MTT assay The cytotoxic effects of the isolated compounds on cultured cells were measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay. In brief, ex vivo expanded EPCs (16104 cells/well) were plated onto gelatin-coated, 96-well plates containing complete EGM-2 medium. After 24 h, the cells were serum starved in EBM-2 medium supplemented with 0.5% FBS for 12 h. They were then incubated with various concentra- tions of phloroglucinol in complete EGM-2 medium for 24 h. These were then incubated with 20 m, MTT solution (5 mg/ml, Amresco) for 4 h. Finally, 200 m, DMSO was added to solubilize the formed formazan, and the amount of formazan formed was determined by measuring the absorbance at 560 nm using a microplate reader. Viability of cells was quantified as a percentage compared to the control, and dose response curves were developed. Migration assay Ex vivo expanded EPCs were cultured in 24-well plates at 1.56105 cells/well as confluent monolayers. The cells were incubated in EGM-2 medium for 24 h. Cells were starved in EBM-2 medium containing 1% FBS for 6 h and wounded. The wounded monolayer was then washed to remove cell debris, incubated for 14 h in 0.1% FBS in EBM-2 media containing various concentrations of phloroglucinol or bevacizumab and VEGF (100 ng/ml, Biobud). The area of the wound was recorded at the indicated time points using a light microscope. Migrated cells were analyzed under a light microscope. Tube formation assay Matrigel (BD Biosciences, Bedford, MA) was dissolved at 4uC overnight, and 96-well plates were prepared with 55 m, matrigel in each well after coating and incubating at 37uC for 30 min. EPCs (26104) in 100 m, complete EGM-2 media were added with various concentrations of phloroglucinol or bevacizumab. After 6,24 h of incubation at 37uC, EPCs’ tube formation was assessed with a photomicroscope, following which each well was photo- graphed at 640 magnification under a light microscope. Tube branches and total tube length were calculated using MacBiopho- tonics Image J software. In vivo tumor growth Male C57BL/6 mice were injected subcutaneously with 56104 LLC cells in DMEM. Drug treatment was initiated the day after tumor injection. LLC tumor-bearing mice (7 per group) were treated for 24 days with 150 m, of 0.94 mg/kg phloroglucinol or the solvent (10% DMSO). Tumor growth was measured with calipers every 3 or 4 days using the following formula: 3 V = height6length6depth (cm ) [41]. We designed the in vivo tumor model of phloroglucinol to identify the possibilities as a tumor preventing drug, as phloroglucinol, a compound from Ecklonia cava, a species of brown alga and can be edible, biosafe natural bioproducts and can be used as a tumor preventing bio- drug. Therefore, we orally administrated phloroglucinol one day after tumor injection to evaluate the effect of phloroglucinol before initiating tumor. Contribution of EPCs during phloroglucinol-mediated tumor angiogenesis To determine incorporation ratio of EPCs into tumor vessel by orally administrated phloroglucinol, the Dil-Ac-LDL-labeling- EPCs (16106 cells) were transplanted into tail vein of LLC- tumor-bearing C57BL/6 mouse (5 per group). Tumor tissue Inhibition of Tumor Angiogenesis by Phloroglucinol samples collected from the experimental animals were fixed 4% paraformaldehyde, rinsed in PBS, transferred to 30% sucrose in PBS at 4uC, and frozen in OCT compound. Frozen tissue sections were treated using goat anti-mouse CD31 antibody overnight at 4uC, followed by staining with FITC conjugated anti-goat antibody to identify capillaries in tumor. In vivo mobilization of circulating EPCs, CD45NEGCD34POScells Male C57BL/6 mice were injected subcutaneously with 56106 LLC cells in DMEM. Phloroglucinol treatment was initiated the day after tumor injection. LLC tumor-bearing mice (5 per group) were treated for 5 days with 0.94 mg/kg phloroglucinol. Control mice received equal amounts of 10% DMSO. Thereafter, mouse circulating EPCs were determined with flow cytometry using the following labels: CD45 (to exclude hematopoietic cell) (BD Pharmingen), hematopoietic/endothelial progenitor cell marker CD34 (BD Pharmingen) antibodies. Enzyme-Linked Immunosorbent Assay (ELISA) Seven-week-old C57BL/6 mice were received subcutaneous injection with LLC cells (16106 cells). At starting day after tumor inoculation, they were orally administered 0.94 mg/kg phloroglu- cinol or DMSO solvent (control group) into LLC tumor bearing mice daily for 7 days. Using blood samples, ELISA was performed with a mouse specific VEGF ELISA kit (Quantikine M, R&D system, Minneapolis, Minn), as indicated in the manufacturer’s protocol. Actin reorganization in response to VEGF EPCs were seeded sparsely onto microscope glass coverslips placed within 24 well culture plates and allowed to grow for 1 day on change of the EGM-2 medium. Cells were starved in EBM-2 medium containing 0.1% FBS for 12 h. Incubated for 3 h in EBM-2 media containing various concentrations of phloroglucinol and VEGF (100 ng/ml, Biobud). Cells were fixed in 4% paraformaldehyde in PBS at room temperature for 20 minutes. Rhodamine-conjugated phalloidin was applied at room temper- ature for 60 minutes. After washing the coverslips in PBS, they were mounted onto object glasses using faramount mounting medium (Dako) and analyzed using confocal microscope (Olym- pus). Flow cytometry analysis Mouse circulating EPCs were determined with flow cytometry using the following labels: CD45 (to exclude hematopoietic cell) (BD Pharmingen), hematopoietic/endothelial progenitor cell marker CD34 (BD Pharmingen) antibodies. Following red cell lysis, circulating EPCs were enumerated using flow cytometry (Becton Dickinson, Franklin Lakes, NJ), using gates to exclude dead cells, debris, and platelets. The percentage of stained cells was finally determined after comparing samples with matched isotype controls. Immunohistochemistry of CD31+ micro vessels Vasculogenesis in the tumor tissue was determined by immunohistochemistry staining of CD31, a marker of endothelial cells. The immunohistochemical reactions were carried out on 5- mm-thick paraffin 4% paraformaldehyde-fixed-embedded sections from the phloroglucinol or the solvent treatment group, and paraffin sections of each tumor were stained for CD31 using a rat anti-mouse CD31 monoclonal antibody (BD Pharmingen). Specific binding of the primary antibody was visualized using PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e33618