Identification of Antiangiogenic Potential and Cellular Mechanisms of

Jul 27, 2017 - College of Pharmacy, Yeungnam University, Gyeongsan-si, Gyeongsangbukdo 38541, Korea. § Natural Products Research Center, Korea Instit...
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Identification of Antiangiogenic Potential and Cellular Mechanisms of Napyradiomycin A1 Isolated from the Marine-Derived Streptomyces sp. YP127 Ji Sun Hwang,†,# Geum Jin Kim,‡,# Hyun Gyu Choi,‡ Min Cheol Kim,§ Dongyup Hahn,⊥,∥ Joo-Won Nam,‡ Sang-Jip Nam,▽ Hak Choel Kwon,§ Jungwook Chin,† Sung Jin Cho,†,○ Hayoung Hwang,*,† and Hyukjae Choi*,‡ †

New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu 41061, Korea College of Pharmacy, Yeungnam University, Gyeongsan-si, Gyeongsangbukdo 38541, Korea § Natural Products Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung, Gangwon-do 25451, Korea ⊥ School of Food Science and Biotechnology, College of Agriculture and Life Sciences, and ∥Institute of Agricultural Science & Technology, Kyungpook National University, Daegu 41566, Korea ▽ Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Korea ○ Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Korea ‡

S Supporting Information *

ABSTRACT: Angiogenesis is the process of new blood vessel formation. Excessive angiogenesis is a critical factor in the progression of cancer, macular degeneration, and other chronic inflammatory diseases. When investigating the effects of crude extracts of cultured marine microorganisms, an extract of the cultured Streptomyces sp. YP127 strain was found to inhibit human umbilical vein endothelial cell (HUVEC) tube formation. Bioassay-guided fractionation and spectroscopic data analyses led to the identification of napyradiomycin A1 (1) as an antiangiogenic component of the extract. Compound 1 inhibited HUVEC tube formation in a concentration-dependent manner. It inhibited endothelial cell proliferation but did not affect human dermal fibroblast proliferation. Compound 1 also suppressed migration and invasion of vascular endothelial cells. In addition, compound 1 suppressed vascular endothelial cadherin expression and increased the permeability of the endothelial cell membrane. These results suggested that compound 1 modulates cell permeability and inhibits the angiogenesis of endothelial cells.

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endothelial cells, are thought to be promising drugs to treat various types of cancer. 7 The U.S. Food and Drug Administration (FDA) has approved several antiangiogenic drugs for cancer treatment, such as bevacizumab (glioblastoma),8 sorafenib (hepatocellular carcinoma and kidney cancer),9 sunitinib (kidney cancer and neuroendocrine tumor),10 and pazopanib (kidney cancer).11 These antiangiogenic drugs block the supply of nutrients and oxygen to cancer cells, whereas conventional anticancer drugs are often considerably cytotoxic. Antiangiogenic drugs can become more effective when used in

ndothelial cells line the inner layer of blood vessels and play an important role in vascular functions such as blood clotting, vasodilation/vasoconstriction, fluid permeation, and angiogenesis.1,2 Angiogenesis is a normal process during organ development or during wound healing. However, excessive angiogenesis accompanies cancer spread and growth, macular degeneration, and other chronic inflammatory diseases, including rheumatoid arthritis and Crohn’s disease.3−5 Enhanced angiogenesis occurs to meet the increasing demands of cancer cells and activated inflammatory cells for nutrients and oxygen. It also plays key roles in the migration and invasion of cancer and inflammatory cells into endothelial cell layers.6 Therefore, angiogenesis inhibitors, which target vascular © 2017 American Chemical Society and American Society of Pharmacognosy

Received: March 10, 2017 Published: July 27, 2017 2269

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combination with conventional chemotherapy.12 In addition, excessive angiogenesis contributes to the pathogenesis of inflammatory diseases affecting various tissues, such as the choroid coat in macular degeneration,13,14 synovial endothelial cells in rheumatoid arthritis,15 and intestinal epithelium in Crohn’s disease.16 Therefore, there is a high demand for novel and specific antiangiogenic compounds. Natural products are a rich source of lead compounds for drug discovery. In particular, a number of natural products derived from marine microorganisms are currently being investigated in drug discovery pipelines, and some of them, such as brentuximab vedotin and marizomib, have been approved by the FDA.17,18 In a preliminary study, 50 crude extracts of marine-derived bacterial strains have been screened for the ability to inhibit human umbilical vein endothelial cell (HUVEC) tube formation. Among the tested samples, an ethyl acetate (EtOAc) extract of the cultured Streptomyces sp. YP127 strain (GenBank accession no. MF102228) potently inhibited HUVEC tube formation. Through bioactivity-guided fractionation, napyradiomycin A1 (1), a chlorinated α-lapachone derivative, was isolated as a potential tube formation inhibitor. Previously, compound 1 was reported to possess cytotoxic,19 antibacterial,20 and nonsteroidal estrogen-receptor antagonizing effects.21 In this study, the effects of compound 1 on HUVEC tube formation, cell migration, cell invasion, and expression of vascular endothelial cadherin (VE-cadherin) were investigated in vitro. In addition, the antiangiogenic activity of compound 1 was described.

[δH 2.70 (brd, J = 8.4 Hz), 2.49 (dd, J = 14.2, 5.0 Hz), 2.42 (dd, J = 14.2, 11.0 Hz), and 1.60 (m)], and five methyl protons [δH 1.60 (s), 1.49 (s), 1.48 (s), 1.30 (s), and 1.17 (s)]. The 13C NMR spectrum of 1 showed resonances for two carbonyl carbons (δC 197.2 and 193.8), two phenolic carbons (δC 164.9 and 164.2), two aromatic methine carbons (δC 110.0 and 108.2), two methinyl vinylic carbons (δC 123.7 and 114.8), six nonprotonated carbons (δC 143.1, 135.1, 131.9, 110.2, 83.6, and 79.0), a tertiary carbon adjacent to a chlorine atom (δC 78.9), a methinyl carbon adjacent to a chlorine atom (δC 58.7), four methylene carbons (δC 42.8, 41.5, 39.8, and 26.0), and five methyl carbons (δC 28.8, 25.8, 22.4, 17.7, and 16.6). In the LCMS chromatogram, the UV spectrum of 1 was similar to those of napyradiomycins with the UV absorption pattern at 255, 299, and 361 nm.20 The specific rotation value of 1 was similar to the literature value {[α]13D +49.1 (c 0.3, MeOH); literature [α]20D +51 (c 0.3, MeOH)}.19 In addition, the spectroscopic data comparison between observed and previously reported data further confirmed that compound 1 was napyradiomycin A1.22 Inhibitory Effects of Streptomyces sp. YP127 Extract and Fraction D on HUVEC Tube Formation. An EtOAc extract of Streptomyces sp. YP127 inhibited HUVEC tube formation at concentrations of 25 and 50 μg/mL (Figure 1). The extract was subjected to vacuum liquid chromatography (VLC), and seven fractions were obtained. The fraction D eluting with 40% EtOAc in hexanes was found to inhibit HUVEC tube formation at concentrations of 25 and 50 μg/mL (Figure 1). Antiangiogenic Effect of 1 in Microvascular Endothelial Cells. To verify the antiangiogenic activity of 1, we examined its effects on vascular endothelial growth factor (VEGF)-induced capillary-like tube formation in vitro. After 24 h of incubation with VEGF, untreated HUVECs showed enclosed tube-like capillaries. However, in the presence of 1, tube formation was significantly reduced in a concentrationdependent manner, and the concentration of 1 at which the inhibition was half-maximal (IC50 value) was calculated to be 10.0 μM (Figure 2). Proliferation of endothelial cells is required to generate new vessels from pre-existing ones during angiogenesis. Hence, we examined the effects of 1 on proliferation of endothelial cells, and cell proliferation was represented as cell confluency (%) covering the culture plate, analyzed by cell images as described in the Experimental Section. We monitored cell growth for 72 h in the presence or absence of 1 and found that this compound at a concentration of 20 μM significantly inhibited HUVEC proliferation, when compared to the extent of proliferation in the control (Figure 3A and B). The concentration of 1 inhibiting endothelial cell growth by 50% (GI50 value) was calculated to be 27.6 μM (Figure 3C). From these results, we noticed that treatment with 1 suppressed cell growth, rather

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RESULTS AND DISCUSSION Structural Identification. In the electrospray ionization mass spectrometry spectrum (ESIMS), compound 1 exhibited a deprotonated [M − H]− ion cluster at m/z 479.3/481.3/483.3 with an intensity ratio of 100:64:10, indicating dichlorination of the molecule. The 1H NMR spectrum of 1 exhibited resonances for two exchangeable protons [δH 11.84 (brs) and 3.61 (brs)], two meta-coupled aromatic protons [δH 7.32 (d, J = 2.4 Hz) and 6.80 (d, J = 2.4 Hz)], two olefinic protons [δH 4.87 (brs) and 4.69 (brt, J = 8.4 Hz)], a methine proton [δH 4.42 (dd, J = 11.0, 5.0 Hz)] next to chlorine, four sets of methylene protons

Figure 1. Inhibitory effects of Streptomyces sp. YP127 crude extract and fraction D on HUVEC tube formation (bar: 500 μm). 2270

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both in the control and in 1-treated cells. To elucidate the mechanism of cell growth inhibition by 1, we checked the expression of Ki-67, a cellular marker of proliferation, which is expressed throughout all active phases of the cell cycle. As shown in Figure 3D, immunostaining showed that Ki-67 expression was not detected in 1-treated HUVECs. This finding suggested that 1 could inhibit cell growth by regulating the expression of Ki-67, which is necessary for cell proliferation. Next, we tested the effect of 1 on the proliferation of human fibroblasts to clarify if 1 selectively affected endothelial cells. As shown in Figure 3E and F, 1 did not affect the proliferation of human dermal fibroblast BJ6 cells. These results strongly suggested that 1 specifically inhibits the proliferation of endothelial cells and suppresses angiogenesis. Effects of 1 on the Migration and Invasion of Endothelial Cells. Migration and invasion of vascular endothelial cells are important events during angiogenesis. Therefore, we used the wound-scratching migration assay to investigate the effects of 1 on VEGF-induced HUVEC migration. After wound-scratching as shown as the blue line, where cells were not present, VEGF-treated endothelial cells migrated and completely covered the wound area within 24 h after scratching (Figure 4A). Meanwhile, treatment with 1 dramatically inhibited the migration of HUVECs, showing a vacant area without cells as blue line. Images were taken every 3 h after wound-scratching, and the mean confluency of the area covered with cells was measured as shown as cell density in the wound area (%) in Figure 4B. We then investigated whether 1 could inhibit the invasive behavior of HUVECs using a Matrigel coating system. Invasive cells spread out into Matrigel, a mixture of extracellular matrix proteins, and these invasive moving cells are called tip cells. Representative tip cells are marked with arrows in Figure 4C. Upon treatment, 1 markedly suppressed HUVEC invasions (Figure 4C). The number of tip

Figure 2. Concentration-dependent inhibition of HUVEC tube formation by 1. (A) Analysis of capillary-like tube formation by Calcein AM staining on live cells (upper) and a phase contrast image (lower) (scale bar: 500 μm). (B) Number of tube formations in compound-treated cells. (C) Inhibited HUVEC tube formation calculated from three branch points by concentration-dependent treatment of 1.

than inducing cell death processes such as apoptosis. To verify this result, dead cells were detected 24 h after treatment of 1 using cell-impermeant dye, which could penetrate into only dead cells, not live cells. As a result in Figure S3, dead cells appearing as green fluorescence-positive cells were very few

Figure 3. Inhibition of HUVEC cell proliferation by 1. (A) Cell proliferation over 72 h. (B) Cell viability measurement at the end point compared to the control group. (C) Evaluation of GI50 value of 1 on the proliferation of HUVECs. (D) Suppressed expression of Ki-67 by treatment of 1 at 20 μM on immunofluorescence assay. (E and F) BJ6 human fibroblast cell proliferation monitoring after treatment with 1. 2271

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Figure 4. Suppressed migration and invasion of HUVEC cells by treatment with 1. (A) Migration of VEGF-stimulated HUVEC cells after treatment with DMSO and 20 μM 1 (scale bar: 300 μm). (B) Quantitative analysis of relative wound density in HUVECs treated with 1 for 24 h under VEGF stimulation. (C) Invasion of VEGF-stimulated HUVEC cells after treatment with DMSO and 20 μM 1 (scale bar: 300 μm). (D) Numbers of tip cells at the edge of the wound after 24 and 48 h of treatment with 1.

cells that were localized at the invasive front was significantly reduced after 24 and 48 h of treatment with 1 by about 43% and 52%, respectively, as compared to their number in control conditions (Figure 4D). These results suggested that 1 regulated angiogenesis by inhibiting VEGF-stimulated endothelial cell migration and invasion. Downregulation of VE-Cadherin Expression and Enhancement of Permeability by 1. Next, we examined the effects of 1 on the expression of PECAM1 and VE-cadherin, which are representative adhesion molecules associated with angiogenesis. Since VE-cadherin and PECAM1 play crucial roles in regulating endothelial barrier function and leukocyte transmigration, they represented good candidate targets that are involved in angiogenesis.23,24 As shown in Figure 5A, incubation with 1 for 24 h significantly suppressed mRNA expression of PECAM1 and VE-cadherin, by 53% and 76%, respectively. This inhibitory effect was even stronger after 48 h of incubation, as PECAM1 and VE-cadherin mRNA expression levels were inhibited by 70% and 94%, respectively. Consistent with the quantitative real-time PCR (qRT-PCR) results, the protein level of VE-cadherin was also decreased after 1 treatment (Figure 5B). Treatment with 1 decreased PECAM1 mRNA expression in endothelial cells, whereas the PECAM1 protein expression level did not statistically differ from the level in untreated cells (data not shown).

Figure 5. Reduced expression of the endothelial cell surface marker VE-cadherin by treatment with 1. (A) Quantitative analysis of PECAM1 and VE-cadherin mRNA expression on 1-treated HUVECs. (B) Time-dependent VE-cadherin protein level changes of HUVECs by treatment with 1.

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Strain Identification and Bioactivity-Guided Isolation. The bacterial strain YP127 was collected from the intertidal area of Mokpo, Chonnam Province of South Korea. This bacterial strain was isolated on SYP agar solid medium (starch 10 g, peptone 4 g, yeast extract 2 g, agar 16 g, seawater 1 L). The bacterial strain YP127 (GenBank accession no. MF102228) was identified as Streptomyces sp. (95.3% similarity in 16S rRNA sequence to Streptomyces sp. 215232), and the strain was stored at the College of Pharmacy of Yeungnam University. After bacterial strain cultivation in solid SYP medium for 15 days, the medium color changed to dark red. Then, cultivation of the bacterial strain YP127 was performed in SYP SW liquid medium (10 L) for 7 days at 25 °C with shaking at 150 rpm. After cultivation, the cultured broth was extracted twice with EtOAc. The EtOAc extract of the bacterial strain YP127 inhibited HUVEC tube formation. The bioactive crude extract was subjected to VLC eluting with hexanes, EtOAc, and MeOH to give seven fractions. Among the seven fractions, fraction D showed inhibitory activity toward HUVEC tube formation. The MeOH-soluble part of fraction D (115.7 mg) was subjected to RP-HPLC (Phenomenex Luna C(18), 22.5 × 250 mm, 6 mL/min, MeCN−H2O = 70:30 → 100:0, UV 210 and 310 nm), and compound 1 (14.1 mg, tR = 38.12 min) was purified. Napyradiomycin A1 (1): pale brownish-yellow oil; [α]13D +49.1 (c 0.3, MeOH); LR-ESIMS m/z 479.3 [M − H]− (C25H30Cl2O5); 1H NMR (CDCl3, 250 MHz) and 13C NMR (CDCl3, 63 MHz) data, see Supporting Information (Table S1, Figures S1 and S2). Cell Culture. HUVECs were purchased from Cascade Biologics (Carlsbad, CA, USA) and cultured in M200 medium supplemented with 2% (v/v) low serum growth supplement (Invitrogen, Waltham, MA, USA) and 1% (v/v) penicillin plus streptomycin (Hyclone, South Logan, Utah, USA) at 37 °C in a humidified atmosphere of 5% CO2. HUVEC cells were cultured in 0.2% gelatin-coated plates (SigmaAldrich, St. Louis, MO, USA), and all experiments used cells from passages six to eight. The human normal fibroblast cell line BJ6 was purchased from Stemgent (Cambridge, MA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone) and 1% (v/v) penicillin and streptomycin. In Vitro Tube Formation Assay. HUVEC cells (6 × 104 cells) were seeded in a 24-well plate precoated with the Matrigel growth factor reduced (GFR) basement membrane matrix (Corning, New York, NY, USA). Then, cells were incubated with various concentrations of 1 and 10 ng/mL recombinant human VEGF (PeproTech, Rocky Hill, NJ, USA). After 24 h, cells were stained with 2 μg/mL Calcein AM (Invitrogen) for 20 min at 37 °C, and images were taken with an EVOS FL cell imaging system (Thermo Scientific, Waltham, MA, USA). Endothelial cell tube formation was quantified by determining the number of new tubes with three branch points. For each condition, three independent experiments were performed, and mean tube numbers were used for statistical analyses. Cell Migration and Invasion Assays. For the cell migration assay, HUVECs were plated at a density of 1 × 105 cells/well in a 96well Essen ImageLock plate, allowed to grow overnight, and then loaded into a 96-pin WoundMaker device (Essen BioScience, Ann Arbor, MI, USA) to make a wound area. The medium was aspirated from each well, and wells were washed twice with phosphate-buffered saline (PBS). Next, the culture medium supplemented with 10 ng/mL VEGF and 1 or DMSO were added and monitored by an IncuCyte ZOOM live-cell imaging system (Essen BioScience) for 24 h. For the invasion assay, the GFR basement membrane matrix was overlaid on the wound area prepared as above, and then, 1-containing culture medium was added. Wound images were automatically acquired at 3 h intervals for a total of 24 h by the IncuCyte ZOOM live-cell imaging system. Wound confluence parameters were calculated using a customized algorithm provided by the IncuCyte ZOOM program. Cell Proliferation Assay. HUVEC and fibroblast proliferation was monitored in real time using the IncuCyte ZOOM live-cell imaging system. Cells were seeded at a density of 2.5 × 103 per well in a 96well plate and treated with 1 at different concentrations, and cell growth was monitored for 72 h. Images of the cell culture plate were taken every 6 h, and cell confluency (%) to cover each area was

VE-cadherin plays a pivotal role in cell−cell junctions, and the disruption of its expression in endothelial cells weakens cell−cell interactions and increases permeability, resulting in vascular leakage.25,26 Therefore, we assessed whether 1 affected cellular permeability of endothelial cells. We found that treatment with 1 at 20 μM increased endothelial cell permeability in a time-dependent manner (Figure 6).

Figure 6. Effects on endothelial cell permeability by treatment of 1 (20 μM).

Collectively, our observations suggested that 1 affected endothelial cell permeability by regulating the expression of VE-cadherin, which could be the mechanism of its antiangiogenic effects. Bioactivity-guided fractionation on the Streptomyces sp. YP127 extract led to the isolation of 1 as an antiangiogenic compound. Napyradiomycins are halogenated meroterpenoids that comprise a seminapthoquinone core with a cyclized prenyl group and monoterpenyl substituents on C-4a and C-10a. Napyradiomycins are divided into three subclasses based on the type of monoterpenoid moiety.22 Recently, napyradiomycin B and its analogues in the corresponding subclass have been reported to exhibit cytotoxic effects in HCT-116 cells by inducing apoptosis.27 While moderate cytotoxicity of 1 against glioblastoma, breast cancer, human non-small-cell lung carcinoma, and liver cancer cells has been reported previously,19 the mechanisms of its cytotoxic actions have not been established yet. The antiangiogenic effect of 1 in HUVECs was observed in this study for the first time. Compound 1 suppressed new blood vessel formation, endothelial cell-specific proliferation, and HUVEC migration and invasion. Compound 1 also decreased the expression of cell adhesion molecules and increased permeability of the endothelial cell membranes. Therefore, 1 could be used as a potential lead in drug discovery for the treatment of cancer and chronic inflammatory diseases.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was measured on a JASCO DIP-100 polarimeter (Jasco, Tokyo, Japan). The 1H and 13C NMR spectra were obtained using a Bruker AVANCE DPX 250 spectrometer (Bruker, Billerica, MA, USA) using CDCl3. LR-ESIMS was performed using an Agilent 6120 series LC-MS system (Agilent Technologies, Santa Clara, CA, USA). Column chromatography was performed with silica gel 60 (230−400 mesh, Merck KGaA, Darmstadt, Germany). Thin-layer chromatography was carried out on silica gel 60 F254 precoated aluminum plates (0.2 mm thickness, Merck KGaA) by visualization under UV light at 254 nm or with spraying 10% ethanolic sulfuric acid. HPLC was carried out on a Gilson system (Gilson Inc., WI, USA) using a Phenomenex Luna C(18) (22.5 × 250 mm) column. 2273

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analyzed as the mean value by the IncuCyte ZOOM program. After 72 h of culture, viable cells were counted using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA), and luminescence activity was measured in a multifunctional microplate reader (Tecan, Infinite M200 Pro, San Jose, CA, USA) according to the manufacturer’s protocol. Cell proliferation data from IncuCyte were analyzed using the IncuCyte ZOOM program and GraphPad Prism 6 (La Jolla, CA, USA). Immunofluorescence Staining. HUVECs were seeded at a density of 4 × 104 cells per well in gelatin-coated 12-well plates and treated with compound 1 for 24 h. For immunostaining, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. Permeabilized cells were blocked with PBS containing 10% normal horse serum for 1 h at room temperature and incubated with an antiKi-67 antibody (Abcam, Cambridge, MA, USA) for 1 h. Then, cells were incubated with a cy3-conjugated secondary antibody to detect the primary anti-Ki-67 antibody, and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) was added to a concentration of 5 M for 10 min for nuclear staining. Fluorescence images were taken by a fluorescence microscope (Zeiss Axio Observer A1, Oberkochen, Germany). RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR. Total RNA of HUVECs treated with 1 or DMSO was extracted with RiboEx (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s protocol. For reverse transcription, 1 μg of total RNA was used and cDNA was generated using an oligo (dT) primer and ImProm-II reverse transcriptase (Promega) in a total volume of 20 μL. VE-cadherin mRNA level was determined by qRT-PCR (Applied Biosystems, Waltham, MA, USA) with SYBR Green (Roche, New York, NY, USA) using a protocol provided by the manufacturer. Human GAPDH primers were used for qRT-PCR to normalize the amount of cDNA used for each condition. The primer sequences were as follows. GAPDH: (F-5′-GGA AGG TGA AGG TCG GAG TCA ACG-3′; R-5′-GTG AAG ACG CCA GTG GAC TCC AC-3′); PECAM1: (F-5′-CGA TGT GGC TTG GAG TCC TGC TG-3′; R5′-CCT TAT AGA ACA GCA TCT GGT GCT GAG-3′); VEcadherin: (F-5′-GGA TTT GGA ACC AGA TGC ACA TTG ATG AAG-3′; R-5′-CAG CCT CTC AAT GGC GAA CAC GTC-3′). Western Blot Analysis. Total protein extracts were prepared from compound 1-treated HUVECs in RIPA (radioimmunoprecipitation assay) lysis buffer (Roche) containing protease inhibitors (Roche). Protein samples were electrophoresed in the 4−12% gradient sodium dodecyl sulfate polyacrylamide gel (Bio-Rad, Hercules, CA, USA), transferred to PVDF membranes (EDM Millipore, Billerica, MA, USA), and incubated with the following primary antibodies: anti-VEcadherin (Abcam) and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). Primary antibody binding was detected using a horseradish peroxidase-conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) and Clarify Western ECL substrate (Bio-Rad). Western blot images were visualized by an ImageQuant LAS4000 imager (GE Healthcare, Pittsburgh, PA, USA). Endothelial Cell Permeability Assay. HUVECs (5 × 105 cells/ well) were seeded in Transwell cell culture inserts in 12-well plates (Corning) precoated with 0.2% gelatin and cultured for 48 h. Then, 1 was applied for another 48 h, and 1 mg/mL FITC-conjugated dextran (Sigma-Aldrich) was added to the upper chamber of the Transwell plate.26 Cellular permeability was assessed by measuring the amount of FITC-conjugated dextran transferred into the lower chamber of the Transwell plate by using a multifunctional microplate reader (Tecan). Statistical Analysis. For statistical analysis, the two-tailed Student’s t test was performed to calculate the statistical significance of the experimental data. The levels of significance were as follows: *p < 0.05, **p < 0.005, and ***p < 0.001.

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1 H and 13C NMR data including chemical shift table and spectra of 1 as well as cell death count by the treatment of 1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +82-53-790-5208. E-mail: [email protected] (H. Hwang). *Tel: +82-53-810-2824. E-mail: [email protected] (H. Choi). ORCID

Jungwook Chin: 0000-0001-6060-0508 Sung Jin Cho: 0000-0002-4786-8830 Hyukjae Choi: 0000-0002-7707-4767 Author Contributions #

J. S. Hwang and G. J. Kim contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (Nos. 2014M3A9D9033717 and 2014R1A1A2057302).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00211. 2274

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DOI: 10.1021/acs.jnatprod.7b00211 J. Nat. Prod. 2017, 80, 2269−2275