D383, and P386 (Figure 5C). Part C interacts with residues S336 and D383 through slight van der Waals interactions. These observations suggest that 1 can fit well into the binding site and inhibit ADAM15 activity by occupying the zinc-binding site. The data showed that 1 tunes autoregulatory splicing events to control ADAM15 expression levels and in turn alters their respective splicing networks (Figure 4). Potential Role of ADAM15 in Methyl Protodioscin (1)-Mediated Pharmacological Effects. The study reported that dioscin has been suggested to induce breast cancer cell apoptosis and inhibit cell prolifeartion by the suppression of MAPK signaling.24 As well, dioscin was mediated through inhibition of both ERK phosphorylation and AKT phosphorylation, thereby inducing cancer cell proliferation and apoptosis both in vitro and in vivo,25−27 and downregulation in the expression of MMPs, promoting VSMC proliferation and migration via the shedding signaling pathway.28,29 The PI3K/AKT signaling pathway also plays a pivotal role in the regulation of VSMC migration and proliferation,30 and disruption of PI3K/AKT and MAPK/ERK signaling led to a profound reduction of neointima formation after balloon injury.14,31 This research provides new evidence that agonist-induced cardiovascular disease is signaled by multiple, nonredundant proteins that play unique physiological roles. We demonstrate that ADAM15 inhibition (by pharmacological means) and gene knockdown (by RNA interference) attenuate VSMCs-induced neointima formation. However, we observed a novel transcriptional regulation of ADAM15 by two other proteins, FAK and ERK. Importantly, protein expression studies confirmed that the

under the same experimental condition. The results indicated that 1 did not significantly influence the protein expressions of a pro-apoptotic factor (Bax), an antiapoptotic factor (Bcl-2), and the ratio of Bax/Bcl-2 (Figure 2C,D). This result suggested that 1 did not induce the apoptosis of A7r5 cells under this experimental condition, which also supported the flow cytometric analysis showing that the concentrations used in the present study did not induce a statistical change in the subG1 phase, a positive index of apoptosis (Figure 2A,B). Methyl Protodioscin (1) Regulates Balloon InjuryInduced Neointima Formation. ADAM15 is thought to play a critical role in many diseases or physiological conditions, such as cancers, rheumatoid arthritis, and cardiovascular diseases.10,22 A previous study showed that upregulation of ADAM15 promotes the migration and proliferation of VSMCs.11 Histopathological analysis of arterial sections suggested that extraarterial treatment of 6 mm 1 observably reduced the index of neointima formation (the ratio of neointima formation and the ratio of neointima-to-media area) compared with that of the sham control (Figure 3A,B). In addition, we found that 1 can markedly reduce the protein expression of ADAM15 within VSMCs and injured arteries (Figures 3C). Binding Conformation and Interactions of Methyl Protodioscin (1) in ADAM15. In the absence of evidence for a direct effect of 1 on ADAM15, we hypothesized that 1 inhibits p-FAK indirectly by targeting ADAM15 (Figure 4). The relative molecular mass of 1 (Mr = 1062) is far larger than the appropriate molecular weight (Mr ≈ 500), which can easily pass the cell membrane.23 Accordingly, we hypothesized that 1 may interact with ADAM15, which was further supported by simulation data of the computation analysis (Figure 5A−C). The docking result shows that 1 is bound across the binding site (Figure 5A). Methyl protodioscin (1) was divided into three parts (parts A−C) to examine their interactions (Figure 5). Part A is located at the zinc-binding site, comprising three histidines: H348, H352, and H358. The main interaction type formed between this site and 1 is a hydrogen bond. The D



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Figure 6. Regulations of 1 on ADAM15 and its downstream signaling molecules. Effect of ADAM15 knockdown was examined in A7r5 cells transfected with ADAM15 siRNA for 24 h (A). A7r5 cells were treated with 1 to analyze the expression levels of ADAM15 and its downstream signaling molecules (B); *p < 0.05 and **p < 0.01 compared with the control group (10% FBS alone). ##p < 0.01 compared with the siRNA group (10% FBS alone).

revealed that pharmacological effects of 1 may act by downregulating the expression or activation of ADAM15 and its downstream signaling pathways. Regulation of these pathways can explain the mechanisms of the inhibitory effects of 1 on VSMC migration and proliferation as well as restenosis (Figure 7). ADAM15 may play an important role as a new indicator protein and may serve as a potential therapeutic target after vessel injury. Effect of Methyl Protodioscin (1) on the Migration of A7r5 Cells. Moreover, in a transwell assay, the data indicated that after 16 h of treatment of 1 (6 and 9 μM) cell migration was markedly inhibited compared with that of the control group (10% FBS group; Figure 8A). In a wound-healing assay,

p-FAK, p-ERK, and MMP-9 protein expressions were decreased by interference of ADAM15 siRNA (Figure 6A). Moreover, compound 1 (9 μM) observably reduced the translational level of ADAM15 protein in A7r5 cells (Figure 6B). Expression or activation levels of downstream molecules of ADAM15, such as FAK, PI3K/AKT, and ERK1/2, were also markedly suppressed in A7r5 cells treated with 1 for 24 h (Figure 6B). The above experimental results provide evidence of molecular mechanisms to support the findings that 1 has inhibitory effects on VSMC proliferation and migration. In summary, our study demonstrated that 1 significantly inhibits VSMC growth and migration and prevents the development of balloon angioplasty-induced neointimal hyperplasia. Experimental results also E



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Plant Material. The roots of Dioscorea nipponica Makino were collected at Sep. Ten in 2009 from Fushun, Liaoning Province, China. They were identified by Mrs. Xiuhong Zhou (Senior Engineer, Forestry Bureau of Yongchun, Quanzhou City, China), and the voucher specimen was deposited at the School of Pharmaceutical Sciences at Xiamen University. Extraction and Isolation. Compound 1 was isolated according to a previous report.33 Briefly, the fresh root of D. nipponica (5 kg) was boiled and refluxed for 2 h with a 60% aqueous ethanol solution (10 L × 3 times). After filtration, the solution was concentrated in vacuo. The extract (600.0 g) was then separated on a Diaion HP-20 column using EtOH−H2O as the mobile phase to yield three fractions (Fr. A−C). Fraction B (70% EtOH−H2O eluent, 102.0 g) was subjected to silica gel column chromatography using stepwise gradient elution with CHCl3−MeOH (100:0−0:100) to obtain eight fractions (Fr. B1−B11). Fr. B5 (19.2 g) was subjected to silica gel column chromatography and eluted with CHCl3−MeOH (8:2) to obtain five fractions (Fr. B5-1−B5-5). Fr. B5-3 (3.4 g) was subjected to octadecylmodified silica (ODS) column chromatography and eluted with MeOH−H2O (3:7−7:3), followed by another elution with MeOH− H2O (6:4), and this elution was further purified with preparative HPLC (Restek Prinnacle DB C18, 5 μm, 250 × 20 mm) and eluted with a 70% aqueous methanol solution to obtain compound 1 (1.1 g, tR 15.5 min). Cell Culture. A7r5 cells (#60082), a cell line isolated from rat smooth muscle of the thoracic aorta, were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Inc., Grand Island, NY, USA.) supplemented with 10% fetal bovine serum (Gibco Inc.) and 1% penicillin−streptomycin (#15140-122; Gibco Inc.). The cells were incubated in a humidified 5% CO2 atmosphere at 37 °C, and the culture medium was replaced every 2 days. Cell Viability Analysis. The cells were seeded in 96-well plates (8 × 103 cells/well) and allowed to adapt overnight. After an additional 24 h of serum starvation in 0.5% FBS, the cells were treated with different concentrations (0.625, 1.25, 2.5, 5, 10, 20, and 40 μM) of 1 in the culture medium containing 10% FBS for 24 h. Subsequently, the culture medium was replaced with fresh medium containing 5 mg/mL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide). After 3 h of incubation, the cells were washed twice with ice-cold 1× phosphate-buffered saline (PBS), and 100 μL of dimethyl sulfoxide was added to each well. The absorbance was measured using a multiplate reader (Anthos 2001; Anthos Labtec, Salzburg, Austria) at a wavelength of 590 nm. A scatter chart (x- and y-axes are serial concentrations of 1 and % of cell viability, respectively) was made using Microsoft Excel software and to get the equation of the trend line of raw data. IC50 value will be calculated, as the value of the y-axis is 50 according to the above equation.35 Flow Cytometric Analysis. A7r5 cells seeded in 10 cm dishes (1.0 × 105 cells/well) were incubated with various concentrations of 1 in DMEM containing 10% FBS for 24 h. After the treatment, the cells were harvested with 0.5% trypsin-EDTA (trypsin-EDTA; Gibco, Canada), washed twice with 10 mL of ice-cold 1× PBS, fixed in sufficient ice-cold 70% ethanol, and stored at −20 °C overnight. The cells were centrifuged, washed twice in ice-cold 1× PBS, and resuspended in 0.4 mL of DNA-staining solution (0.4 mg/dL of propidium iodide, 1% Triton X-100, and 0.1 mg/mL of RNase A). The cells were incubated at 37 °C, protected from light for a minimum of 30 min, and analyzed within 2 h. Flow cytometry was conducted using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The distribution of cell cycle phases (sub-G1, G0/G1, S, and G2/M) was analyzed using the ModFit LT Program (Verify Software House, Topsham, ME, USA). Animal Study. A total of 24 male rats (3 to 4 months old) weighing 300−350 g were purchased from BioLASCO (Taipei, Taiwan). Rats were divided randomly into the following four groups (n = 6 rats/ group): sham control group, balloon injury (BI) + PBS, BI + 3 μM 1, and BI + 6 μM 1. PBS or 1 was dissolved in ice-cold 30% (w/v) pluronic-F127 gel. All the animals were housed in a 12 h light/dark

Figure 7. Schematic overview of the signaling networks regulated by methyl protodioscin (1) in VSMCs. ADAM15, a disintegrin and metalloprotease 15; FAK, focal adhesion kinase; MMP-9, matrix metalloproteinase-9; PI3K, phosphoinositide 3-kinase; AKT, v-akt murine thymoma viral oncogene homologue.

an analysis of the results revealed that migration of A7r5 cells was significantly reduced after 18 h treatment with 9 μM 1 compared with that of the control group (10% FBS group; Figure 8B). Methyl Protodioscin (1) Regulates the Enzymatic Activity and Protein Expression of Gelatinases in A7r5 Cells. In addition, extracellular FAK activity is a prerequisite for the ECM via interaction with integrin.32,33 In our study, 1 significantly reduced protein expression and activation of gelatinases in A7r5 cells (Figure 9). The condition medium of A7r5 cells treated with 1 was harvested to investigate the activities of gelatinases (MMP-2 and MMP-9) using gelatin zymography. Methyl protodioscin (1) (6 and 9 μM) observably inhibited MMP-9 activities (Figure 9A). Moreover, compound 1 significantly reduced the protein expression of MMP-2 and MMP-9 in A7r5 cells (Figure 9B). Collectively, our data clearly suggest that 1 is able to decrease the ADAM15-mediated pathway function.

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EXPERIMENTAL SECTION

General Experimental Procedures. Column chromatography: SiO2 (200−300 mesh, Qingdao Haiyang Chemical Industry) and ODS (40−63 μm; Merck, Darmstadt, Germany). HPLC: MPD was analyzed by an Agilent 1100 (column: Phenomenex Gemini, C18, ⦶ 250 mm × 4.6 mm, 5 μm, MeOH−H2O = 7:3, flow rate: 1 mL/min, detective wave: 208 nm) and a Shimadzu Prominence LC-20A preparative liquid chromatograph, with LC-20AT pumps, an SPD-20A UV detector (Shimadzu Co., Japan), and a Restek Prinnacle DB ODS column (250 mm × 10 mm, 5 μm). NMR spectra: BrukerAvance 600 III spectrometer; δ in ppm, with TMS as internal standard. ESI-MS: Q Exactive mass spectrometer (Thermo Scientific). F



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Figure 8. Effect of methyl protodioscin (1) on A7r5 migration. Cell migration was examined using a transwell assay (A) and a wound-healing assay (B). Pictures were taken under a microscope at 100-fold magnification; *p < 0.05 and **p < 0.01 compared with the control group (10% FBS alone). cycle with free access to food and water. All animal procedures were performed by a trained operator of the Institutional Animal Care and Use Committee of China Medical University in accordance with the committee’s guidelines. Angioplasty of the rat carotid artery was performed as previously described.34−36 The balloon catheter (2F Fogarty; Becton-Dickinson, Franklin Lakes, NJ, USA) was introduced through the right external carotid artery into the common carotid artery, and the balloon was inflated at 1.3 kg/cm2 using an inflation device. We inflated the balloon by pushing and pulling through the lumen three times to damage the vessel. After surgery, the arterial adventitia of the balloon-injured carotid artery was coated with 30% (w/v) pluronic-F127 gel (with PBS or 1). Two weeks after the balloon injury, the rats were sacrificed. For morphological examination, the right common carotid arteries were collected and then fixed in 75% ethanol and embedded in the Parafilm block. Embedded vessel tissues were cut into slices 5−10 μm thick (n = 6 slices/tissue block), and then the areas of intimal and media layers as well as their area ratio in each arterial section were analyzed by using ImageJ software (NIH, Bethesda, MD, USA). The slices were stained with hematoxylin and eosin Y (Merck, Whitehouse Station, NJ, USA). The manifestation of vessel restenosis was presented as the ratio of the neointima-to-media area. The animal experiments were approved by the Institutional

Animal Care and Use Committee of China Medical University (approval ID: 100-49N). Transwell Assay. A7r5 cells (1.2 × 104 cells/well) were directly seeded on the inner surface of the upper chamber (ThinCerts cell culture inserts with 8 μm pore size; #662638; Greiner Bio-One Inc., Monroe, NC, USA). After 2 h for cell attachment, the culture medium was replaced with new serum-free medium with 1 in the upper chamber, and the lower chamber was filled with culture medium with 10% FBS and 1. After incubation at 37 °C for 16 h, the cells were fixed with 37% formaldehyde for 15 min, followed by staining with crystal violet for 15 min. The number of cells was then counted in five random fields using a microscope. The cell migration was evaluated according to the average number of cells for each group (migrated cells (%) = migrated cell number in the tested group/total cell number in the control group × 100%). Wound-Healing Assay. Cells were cultured in a 12-well plate (1.2 × 104 cells/well) and grown to 90% confluence. After 24 h of serum starvation, the cell monolayer was scratched with a 200 μL pipet tip to create a lengthwise wound. Next, the cells were rinsed with 1× PBS and treated with various concentrations (3, 6, and 9 μM) of 1. The cells that invaded the scratched region were counted at 0 and 24 h after treatment. The number of migrated cells within the denuded G



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Figure 9. Regulation of methyl protodioscin (1) on gelatinases (MMP-2 and MMP-9). The enzyme activities and protein expressions were examined using gelatin zymography (A) and immunoblotting (B). *p < 0.05 and **p < 0.01 compared with the control group (10% FBS alone).

Table 1. Morphometric Analysis of Arterial Sections groupa

area 2

intima (μm ) media (μm2) lumen (μm2) I/M ratio

sham control

BI + PBS

BI + 3 μM of 1

BI + 6 μM of 1

(n = 5)

(n = 6)

(n = 5)

(n = 5)

800.8 1629.4 4430.2 0.48

± ± ± ±

b

116.1 170.5 282.6c 0.03c

3346.4 1462.7 6809.1 1.98

± ± ± ±

556.7 393.9 820.8 0.36

1828.4 1902.0 4730.4 0.92

± ± ± ±

199.3 591.8 573.9b 0.10

912.4 1164.2 3076.6 0.34

± ± ± ±

92.0b 502.7 441.2c 0.15b

BI, balloon injury. Data are expressed as mean ± SEM. bp < 0.05 compared with the control group (BI + PBS). cp < 0.01 compared with the control group (BI + PBS). a

24 h. Then, the gels were stained with the staining solution (0.125% coomassie blue R-250, 50% methanol, and 10% acetic acid) for 2 h and then destained using 30% methanol and 10% acetic acid. Enzyme activities were identified as bright bands against the dark blue background and quantified using Multi Gauge software. The relative gelatinase activity was calculated according to the following equation: the value expression (%) = (treated group/control group) × 100%. Western Blot. A7r5 cells were seeded on Petri dishes and incubated with 3, 6, and 9 μM 1 for 24 h under culture medium. The cells were lysed in the protein extraction buffer containing 2% SDS, 50 mM dithiothreitol, and 62.5 mM Tris-HCl at pH 6.8, followed by incubation at 95 °C for 5 min. The samples were separated using SDSPAGE, transferred to PVDF membranes, blocked with 5% nonfat dry milk in 0.05% TBST (Tris-base saline containing 0.05% Tween-20) for

zone was quantified based on three independent experiments. Photos of the migrated VSMCs cells were taken under a microscope (100-fold magnification). Cell migration was measured as the percentage of wound closure [wound closure (%) = the area of scratched region at the end of the study/the area of initial scratched region × 100%]. Gelatin Zymography. A7r5 cells seeded in 10 cm dishes (1.0 × 105 cells/well) were incubated with various concentrations of 1 in DMEM containing 10% FBS for 24 h (n = 3). After the procedure, the culture medium was mixed with 8 μL of 5× nonreducing loading buffer (12.5% bromophenol blue, 10% SDS, 0.5 M Tris-HCl pH = 6.8, and 50% glycerol) and then dissolved in 10% slab SDS-PAGE gels with 0.1% gelatin. After electrophoresis, gels were soaked in 2.5% Triton X-100 for 1 h, then incubated in developing buffer (0.01% NaN3, 10 mM CaCl2, and 40 mM Tris-HCl pH = 8.0) at 37 °C for H



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1 h, and then probed with the desired antibodies against ADAM15 (GTX101599; GeneTex), phosphorylated-FAK (#8556; Cell Signaling Technology), PI3 kinase (#4257; Cell Signaling Technology), phosphorylated-Akt (#4060; Cell Signaling Technology), phosphorylated-Erk1/2 (#4370; Cell Signaling Technology), MMP-2 (#4022; Cell Signaling Technology), MMP-9 (#ab38898; abcam), Bax (#ab7977; abcam), Bcl-2 (#ab7973; abcam), or β-actin (#sc-47778; Santa Cruz Biotechnology Inc.) overnight at 4 °C. Next, the secondary antibodies (1:1000 dilution) conjugated with horseradish peroxidase were applied for 1 h at room temperature. The immunoreaction bands were visualized using Amersham ECL Plus Western blotting detection reagents (GE Healthcare Bio-Sciences, Bucks, UK) according to the manufacturer’s instructions, and the images were acquired using the Fujifilm LAS-4000 system (San Leandro, CA, USA). All data were normalized to the band density of β-actin content. The relative protein expression was also calculated using the following equation: relative protein expression (%) = (treated group/control group) × 100%. Mathematical Model. While gene regulatory networks can provide a static picture, the dynamics of molecular interactions in time and space plays a key role in the behavior of cells and organisms. Dynamical simulations have been mostly based on models created by the deep curation approach rather than by the data-driven approach. This is because deep curation captures causality, stoichiometry, and mechanisms of interactions, which are mandatory in dynamical simulations. Simulation is important in verifying biological models and predicting biological behaviors. After an initial model is created based on a set of hypotheses, dynamical simulations examine whether the model behaves like a real biological system. In the last years computing models of complex biological systems have been developed with varying degrees of success. Among others the S-system39 has been used widely as a standard numerical method in many successful cases.40 Three models (the S-system mode, Muchrlis− Menten model, and mass-action model) to describe gene regulation of the flowing transition process in Arabidopsis were compared. The results suggest that the S-system model has the best performance. In this study, applications of the S-system model to describe drug development and mathematical modeling are positioned to play an essential role, especially in the integration and analysis of quantitative and often heterogeneous experimental data. The S-system model is a general framework for modeling and analyzing nonlinear systems. It is based on the biochemical systems theory (BST).39 BST has been shown to provide a consistent mathematical framework for representing and analyzing biological systems. The S-system model represents the gene regulatory network as a set of differential equations in the general format M

i ,j

M

performance of iGEMDOCK was similar to that of other docking methods, such as DOCK, FlexX, and GOLD.43 Statistical Analysis. The data were expressed as the mean ± SD (standard deviation). In vitro experiments were conducted in triplicate (n = 3). The data were statistically analyzed with one-way ANOVA. A p-value < 0.05 was considered significantly different from the control.

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Corresponding Authors

*Tel (H.-F. Chen): +86-592-218-7225. E-mail: haifeng@xmu. edu.cn. *Tel (C.-H. Wu): +886-2-27361661, #6100. E-mail: chhswu@ tmu.edu.tw. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the China Medical University (CMU94-032, CMU95-329, CMU96-084, and CMU97-084), Taipei Medical University (TMU102-AE1-B15), and Ministry of Science and Technology (MOST 103-2320-B-038-008 and MOST 104-2320-B-038-026).

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REFERENCES

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i ,j

Ẋi = αi ∏ X jg − βi ∏ X jh , i = 1, 2, ..., M j=1

AUTHOR INFORMATION

j=1

Here, Xi represents the concentration of gene i, αi and βi are nonnegative rate constants, and gi,j and hi,j are real-valued kinetic orders for production and degradation terms, respectively. M represents the number of genes. If gi,j > 0, gene j will induce the expression of gene i. On the contrary, gene j will inhibit the expression of gene i if gi,j < 0. The variable hi,j has similar effects in controlling the gene degradation compared to gi,j. In the present study, the range for αi and βi falls between −10 and 10; that for hi,j and gi,j falls between −2 and 2. Molecular Docking. We used molecular docking to investigate the binding mechanism between 1 and ADAM15. Because the crystal structure of ADAM15 is unavailable in the Protein Data Bank (PDB), a homology modeling approach was used to model the structure of ADAM15.41 The structure (PDB code 3G5C) of human ADAM22 with the highest sequence identity of 34% (E-value: 5 × 10−83) was selected as a structure template. The compound structure of 1 was generated using the CORINA software.42 Then, 1 was docked into the modeled structure of ADAM15 using our in-house docking tool, iGEMDOCK. iGEMDOCK can provide and visualize protein− compound interactions, including electrostatic, hydrogen-bonding, and van der Waals interactions. Our previous studies showed that the I



Article

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