| Objective: Chronic inflammation plays an important role in the development of lung cancer by releasing different kind of oxidizing medium and inflammatory cytokines. In the early stage of tumorigenesis, inflammatory cytokines not only induce cell proliferation, but also lead to oxidative stress in the form of reactive oxygen species(ROS) formation to cause DNA damage and mutations. Recently, we found oral administration of AFG1-induced chronic inflammation may contribute to lung tumorigenesis. AFG1-induced chronic inflammation can cause alveolar type II cell(AT-II) oxidative damage and proliferation. In the chronic inflammatory environment, increased expression of TNF-α and IL-6, and up-regulation of NF-κB p65 and p-STAT3 were observed. Furthermore, we found that TNF-α upregulated CYP450 expression to promote the metabolism of AFG1 through NF-κB pathway, and then enhanced oxidative DNA damage in 549 cells. The results suggest that inflammatory cytokines may play a critical role in the metabolism of AFG1 and oxidative DNA damage in AT-II cells in AFG1-induced chronic inflammatory environment.IL-6 is an important tumor promoting cytokine, and involved in a variety of malignant transformation, cell proliferation and metastasis in the stage of tumor progression and promotion. However, the function of IL-6 in carcinogen-induced DNA damage is unclear. Our previous studies have shown that AFG1 induced oxidative DNA damage in AT-II cells, however the role of IL-6 in the metabolism of AFG1 as well as AFG1-induced cell damage needed to be investigated in the chronic inflammation microenvironment.In order to explore the role of IL-6 in AFG1-induced oxidative DNA damage in AT-II cells in AFG1-induced inflammation microenvironment, we applied the A549 cell line as a model of human AT-II cells and treated it with AFG1 and IL-6 together to mimic an AFG1-induced inflammatory response in vitro. We will explore the effect of IL-6 on AFG1 activation-related enzyme Cyp450, as well as AFG1-induced DNA damage in A549 cells.Methods:1 Cell culture and treatmentThe A549 cell line as a model of human AT-II cells are grown in RPMI-1640 medium supplemented with 10% new-born calf serum(NBCS), streptomycin(100 μg/ml) and penicillin(100 U/ml).A549 cells in logarithmic growth phase were randomly divided into solvent control group and the experimental group. The experimental group were then treated with 4ug/ml AFG1 alone or treated with 4ug/ml AFG1 and 20ug/ml IL-6 combination for 24 h. Solvent control group were treated with DMSO at a concentration of 0.08%(n=3).2 FCMWe compared ROS generation in A549 cells on different time points upon AFG1-treatment or AFG1+IL-6- treatment by FCM assay.3 IHCBalb/c mice were treated by repeated oral administration of AFG1(three times weekly) for 6 months,and the expression of Cyp1A2,Cyp2A6,Cyp2A13 were measured in mice lung tissues.4 Western blotAfter treated with AFG1 alone or treated with AFG1 and IL-6 together for 24 h, the expression of SOD2, γH2AX, ATM, ATR, Cyp450, Phospho-Stat3, BCL-XL,Cyclin D1,Bax at protein level in A549 cells was determined by Western blot.5 Real-time PCRAfter treated with AFG1 alone or treated with AFG1 and IL-6 together for 24 h, the expression of IL-1β, IL-6, IL-10, IL-12, TNF-α, Cyp1A2, Cyp2A6, Cyp2A13, Cyp3A4 at m RNA level in A549 cells was determined by Real time PCR.6 MTTAfter treated with AFG1 alone or treated with AFG1 and IL-6 together for 24 h, the A549 cells survival rate were detectioned by MTT assay.7 Treatment in A549 cells with STAT3 inhibitorsA549 cells in logarithmic growth phase were preincubated for 30 min with 1μM Cpd188(STAT3 inhibitor). Then the cells were treated with 4ug/ml AFG1 and 20μg/ml IL-6 for 24 h.8 si RNA transfectionCells were reversely transfected with Negtive control si RNA(NC si RNA) or si RNA targeting Stat3 at a concentration of 100 pmol respectively using Lipofectamine 2000(Invitrogen) according to the manufacturer’s instructions. And 24 h post transfection, cells were washed with PBS and subsequently treated with 4ug/ml AFG1 and 20ug/ml IL-6 for 24 h. Afterwards, cells were harvested and assayed by Western blot and flow cytometry.9 Statistical analysisThe results were presented as means ± SD. Data were analyzed using one-way analysis of variance(ANOVA) analysis. Values were considered statistically significant when P<0.05.Results: 1 The combination of AFG1 and IL-6 together enhanced oxidative DNA damage in A549 cellsWe found the intracellular ROS generation in A549 cells treated with AFG1 and IL-6 together increased at 3 h, while that in AFG1 alone started to increase at 6 h after treatment. SOD-2 and γH2AX expression were increased in A549 cells treated with AFG1 and IL-6 together for 6 h, while AFG1 alone did not affect γH2AX and SOD-2 expression(P<0.05). After 24 h treatment, we found combination of AFG1 and IL-6 enhanced γH2AX, SOD-2, ATM, ATR expression than AFG1 alone(P<0.05). The results obviously suggest that IL-6 could enhance AFG1-induced oxidative stress to promote DNA double-strand breaks in AT-II cells. 2 The combination of AFG1 and IL-6 together increased inflammatory cytokines expression in A549 cellsWe found that AFG1 alone increased TNF-α, IL-1a, IL-6, IL-10, IL-12, and MIP-2 m RNA expression in A549 cells. The combination of AFG1 and IL-6 increased IL-1β, IL-6, IL-10 and IL-12 expression in A549 cells, compared to AFG1 alone(P<0.05). The results suggest that IL-6 could enhance AFG1 induced activatin of AT-II cells to promote inflammatory cytokines production, which may contribute to lung chronic inflammatory responses. 3 The expression of Cyp1A2, Cyp2A6 and Cyp2A13 in AFG1-induced chronic inflammatory environmentWe detected Cyp1A2, Cyp2A6 and Cyp2A13 expression in lung tissues of mice after oral admination of AFG1 for 6 months. We found higher level expression of Cyp1A2, Cyp2A6 and Cyp2A13 in AT-II cells in AFG1-induced inflammatory lung tissues. The results suggest that AFG1-induced inflammation induced Cyp450 expression in AT-II cells. 4 Combination of AFG1 and IL-6 together increased Cyp450 expression in A549 cellsWe found AFG1 alone only increased Cyp1A2 expression at m RNA and protein level, but did not affect Cyp2A6 and Cyp2A13 expression. However, the combination of AFG1 and IL-6 together increased Cyp1A2, Cyp2A6 and Cyp2A13 expression in A549 cells than AFG1 alone(P<0.05). The results suggest that IL-6 may enhance the metabolism of AFG1 in A549 cells by up-regulating Cyp450 expression in AFG1-induced chronic inflammatory environment. 5 Effect of STAT3 inhibition on oxidative DNA damage and Cyp450 expression in A549 cells treated by AFG1 and IL-6 coordinately 5.1 Effect of STAT3 specific inhibitors(Cpd188) on oxidative DNA damage and Cyp450 expression in A549 cells triggered by AFG1 and IL-6 coordinatelyTo further explore whether IL-6 enhances AFG1-induced oxidative DNA damage and contributes to Cyp450 expression in A549 cells through STAT3 pathway, we investigated Cyp1A2, Cyp2A6, Cyp2A13 and γH2AX expression in 549 cells pretreated with Cpd188. We found blocking STAT3 pathway significantly inhibited the expression of Cyp1A2, Cyp2A6 and γH2AX in A549 cells treated with AFG1+IL-6 together(P<0.05). The results suggest that IL-6-activated STAT3 pathway upregulates Cyp450 to promote the metabolism of AFG1, and then enhances oxidative DNA damage in AT-II cells. 5.2 Effect of STAT3 si RNA transfaction on oxidative DNA damage in A549 cells triggered by AFG1 and IL-6 coordinatelyFCM results showed that the ROS generation in A549 cell treated with Stat3 si RNA+AFG1+IL-6 was significantly decreased compared with that in A549 cells treated with AFG1+IL-6(P<0.05). The results suggest that IL-6 enhanced AFG1-induced ROS generation in AT-II cells through STAT3 pathway.Western blot results showed that blocking STAT3 pathway by Stat3 si RNA significantly inhibited the expression of Cyp1A2, Cyp2A6 and γH2AX in A549 cells treated with AFG1+IL-6 together(P<0.05). The results provide supports that IL-6 upregulates Cyp450 to promote the metabolism of AFG1, and then enhances oxidative DNA damage in AT-II cells through STAT3 pathway. 6 Effect of AFG1 and IL-6 on A549 cells survivalMTT results showed no significant difference between AFG1 treatment group and AFG1+IL-6 treatment group(P>0.05). Western blot results showed that the combination of AFG1 and IL-6 increased the expression of BCL-XL, Cyclin D1 and Bax. The results suggest that AFG1-injuried AT-II cells with DNA damage may acquire proliferation ability triggered by IL-6.Conclusion:1 IL-6 could enhance AFG1-induced oxidative stress to trigger DNA double-strand breaks in AT-II cells.2 IL-6 may enhance the metabolism of AFG1 in A549 cells by up-regulating CYP450 expression in AFG1-induced chronic inflammatory environment.3 IL-6 upregulates Cyp450 to promote the metabolism of AFG1, and then enhances oxidative DNA damage in AT-II cells through STAT3 pathway.4 In AFG1-induced inflammatory microenvironment, the combination of AFG1 and IL-6 increased oxidative DNA damage in AT-II cells, which may contribute to AFG1-induced lung tumorigenesis in tumor initiation stage. |
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