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-a and TNF-a were analyzed by western blotting assay. It was found that the levels of TNF-a and IFN-a remained unchanged in A498 xenografts after IL-22 treatment compared with that of controls (p.0.05). TNF-a was originally described as an endotoxin- induced, macrophage-derived protein with different effects depending on the tumor type to which it is administered [42]. The results of minimal changes of TNF-a and IFN-a in A498 xenografts tissue after IL-22 exposure suggest minimal immuno- cytes 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 [26]. 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 [27]. 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 [32]. 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 [28]. 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 [30]. 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 [30]. 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. Interleukin22 Suppress Renal Cell Carcinoma Growth 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. Materials and Methods Cell culture The human A-498RCC cell line was purchased from Cell Bank of China Union Medical University (Beijing, China) and cultured in 5% CO2 humidified atmosphere at 37uC in complete RPMI- 1640 medium (GIBCO, UK) supplemented with 25 mM HEPES, 2 mM L-glutamine, 1% non-essential amino acids, 100 mg/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS). Growth inhibition assay Exponentially growing A498 cells were seeded at 1,000 cells/ well in a 96-well microculture plate. Multi-dosage of recombinant human interleukin 22 (rhIL-22, ADL), 10 ng, 20 ng, 50 ng and 100 ng respectively, were added after cell adhesion for 4 hrs followed by continuous incubation for 3 days. Cells treated with PBS served as controls. 20 ml MTT (5 mg/ml) was added to each well and the plate was incubated at 37uC for another 4 h, after which 10% HCl-SDS (100 ml) was added to each well. The plate was kept at 4uC overnight. Cells were then harvested and cell proliferation was determined using a 96-well plate reader to record the absorbance at 570 nm. The percentage inhibition of A498 cells was calculated using the following formula: % inhibitio- n = (absorbance of control well2absorbance of IL-22 well)/ absorbance of control well6100%. Monoclonal human IL-22 antibody (300 mg/well, R&D) was used to confirm the contribution of IL-22 to A498 cells growth. The percentage inhibition of A498 cells treated with different doses of rhIL-22 was re-calculated with the similar procedures. Growth inhibition activity of IL-22 on A498 xenografts. BALB/C nude mice provided by SLAC Laboratory Animal (Shanghai, China), were studied after approval from the Medical ethics committee of Beijing Friendship Hospital, Capital Medical University. Three- to four- week-old mice were maintained in high-efficiency particulate air- filtered cages in a pathogen-free facility. A498 cells were washed once and resuspended in serum-free medium. 56106 cells in matrigel (BD Biosciences, San Jose, CA) were injected into the neck region; mice were examined the day after injection. When the tumor size 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 5 weeks; the tumor volume was determined by measuring the maximal (a) and minimal (b) diameters using a caliber and calculated by using the formula a6b2. After five weeks, mice were sacrificed under deep anesthesia and the final volumes of the tumors were measured. Flow cytometric analysis of cell cycle status A498 cells (16104) were seeded on 10-cm dishes and incubated for 24 hrs in serum-free medium. Cells were then treated with 25, 50, 100, 200 or 400 ng/ml of rhIL-22 for 24 hrs or with PBS as control. The cells were then collected, fixed with 70% ethanol at 220uC overnight, washed 3 times with PBS and incubated at PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20382