Inhibition of Tumor Angiogenesis by Phloroglucinol Figure 2. Effect of various concentrations of phloroglucinol on the migratory activity of EPCs in a wound healing assay. (A) Ex vivo cultured outgrowth ECs were subjected to a wound healing migration assay. Bar: 500 mm. EPCs were wounded and treated with 100 ng/ml of VEGF with or without 20 `ıM or 100 `ıM of phloroglucinol or a vehicle. (B) Bar graph represents the number of migrated cells. Fields were chosen randomly from various section levels to ensure objective sampling. In response to phloroglucinol, the VEGF-induced migratory activity of EPCs was significantly decreased. doi:10.1371/journal.pone.0033618.g002 tube-forming activity of EC outgrowths, and the mobilization of EPCs into the peripheral circulation. Among the proposed pivotal mediators of tumor-mediated neovascularization, VEGF signaling has been identified as the central signaling axis regulating proliferative activities, migration, and homing/incorporation of EPCs into sites of ischemic tissue [28,29]. Thus, a wide range of preclinical strategies for the VEGF pathway has been shown to hinder tumor growth and angiogenesis [29,30]. Increasing evidence shows that a VEGF-neutralizing antibody (bevacizumab) and VEGF receptor tyrosine kinase inhibitor (RTKIs), including sorafenib and sunitinib, are clinically approved anticancer treatments that target the VEGF/VEGF receptor axis [31,32,33]. Importantly, as shown in Figure S2, phloroglucinol more effectively inhibited tube forming capacity and migratotory capacity in low concentration as compared to those of bevacizu- mab. Based on the present findings, we concluded that phloroglucinol showed more superiority of phloroglucinol over well established, clinically proven antiangiogenic drug- bevacizu- mab, partly affecting in vitro bioactivities of EPCs in intracellular VEGF-mediate signal cascades, although bevacizumab can only modulate surface-mediated VEGF signaling. This is the first report that phloroglucinol inhibits multiple VEGF-dependent EPC bioactivities, including the VEGF-depen- dent tube-forming capability and VEGF-dependent migratory capacity of EPCs in vitro. Further, this compound effectively abrogates VEGF-dependent neovessel formation in vivo, leading to delayed tumor progression and angiogenesis, partly through the modulation of EPCs in vivo. PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e33618