Figure 5. The regulation of STAT1 and ERK1/2 pathways by rhIL-22. A. p-STAT1 and p-ERK1/2 levels after 50 ng/ml rIL-22 exposure. B. p-STAT1 and p-ERK1/2 levels 15 minutes after administration of various doses of rhIL-22. N = 3. doi:10.1371/journal.pone.0020382.g005 treatment (87.2%63.6% decrease relative to control) compared with that of negative control siRNA (Fig. 6) and significant lower growth inhibition rates of these cells (11.3%, 15.7%, 17.2% and 21.4%) were observed when treated with 10, 20, 50 and 100 ng/ ml of rhIL-22compared with that of A498 cells without siRNA transfection. Although the phosphorylation of ERK1/2 was still attenuated during this procedure, the absolute contribution of STAT1 pathway to the growth inhibition of A498 cells was observed via the limited revision of these cells’ growth inhibition by dephosphorylation ERK1/2 alone when STAT1 was knocked down. Moreover, the cells transfected with negative control siRNA had a comparative growth inhibition rate after exposure to rhIL- 22 with that of A498 cells (Fig. 1A). Lack of apoptosis of A498 cells after IL-22 exposure We next determined whether inhibition of tumor growth by IL- 22 involves the induction of apoptosis. We used FACScan to measure annexin V binding in A498 cells treated with doses of rhIL-22 ranging from 0 to 400 ng/ml for times ranging from 24 to Interleukin22 Suppress Renal Cell Carcinoma Growth Figure 6. STAT1 and ERK1/2 pathways in A498 cells transfected with STAT1 siRNA by western blot assay. The expression of p-STAT1 decreased by 87.2%63.6% in STAT1 siRNA transfected cells at the 30th minute after IL-22 treatment and maintained in a lower level compared with that of negative control siRNA (P,0.05). N = 2. doi:10.1371/journal.pone.0020382.g006 72 hrs. The analysis showed that the fraction of annexin-V- positive/ PI-positive cells did not change significantly after rhIL-22 treatment, even at a dose of 400 ng/ml rhIL-22 and an exposure time of 72 hrs (Fig. 7). Taken together with the results of the FACScan analysis of the PI-stained cells, these data suggest that IL-22 treatment induces cell cycle arrest in the G2/M phase but does not induce apoptosis in A498 cells. Discussion In the current study, we investigated the effects of IL-22, a member of the IL-10 cytokine family, on human RCC cell line A498 cells in vitro and in vivo and studied the possible mechanisms underlying the anti-RCC tumor effects of the cytokine. First, we found that IL-22 receptors are expressed in A498 cells and that IL- 22 dose-dependently suppresses A498 cell growth and inhibits A498 xenograft growth. Second, we observed that IL-22 induced G2/M cell cycle arrest occurs without cell apoptosis. Moreover, we showed that the phosphorylation of STAT1 is increased and the phosphorylation of ERK1/2 is attenuated in A498 cells exposed to IL-22. Indeed, the growth inhibition of A498 cells exposed to IL-22 was partially revised to a relatively lower level after STAT1 was knocked down, indicating that STAT1 pathway plays a more important role in the growth inhibition of A498 cells exposed to IL-22 than ERK1/2 pathway does. We also showed that IL-22 did not enhance the anti-tumor immunity effectively in vivo by increasing the expression of IFN-a and TNF-a in A498 cell xenografts. These results suggest that the anti-A498 tumor effects of IL-22 are directly mediated by up-regulation of STAT1 and G2/M cell cycle arrest rather than by inducing apoptosis and inflammatory cytokines. Epidemiological studies have shown that the 5-year survival rate of advanced RCC patients is only 9.5% [2]. RCC is resistant to chemotherapy, and chemotherapeutic agents alone achieve a clinic response rate of less than 10%. Treatment regimens involving the administration of IFN-a have been used in patients with RCC with therapeutic response rates around 4–33% [29]. The low response rate, toxicity associated with high-doses regimens and low long-term survival rate of immunotherapy emphasize the necessity of finding a new agent to deal with RCC; the current lack of therapeutic agents for RCC that fulfill the basic PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20382