Abstract
BackgroundH. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models.MethodsTo investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays.ResultsFollowing infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models.ConclusionOur findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.
Highlights
H. pylori infection is the main risk factor for gastric cancer
Reconstitution of Trefoil Factor 1 (TFF1) decreases H. pylori‐mediated activation of NF‐κB and Signal transducer and activa‐ tors of transcription (STAT3) in gastric cancer cells In this study, we investigated the role of H. pylori in regulating Nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) and STAT3
Our results demonstrated an early induction of p-NF-κB-P65 (Ser536) at 1 h, whereas p-STAT3 (Y705) was delayed and increased at 24 h time point, following infection with H. pylori strain 7.13 or J166
Summary
H. pylori infection is the main risk factor for gastric cancer. Helicobacter pylori (H. pylori), a gram-negative bacterium, has been considered an important environmental risk factor for gastric cancer, classified by the World Health Organization as class I carcinogen [2, 3]. Chronic infection with H. pylori plays an important role in activating several pro-inflammatory signaling. NF-κB and STAT3 are among the major pro-inflammatory pathways activated during carcinogenesis [11]. NF-κB is a transcriptional factor which is constitutively activated in gastric cancer [12], and its activation is mediated via cagA after H. pylori infection [13]. Activation of NF-κB induces release of several pro-inflammatory cytokines and chemokines that are involved in proliferation, angiogenesis, invasion and blockade of apoptosis [14]. STAT3 is another transcriptional factor that is activated by cytokines and growth factor-induced signaling [15]. A number of recent reports demonstrated functional interaction between NF-κB and STAT3 transcriptional activities for maximum induction and activation of cytokines [16]
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