Resveratrol in IL-2 Therapy against Melanoma Figure 9. Resveratrol enhances the expression of FoxO1 and the cytolytic susceptibility of melanoma. Panel A and B: C57BL/6 mice were implanted s.c. with B16F10 melanoma cells (26105 cells/mouse) and orally administered with resveratrol. On day 9, mice were treated with IL-2 as described in Fig. 1. The mice were sacrificed for evaluation of VLS on day 12. The primary tumors in the skin were resected and weighed. Total RNA was extracted. Expression of FoxO1 was detected by QPCR (A). The relative expression was normalized to the endogenous 18S. The mean 6SEM of weight is shown (B). Panel C: B16F10 cells were cultured with vehicle (Veh), IL-2 (IL) (1000 units/ml), or resveratrol at the indicated dose for 24 h. Total RNA was extracted. Expression of FoxO1 was detected by QPCR. Panel D: LAK cells were generated by culturing splenocytes from na¨ıve mice with IL-2 (1000 units/ml) for 48 h. B16F10 cells were incubated with resveratrol (25 mM) for 2 h and labeled with 51Cr. The labeled B16F10 cells were then added to the LAK cells. After 4 h, the release of 51Cr was measured and percent cytotoxicity was calculated. Statistical analysis was performed at each effector to target ratio and showed p,0.001. doi:10.1371/journal.pone.0035650.g009 are able to overcome the suppressive activity of MDSC against the immunity of the host that eventually leads to the tumor regression. Discussion Our results support a potential use of resveratrol in HDIL-2 treatment against melanoma and revealed some mechanisms. The resveratrol treatment effectively inhibited the development of VLS that is the most severe side-effect from HDIL-2 therapy. Meanwhile, the co-treatment with resveratrol and HDIL-2 promoted the efficacy of the tumor therapy arisen from either resveratrol or HDIL-2 treatment. Resveratrol played a differential role in the protection of EC injury and killing of the tumor. On the one hand, resveratrol prevented ECs from the ultrastructural damage and apoptosis; on the other hand, it increased the tumor killing by enhancing the susceptibility of the tumor to the cytotoxicity killer cells. To understand how resveratrol played the suppressive role in the development of VLS, we focused on the study of Treg and MDSC, the two main populations of the immune suppressors. We found that resveratrol induced the expansion of MDSCs that can suppress the cytolytic killing of ECs and the development of VLS while the number of Treg decreased at the same time. Previous studies from our lab and the others also showed that resveratrol inhibited the production of TGF-b and the expansion of Treg in the tumor-bearing mice while it induced the expansion of MDSCs that suppressed the development of chronic colits [33,34]. These results demonstrate that MDSC is the predominant suppressor cells than Treg in the development of VLS. It could be true since MDSCs are the dominant suppressor cells that induce the tumor escape than Treg [35]. This conclusion could be corroborated by the fact that HDIL-2 treatment can induce the expansion of Tregs but they are not able to prevent the development of VLS [12]. There are conflicting reports on whether MDSC are involved in the induction of Treg [13]. It was also reported that both Treg and MDSC could expand in the tumor-bearing mice, and that the expansion of the two populations were not related [36]. Here, we showed that IL-2 treatment induced both MDSC and Treg expansion. However, resveratrol treatment only induced MDSC expansion while it inhibited Treg expansion. Therefore, our current study demonstrates a differential role of resveratrol on Treg and MDSC. The expansion of MDSC in HDIL-2 immunotherapy was found in patients with renal cell carcinoma (RCC) therapy. In these patients, an increased secretion of Arg1 in the patients blood was also detected [37,38]. However, the role of MDSC in the development of HDIL-2-induced VLS and their functionality has not been studied before. In the current report, we found that HDIL-2-induced MDSCs from spleen were not suppressive. This is not surprising considering that MDSCs from spleen of na¨ıve mice or tumor-bearing mice do not possess the suppressive PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35650