chemopreventive effects [21,22]. Our lab has reported that resveratrol suppresses tumor growth by inducing apoptosis in tumor cells through aryl hydrocarbon receptor (AhR) and by reciprocal regulation of SIRT1 and NF-kB signaling [23]. For melanoma, experiments showed that resveratrol can enhance chemical cytotoxicity to the tumor, and suppress tumor growth by inducing cell-cycle interruption and apoptosis [24,25]. Neverthe- less, effects of resveratrol on the development of VLS in HDIL-2 therapy against melanoma have not been studied. In this report, we studied the role of resveratrol in the development of VLS in B16F10 melanoma-bearing mice and revealed its role in protection of the endothelium and suppression of metastasis. We also discovered a novel functional characteristic of resveratrol in induction of suppressive activity of MDSC and expression of FoxO1 transcripts in context of suppressing the development of VLS and the growth and metastasis of B16F10 melanoma. Materials and Methods Ethics statement, mice and recombinant IL-2 Female C57BL/6 (6–8 wk of age) mice were purchased from NIH. All animals were housed in University of South Carolina Animal Facility. All animal procedures were performed according to NIH guidelines under protocols approved by the Institutional Animal Care and Use Committee of the University of South Carolina. Recombinant IL-2 was provided by the NCI Biological Resources Branch (Rockville, MD). All antibodies were purchased from Biolegend. 3H-thymidine and 51NaCr04 were purchased from MP biomedical. Resveratrol was purchased from Sigma or Supelco (Bellefonte, PA). Induction and quantification of VLS VLS was induced by IL-2 according to the established method from our lab [12,26,27,28]. On day 0, groups of 4–5 mice were injected intraperitoneally with 75,000 units of IL-2 or PBS as Figure 1. Decreased VLS in resveratrol-treated non-tumor mice. Non-tumor bearing mice were given HDIL-2 and/or resveratrol. The vascular leak in the lungs and liver as well as TNF-a in the serum were measured. A. VLS in lung. B. VLS in liver. C. The TNF-a level in the serum was measured by sandwich ELISA. doi:10.1371/journal.pone.0035650.g001 Resveratrol in IL-2 Therapy against Melanoma a control, 3 times a day for 3 consecutive days. On day 4, the mice received one injection and 2 h later were injected intravenously with 0.1 ml of 1% Evan’s blue in PBS. After 2 h, the mice were exsanguinated under anesthesia, and the hearts were perfused with heparin-PBS until lungs and livers were blanched. The lungs and livers were harvested and placed in formamide at 37uC overnight. The Evan’s blue in the organs was quantified by measuring the absorbance of the supernatants at 650 nm with a spectrophotom- eter. For administration of resveratrol, mice were administered resveratrol (100 mg/kg body weight) on day -1 by gavage, and thereafter once a day 1 h before the first IL-2 injection during day 0 to day 4. The VLS seen in IL-2-treated mice was expressed as percentage of increase in extravasation of Evan’s blue when compared with that of the PBS-treated controls and was calculated as: [(OD650 in the organs of IL-2-treated mice)-OD650 in the organs of PBS-treated controls)]/(OD650 in the organs of PBS- treated controls) 6100. Each mouse was individually analyzed for vascular leak, and data from 4–5 mice were expressed as mean 6SEM. Tumor cell implantation, HDIL-2 and resveratrol treatment Mice were injected with 56105 B16F10 cells intravenously or implanted with 26105 B16F10 cells subcutaneously on day 0. The B16F10 melanoma cell line was purchased from American Type Culture Collection (Manassas, VA) and maintained as recom- mended by the supplier [26]. We started to treat mice with resveratrol on day 1 and induce VLS with HDIL-2 on day 9 as described above. On day 12, mice were sacrificed to evaluate VLS and tumor metastasis in lungs. Black nodules were counted under a dissecting microscope on the surface of the lung specimens. These nodules exhibited characteristic histological features of metastatic melanomas (data not shown). The area of each nodule was measured with software Image Pro. The area of each nodule was normalized to the whole area of the same lobe and multiplied by 1000. The average area of the normalized measurement was present. For the primary tumors in the skin, they were resected and weighed, and then used for the total RNA extraction. Flow cytometry studies of MDSC and Treg For flow cytometry analysis, splenocytes were prepared. Red blood cells were lysed using red blood cell lysing buffer (Sigma). Lung and liver infiltrating inflammatory cells were isolated using Percoll gradient centrifugation (detailed below). Cells were re- suspended in staining buffer (PBS containing 2 mM EDTA and 2% fetal bovine serum) and pre-incubated for 10 min at room temperature with purified anti-CD16/32 antibody to block non- specific binding of antibodies to Fcc receptors. For analysis of MDSC, cells were then incubated with FITC-conjugated anti- mouse CD11b and PE-conjugated anti-mouse Gr-1 mAbs in the staining buffer for 30 min at 4uC. For analysis of Treg, cells were incubated with FITC-conjugated anti-mouse CD4 mAb in the staining for 30 min at 4uC, then fixed and permeabilized with FoxP3 fix/perm buffer at room temperature in the dark for 20 min. The cells were washed once in FoxP3 perm buffer and continued to stay in FoxP3 perm buffer at room temperature for 15 min. Cells were spun down and re-suspended in 100 ml fresh FoxP3 perm buffer. Cells were then incubated with PE-conjugated anti-mouse FoxP3 mAb at room temperature in the dark for 30 min. After incubation with the mAbs, cells were washed twice in the staining buffer and re-suspended in the staining buffer. The staining was analyzed with a CXP500 flow cytometer with post- acquisition data analysis using CXP500 software (Beckman Coulter). All antibodies were purchased from Biolegend. PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35650