Some studies support the notion that IL-22 may play different roles in different tumor cells. Although the growth of Colon 26/ IL-22 tumors in syngeneic mice did not differ from that of parent tumors, survival of mice inoculated with Colon 26/IL-22 tumors was significantly prolonged compared with the survival of mice inoculated with parent tumors [26]. IL-22 inhibited the growth of human mammary adenocarcinoma EMT6 cells both in vivo and in vitro [27]. In some respects IL-22 acts synergistically with tumor necrosis factor-a, IL-1b, and IL-17. For example, IL-22 was highly expressed in non-small cell lung carcinoma, and the overexpres- sion of IL-22 protected lung cancer cell lines from serum starvation-induced and chemotherapeutic drug-induced apoptosis; furthermore, administration of IL-22-RNAi significantly inhibited human lung tumor cell growth in BALB/c nude mice [28]. Despite these intriguing results, the functions of IL-22 are not clearly understood. In the present study, we investigated the effects of IL-22 on human RCC cell line A498 cells in vitro and in vivo and studied the possible mechanisms underlying the anti-tumor effects of this cytokine. We found that IL-22 dose-dependently suppresses A498 cell growth and that it inhibits the growth of A498 xenografts. We also found that IL-22 induces G2/M cell cycle arrest without causing cancer cell apoptosis. In addition, we showed that the phosphorylation of STAT1 is increased and the phosphorylation of ERK1/2 is attenuated in A498 cells exposed to IL-22. The expression of tumor necrosis factor (TNF)-a and interferon (IFN)– a were not increased vigorously in A498 xenografts treated with IL-22. These results suggest that the anti-RCC effects of IL-22 are directly mediated by regulation of the STAT1 signaling pathways and G2/M cell cycle arrest rather than by induction of apoptosis or inflammatory cytokines. Results IL-22 inhibits the growth of A498 cells in vitro We used MTT assay to investigate the effects of rhIL-22 on A498 cells and found that the final absorbance values at 570 nm were 0.1254, 0.1092, 0.0945 and 0.0825 respectively after treated with rhIL-22 at doses of 10, 20, 50 and 100 ng/ml. The corresponding inhibition rates of A498 cells were 16.4%, 27.2%, 37.0% and 45% respectively and significantly lower than the control. (Fig. 1A). The IC50 value was approximately 115 ng/ml. These results indicate that rhIL-22 dose-dependently suppresses the growth of A498 cells. Moreover, to confirm the contribution of IL-22 on growth inhibition of A498 cells, monoclonal human IL- 22 antibody was used to neutralize IL-22. Interestingly, the growth inhibition of A498 cells was revised to 9.7%, 10.4%, 9.4% and 11.3% with corresponding absorbance values of 0.1535, 0.1523, 0.1540 and 0.1508 respectively (Fig. 1A). IL-22 suppresses tumor proliferation in A498 cell-bearing mice To determine whether IL-22 has anti-tumor effects in vivo, 56106 A498 cells were injected (s.c.) into the neck regions of BALB/C nude mice. In the course of the experiment, we found that the average volume of tumors was about 100 mm3 three weeks after injection. The mice were treated with rhIL-22 (0.5 mg/ day) for seven days; control mice that also received tumor cell injections were treated with PBS. Tumor diameter was measured once per week after rhIL-22 injection for five weeks. RCC xenografts were proved by the slice pathology without significantly infiltrating of leukocytes. The average volume of the tumors of rhIL-22-treated mice was significantly smaller than that of the tumors of control mice at each time point (Fig. 1B). Interleukin22 Suppress Renal Cell Carcinoma Growth Figure 1. Suppression of A498 cell growth in vitro and in vivo by rhIL-22. A. IL-22 suppressed the growth of A498 cells in vitro in a dose-dependent manner. The growth inhibition of A498 cells exposed to IL-22 was revised by IL-22 antibody neutralization or by STAT1 specific siRNA transfection compared with the control (*P,0.05). Compared with A498 cells transfected with STAT1 siRNA, the growth inhibition of A498 cells treated with IL-22 antibody was not statistically different after IL-22 exposure (P.0.05). N = 4 and mean6SD for all groups. B. Suppressive effect of rhIL-22 on A498-cell-bearing xenografts in mice. When tumors reached 100 mm3, rhIL-22 was injected into the tail vein 0.5 mg /day for 7 days; control mice were injected with the same volume of PBS. Tumor size was measured with a caliper each week for five weeks; tumor volume was determined by measuring the maximal (a) and minimal (b) diameters using a caliber and calculating using the formula a6b2. After five weeks, mice were sacrificed under deep anesthesia and the final volume of the tumors was measured. *P,0.05 compared with control. N = 24 and mean 6 SD for all groups. doi:10.1371/journal.pone.0020382.g001 To further understand the modulations in cytokines expression in A498 xenografts after rhIL-22 treatment, blotting of TNF-a and IFN-a was carried out. We found that both cytokines were barely upregulated after rhIL-22 exposure compared with control (p.0.05), indicating the suppression of A498 cell xenografts dose not involve these cytokines (Fig. 2). IL-22 induces cell cycle arrest in the G2/M phase We investigated the effects of rhIL-22 on cell cycle regulation in A498 cells. FACScan analysis of propidium iodide (PI)-stained A498 cells revealed that after exposure to 50 ng/ml rhIL-22 for 24 hours, significantly more cells accumulated in the G2/M phase PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20382