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-α and TNF-α 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% 
. 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-α have been used in patients with RCC with therapeutic response rates around 4–33% 
. 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 and necessary criteria for clinical treatment demand the development of more effective drugs to combat this disease.
IL-22, which in humans is mainly produced by Th22-, Th1- and Th17-cells, is strongly involved in immune regulation and inflammatory responses 
. IL-22 plays an important role in inflammatory processes through up-regulation of acute phase reactions and of pancreatitis-associated protein 
. However, there is some debate about the effects of IL-22 on tumors. We suppose that IL-22 may play different roles in different tumors and that discrepancies in experimental results may be due to different cell types and diseases. The results of the present study support the idea that IL-22 suppresses the growth of A498 cells both in vivo
and in vitro
The functional IL-22 receptor complex consists of two receptor chains, IL-22R and IL-10R2. IL-22R2 is broadly expressed in various tissues 
, and the expression of the IL-22R chain determines whether a cell will be a target of IL-22. In our study, we detected measurable expression of IL-22R in A498 cells and showed that IL-22 suppressed the growth of RCC cells in a dose-dependent manner. Similar effects were obtained in A498 xenograft tumors.
Although IL-22 is produced by immune cells, unlike other cytokines it does not affect immune cells directly but instead regulates the functions of their target cells, increasing the innate immunity of tissue cells, protecting tissues from damage and enhancing their regeneration. The signal transducer and activator of transcription 1 (STAT1) plays a critical role in carcinogenesis by mediating various biological responses and has been implicated as a tumor suppressor 
. Both carcinogen-induced and transplanted tumors are increased in STAT1 knockout mice cpmpared with wild-type control mice 
. Zhu demonstrated that SNPs in the STAT1 gene (homozygotes of the minor alleles at SNP rs867637, rs3771300, or rs2280235) are associated with a risk of developing hepatocellular carcinoma in Chinese people 
. While, Specific inhibition of ERK1/2 phosphorylation by a variety of natural and synthetic compounds has been shown to be effective in anticancer strategy in the treatment of breast cancer 
.To better understand the molecular mechanisms that underlie the anti-RCC tumor effects of IL-22, we assessed the phosphorylation of STAT1 and ERK1/2 in A498 cells after rhIL-22 treatment. Our western blotting assays showed that p-STAT1 was founded in A498 cells at the 5th
minute and reached its peak level 30 minutes after rhIL-22 exposure. While ERK1/2 was dephosphorylated in response to rhIL-22; this effect was maximal 30 min after rhIL-22 exposure. It suggests that rhIL-22 activates STAT1 and inhibits ERK1/2 pathways in A498 cells in a time- and dose-dependent manner and that the biological functions of the receptors for these pathways show time-dependent STAT1 activation and ERK1/2 deactivation evoked by rhIL-22. Concerning the concentration of STAT1 protein in A498 cells after IL-22 exposure was stable independent on the vary dose of IL-22 and longer exposure time, we believe that IL-22 induce STAT1 pathway activating directly rather than act on gene level. Hence, the STAT1-ERK1/2 pathway participates, at least in part, in the inhibition of A498 cell growth that occurs after IL-22 binding to and activation of IL-22R. To further understand the role of STAT1-ERK1/2 pathway in the growth inhibition of A498 cells exposed to IL-22, the expression of STAT1 was deleted in A498 cells by STAT1 specific siRNA. The phosphorylation STAT1 was decreased by 87.2% after treated with IL-22 compared with the controls and independent to dephosphorylated ERK1/2 pathway.The results show that the growth inhibition of A498 cells with STAT1 deletion was only partially revised in these cells treated with IL-22 compared with the controls (p>0.05), which indicates that the activation of STAT1 pathway is the major mechanism involved in the growth inhibition of A498 cells rather than dephosphorylation of ERK1/2. Dysfunctional STAT1 may contribute to cancer development and progression, while at the same time the ERK1/2 pathway regulates a common set of cell death regulators such as BCL-2, BCL-XL and BIM, indicating that it plays a role in cell cycle control 
. ERK1/2 inhibitors would therefore be expected to function as anti-tumor agents 
. Concerning the lack of contribution of ERK1/2 pathway to the growth inhibition of A498 cells exposed to IL-22, it can be hardly concluded that IL-22 could serve as a reasonable ERK1/2 inhibitor for A498 cells. (Figure S1
: a schematic diagram for STAT1-ERK1/2 pathway work in this procedure.)
We propose that the STAT1 pathway is activated in A498 cells after IL-22 treatment. Because activation of the STAT1 pathway usually leads to apoptosis, we further investigated whether IL-22 suppresses the proliferation of RCC cells by inducing apoptosis or cell-cycle arrest. Our results showed that IL-22 did not induce apoptosis in A498 cells but instead was associated in a dose-dependent manner with cell cycle arrest in the G2/M phase.
Our results also showed a tumor growth inhibition effect in vivo
when exposure to IL-22. After treated with IL-22, the A498 xenografts were significantly smaller than that of control mice. In this study, we used BALB/c nu mice as tumor-bearing host and the tumors were confirmed in pathology. To further understand the function of inflammatory cytokines in this procedure, IFN-α and TNF-α were analyzed by western blotting assay. It was found that the levels of TNF-α and IFN-α remained unchanged in A498 xenografts after IL-22 treatment compared with that of controls (p>0.05). TNF-α was originally described as an endotoxin-induced, macrophage-derived protein with different effects depending on the tumor type to which it is administered 
. The results of minimal changes of TNF-α and IFN-α in A498 xenografts tissue after IL-22 exposure suggest minimal immunocytes recruitment in this procedure. The tumor infiltration leukocytes were rare in both IL-22 treatment tumors and controls probably due to the adoption of athymic BALB/c nu mice as the xenograft host. Hence, we reasoned that IL-22 act as an immediate anti-A498 cell actor in this procedure rather than a promoter cellular immunity. However, we do not deny that other unknown anti-A498 cells mechanisms take part in this procedure and further research in this field is necessary.
Several studies focusing on the effects of IL-22 on cancer cells have shown similar results. IL-22 could significantly prolong the survival of mice bearing Colon 26 cells, though it did not inhibit the growth of colon cancer cells 
. IL-22 could reduce the growth of mammary adenocarcinoma (EMT6) cells; this growth reduction was associated with the inhibition of ERK1/2 and Akt phosphorylation and the induction of G2/M phase cell cycle arrest 
. Interestingly, in contrast to its protective role in some cells, IL-22 seems to serve as a tumor promoter in other human tumor cells, likely as a result of its regulation of different intracellular signaling pathways in different cell types. The growth of HepG2 human hepatocellular carcinoma cells was promoted by IL-22 through the activation of STAT3, ERK1/2 and the induction of antiapoptotic proteins 
. IL-22 also protected lung cancer cell lines from serum-starvation-induced and chemotherapeutic drug-induced apoptosis by activation of STAT3 and its downstream antiapoptotic proteins and inhibition extracellular signal-regulated kinase 1/2 
. Based on these results, we presume that the different observed effects of IL-22 depend on its activation of different cell signaling pathways in different tissue types. Recently, the association between IL-22 genetic polymorphisms and the risk of colon cancer was investigated; it shows that one haplotype containing the rs1179251 G allele with the incidence of 21.03%, which enhance the IL-22 exposure, gave an estimated 52% increase in risk of colon cancer for individuals 
. However, the SNP (rs1179251) is a non-coding (intronic) SNP. Therefore, it is uncertain whether IL-22 plays the role of a potential promoter in colon cancer 
. Due to the controversial biological effects of IL-22 in tumor cells, it is necessary to carry out further research on this topic.
In summary, we have demonstrated that IL-22 inhibits the growth of A498 cells (RCC) both in vivo and in vitro, at least partially by activating the STAT1 pathway directly rather than acting on gene level in these cells. The anti-A498 cells effects of IL-22 were associated with cell cycle arrest in the G2/M phase. Apoptosis and immunocytes recruitment were not found to be involved in this process. We suggest that IL-22 may be an effective agent for human RCC A498 cells growth inhibition; further, the STAT1 pathway could be an interesting target for improving patient responses in RCC. The data presented here support this conjecture, although additional experimental on other RCC cell lines and clinical evidence will be required to advance this hypothesis.