vivo murine matrigel plug assay. In the in vivo Lewis lung carcinoma (LLC)-tumor-bearing mice model, we showed that oral adminis- tration of phloroglucinol significantly inhibited tumor growth and angiogenesis as well as the mobilization of circulating EPCs, CD452/CD34+ progenitor cells. This report highlights a novel role for phloroglucinol as a modulator of EPC bioactivities and suggest that it might be a potential cancer prevention drug. Results Characterization of EPCs from human umbilical cord blood (HUCB) EPCs have been isolated from HUCB mononuclear cells (MNCs). Fluorescent staining was used to detect double positive cells binding of fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-1 (UEA-1) lectin and dioctadecyl-3,3,39,39- tetramethylindo carbocyanine (Dil)-labeled acetylated low density lipoprotein (data not shown). Immunophenotyping further re- vealed that ex vivo expanded EPCs expressed endothelial cells lineage surface antigens, CD31, VEGFR-2 (KDR), von Will- ebrand factor (vWF), eNOS, p-eNOS and p-Akt (Fig. 1B). Effect of phloroglucinol on cell toxicity of EPCs In order to investigate cytotoxity of phloroglucinol in EPCs, cell viability assay was performend. As shown in Fig. 1C, Phloroglu- cinol did not reduce cell viability in EPCs at doses below 100 mM for 24 h. Therefore, concentrations of phloroglucinol ranging from 2 to 100 mM were selected for study on bioactivities of EPCs and tumor angiogenesis. Phloroglucinol inhibits the VEGF-induced migration of EPCs Considering that BM mobilization kinetics of EPCs into peripheral blood (PB) is generally initiated by VEGF signaling, phloroglucinol may modulate the VEGF-induced migratory capability of EPCs. To test this idea, we next examined the effect of phloroglucinol on the migratory capability of EPCs using the wound healing assay. As shown in Figure 2A and 2B, induction of VEGF significantly repaired the wounded monolayer of EPCs. In contrast, phloroglucinol significantly reduced the VEGF-induced wounded area in a dose-dependent manner. Phloroglucinol inhibits the tube-forming capacity of EPCs We further identified the effect of phloroglucinol on the de novo capillary-like tubular formation of circulating progenitor cells (Fig. 3A). Treatment with phloroglucinol resulted in significant reduction in the number of branches and length of EPC tubes in a dose-dependent manner (Fig. 3B and 3C). Phloroglucinol suppresses tumor growth and tumor angiogenesis In order to explore whether daily oral administration of phloroglucinol can suppress tumor growth and tumor-induced angiogenesis, we generated in vivo LLC tumor-bearing mice. To do this, we injected LLC tumor cells into male C57BL/6 mice, following which they were orally administered 0.94 mg/kg phloroglucinol (experimental group) or DMSO solvent (control group) daily for 24 days (Fig. 4A). At the time of death, all the mice treated with the vehicle only had a large tumor volume reaching 3 2.1060.309 cm . A significant decrease in swelled tumor mass (1.06260.341 cm3) was observed when LLC cells (56104) were injected into a mouse flank together with phloroglucinol (Fig. 4B). To further determine the direct effects of phloroglucinol on tumor- Inhibition of Tumor Angiogenesis by Phloroglucinol induced angiogenesis, we analyzed the capillary density of the peritumoral region of each group by staining sections with CD31 antibodies. As shown in Figure 4C and 4D, treatment with phloroglucinol led to a significant reduction in the number of CD31+ capillary microvessels in the peritumoral region, suggesting that phloroglucinol might suppress tumor-induced angiogenesis in vivo. In order to investigate the direct role of phloroglucinol during EPC –mediated tumor angiogenesis, we analyzed the number of incorporated EPC in tumor vessel following orally administration of phloroglucinol. As shown in Figure S1, phloroglucinol significantly inhibited EPC incorporation into tumor-mediated vessels when isolated labelled EPCs applied into tail vein of tumor bearing mice, suggesting that phloroglucinol effectively modulated the contribution of EPC during tumor angiogenesis, partly regulating migratory capabilities of EPC. Effect of phloroglucinol on mobilization of circulating EPCs, CD45NEGCD34POScells Since orally administered phloroglucinol in tumor-bearing mice exerts a significant inhibitory effect on the mobilization of circulating EPCs, we performed additional experiments to determine the effect of phloroglucinol treatment on the mobiliza- tion of EPCs from the bone marrow (BM) niche into circulating blood. Following subcutaneous transplantation (56106 cells) of LLC into the thighs of C57BL/6 mice, they were orally administrated 0.94 mg/kg phloroglucinol for 5 days (Fig. 5A). As shown in Figure 5B, the number of CD45NEGCD34POS cells, i.e., circulating EPCs, significantly increased compared to in normal mice. Importantly, oral administration of phloroglucinol for 5 days resulted in a significant reduction in the number of CD45NEGCD34POS EPCs circulating in PB. Phloroglucinol inhibits VEGF-induced in vivo angiogenesis In order to investigate the reason for phloroglucinol’s anti- angiogenic activity in the in vivo angiogenesis model, we performed a matrigel plug assay (Fig. 6A). As shown in Figure 6B, the group with VEGF-loaded plugs yielded a red image, indicating an abundance of red blood cells in the newly formed vessels, while plugs with matrigel alone or with 0.94 mg/kg and 9.4 mg/kg phloroglucinol yielded light yellow images, indicating compara- tively less blood vessel formation. These results suggest that phloroglucinol significantly reduces VEGF-dependent neovessel formation. To further examine the effect of phloroglucinol on capillary density, we performed immunohistochemical analysis by staining of CD31+ microvessels (Fig. 7A). As shown in Figure 7B, there was a significant decrease in the density of microvessels in the group with plugs with phloroglucinol plus VEGF as compared to the group with plugs with VEGF only. Discussion Tumor-mediated neovascularization refers to the formation of new blood vessels from pre-existing vessels via the migration and proliferation of endothelial cells. This process requires multiple pathophysiological steps during the process of blood vessel formation, and it eventually contributes to tumor growth and angiogenesis [17,18]. BM-derived EPCs are reported to play a significant role in tumor angiogenesis [19,20]. Emerging evidence indicates impairments in the homing activities of BM-derived endothelial and hematopoietic precursor cells significantly blocks tumor angiogenesis and growth by decreasing the growth of early tumors. Blocking occurs because of interference in the angiogenic switch, which promotes tumor neovessel formation during tumor PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e33618