Resveratrol in IL-2 Therapy against Melanoma Figure 5. Resveratrol protects endothelial cells from apoptosis. Lungs from PBS, resveratrol, IL-2 or IL-2+resveratrol-treated mice were examined for apoptosis with TUNEL assay as described in Materials and Methods. Apoptotic cells are depicted by brown stained nuclei (arrow). The histogram shows the quantification of apoptotic cells. Original magnification, 6400. doi:10.1371/journal.pone.0035650.g005 Multiple Comparision Test was applied using Graphpad Prism 5 software. Comparisons were considered significant at p#0.05. Results Resveratrol prevents VLS while maintaining the effectiveness of HDIL-2 treatment HDIL-2 has been shown to have significant efficacy against advanced stages of melanoma. However, HDIL-2 treatment is accompanied by significant toxicity including VLS which limits its therapeutic efficacy [1,2,3,4,5]. In the current study, we first evaluated whether resveratrol plays any role in the prevention of HDIL-2-induced VLS. For this purpose, we induced VLS in normal mice with HDIL-2. Resveratrol was given to mice one day before HDIL-2 treatment and once a day during the treatment until the day of sacrifice of mice. Four sets of experiments were established: IL-2 treatment (IL), resveratrol treatment (Res), IL-2 + resveratrol co-treatment (IL+R), and PBS treatment (PBS). The results showed that the vascular leak was dramatically suppressed by resveratrol treatment. Serum levels of TNF-a were also significantly decreased in resveratrol treatment group (Fig. 1). These results demonstrated that resveratrol can prevent IL-2- induced VLS. Next, we tested whether resveratrol exerts a similar effect in melanoma-bearing mice. To this end, C57BL/6 mice were injected i.v. with B16F10 melanoma cells (56105 cells/mouse). On day 9, mice were treated with IL-2 or resveratrol as described above. The mice were euthanized for evaluation of VLS on day 12. Our results showed that resveratrol significantly inhibited VLS in tumor-bearing mice both in the lungs and liver to a similar extent when compared to that seen in the non-tumor-bearing mice (Fig. 2A and 2B). The metastasis and tumor growth in lungs (seen as black nodules) were significantly inhibited by IL-2 or IL2+resveratrol co-treatment. Moreover, IL2+resveratrol co- treatment was more effective in inhibiting tumor metastasis than IL-2 treatment alone (Fig. 2C–2E). These results indicated that PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35650