Expression and Polymorphism of Toll-Like Receptor 4 and Effect on NF-κB Mediated Inflammation in Colon Cancer Patients

Our aim was to evaluate the association between the expression and the polymorphism of TLR4/NF-κB pathways and colon cancer. TLR4 (rs4986790, rs10759932, rs10759931 and rs2770150) were genotyped in blood samples from Colorectal patients and healthy controls. TLR4 and cytokines inflammatory expression were evaluated by real time PCR on 40 matching normal and colon tissues and the protein level by Immunohistochemistry. The high level of TLR4 expression in colon cancer tissues is mainly due to infections by bacteria in the human colon and leads to induction of an acute secretion of inflammatory cytokines mediated by NF-κB. Also, we report here a clear evidence for an association between TLR4 rs10759931 polymorphism (OR = 0.086, CI: 0.04–0.18, P = <0.00001). This polymorphism affects the entire population without being specific to either gender or to any age group. In contrast, the rs2770150 is associated with colon cancer in women aged over 50 years and is closely linked with the decreased levels of female sex hormones during the post-menopausal period (OR = 0.188, CI: 0.074–0.48, P = <0.00084). rs10759932 and rs4986790 appear to have any association with colon cancer. Our data suggest that TLR4 SNPs could possibly serve as biomarkers for decision making in colon cancer treatment.     

TBK1 Kinase Addiction in Lung Cancer Cells Is Mediated via Autophagy of Tax1bp1/Ndp52 and Non-Canonical NF-κB Signalling

K-Ras dependent non-small cell lung cancer (NSCLC) cells are ‘addicted’ to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC.     

Induction of Peroxiredoxin 1 by Hypoxia Regulates Heme Oxygenase-1 via NF-κB in Oral Cancer

Overexpression of peroxiredoxin 1 (Prx1) has been observed in numerous cancers including oral squamous cell carcinoma (OSCC). The precise molecular mechanism of up-regulation of Prx1 in carcinogenesis, however, is still poorly understood. The objective of this study is to investigate the relationship between Prx1 and hypoxia, and potential mechanism(s) of Prx1 in OSCC cell line SCC15 and xenograft model. We treated wild-type and Prx1 knockdown SCC15 cells with transient hypoxia followed by reoxygenation. We detected the condition of hypoxia, production of reactive oxygen species (ROS), and expression and/or activity of Prx1, heme oxygenase 1 (HO-1) and nuclear factor-kappa B (NF-κB). We found that hypoxia induces ROS accumulation, up-regulates Prx1, increases NF-κB translocation and DNA binding activity, and down-regulates HO-1 in vitro. In Prx1 knockdown cells, the expression level of HO-1 was increased, while NFκB translocation and DNA binding activity were decreased after hypoxia or hypoxia/reoxygenation treatment. Moreover, we mimicked the dynamic oxygenation tumor microenvironment in xenograft model and assessed the above indices in tumors with the maximal diameter of 2 mm, 5 mm, 10 mm or 15 mm, respectively. Our data showed that tumor hypoxic condition and expression of Prx1 are significantly associated with tumor growth. The expression of HO-1 and NF-κB, and NF-κB DNA binding activity were significantly elevated in 15 mm tumors, and the level of 8-hydroxydeoxyguanosine was increased in 10 mm and 15 mm tumors, compared to those in size of 2 mm. The results from this study provide experimental evidence that overexpression of Prx1 is associated with hypoxia, and Prx1/NF-κB/HO-1 signaling pathway may be involved in oral carcinogenesis.     

GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-κB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-κB activation by H-Ala-D-γGlu-DAP (γTriDAP). GEF-H1 is essential for NF-κB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens.     

Anti-neuroinflammatory effects of grossamide from hemp seed via suppression of TLR-4-mediated NF-κB signaling pathways in lipopolysaccharide-stimulated BV2 microglia cells

Rac1 belongs to the Rho family of small GTPases and regulates actin cytoskeleton reorganization. T2R4 is a bitter taste receptor belonging to the G protein-coupled receptor family of proteins. In addition to mediating bitter taste perception from the tongue, T2R4s are found in extra-oral tissues, e.g., nasal epithelium, airways, brain, testis suggesting a much broader physiological function for these receptors. Anti-malarial drug and a bitter tasting compound, quinine, is a known agonist for T2R4, whereas BCML (Nα,Nα-Bis(carboxymethyl)-l-lysine) acts as an inverse agonist. Using western blot and Ca⁺⁺ mobilization assays, the effects of quinine on Rac1 activity in HEK293T cells stably expressing T2R4/Gα_16/44, T2R4, or Gα_16/44 and transiently transfected with HA-Rac1 were investigated. Quinine treatment caused a significant reduction in the amount of active Rac1, whereas in the presence of BCML, quinine failed to cause any significant change in active Rac1. No significant change in Rac1 activity was observed in BAPTA-AM plus quinine-treated Gα_16/44 cells, suggesting possibility of a pathway in addition to the canonical Ca⁺⁺-dependent pathway. A noticeable role for Gα_16/44 independent of T2R4 is observed in quinine-mediated Rac1 inactivation. Further, a significant difference in quinine-induced Ca⁺⁺ response in T2R4/Gα_16/44 or T2R4 cells was observed validating the partial role of calcium and importance of Gα_16/44. This study is the first to show an inhibitory downstream action of a T2R4 agonist on Rac1 function. Further investigation will help in better understanding the downstream signal transduction network of T2R4 and its extra-oral physiological roles.     

大黄多糖抑制脂多糖引起的TLR-4/NF-κB通路活化

目的:探讨唐古特大黄多糖(Rheum tanguticum Polysaccharid,RTP)对脂多糖(Lipopolysaccharide,LPS)刺激上调人结肠癌细胞HT-29中TLR-4/NF-κB通路活性的影响及其机制。方法:将HT-29细胞分为对照组,LPS处理组(1μg/mL,作用30 min,1 h),RTP(1mg/mL,提前LPS 30 min给予)+LPS处理组(1μg/mL,分别作用30 min,1 h),采用免疫荧光法观察NF-κB的细胞分布情况;Western Blot法检测HT-29细胞中IκB-α,磷酸化IκB-α的蛋白变化,以及细胞膜上TLR-4的水平。结果:RTP可抑制LPS刺激引起的HT-29细胞的NF-κB核转位;可有效抑制IκB-α降解及IκB-α的磷酸化;可下调细胞膜上TLR-4的表达。结论:RTP可能通过抑制LPS刺激引起的TLR-4向细胞膜分布,从而抑制了NF-κB信号通路的活化。     

WTAP facilitates progression of endometrial cancer via CAV-1/NF-κB axis

The N⁶-methyladenosine (m⁶A) modification is one of the most prevalent methylations in eukaryotic messenger RNA (mRNA), and it is essential for the development of many important biological processes such as multiple types of tumors. One of the most important enzymes catalyzing generation of m⁶A on mRNA is Wilms' tumor 1-associating protein (WTAP); however, the potential role of WTAP in endometrial cancer (EC) still remains unknown. Here, we investigated WTAP expression level in cancer tissue and paracancerous tissue from an EC patient. Subsequently, WTAP was knocked down by small interfering RNA in EC cell line of Ishikawa and HEC-1A, respectively. Cell proliferation, migration, and invasion were studied. The expression of caveolin-1 (CAV-1) was detected by quantitative polymerase chain reaction (qPCR). The enrichments of m⁶A and METTL3 on CAV-1 were detected using RNA immunoprecipitation-qPCR. The activity of nuclear factor-κB (NF-κB) was studied using Western blot. We observed that WTAP was dramatically upregulated in the cancer tissue, and there was an enhancement in cell proliferation, migration, and invasion and a decrease in EC apoptosis in vivo and in vitro, which indicated higher tumor malignancy and worse survival outcome. After WTAP was knocked down in EC cells, CAV-1 was significantly upregulated and the enrichments of m⁶A and METTL3 at 3′-untranslated region (UTR) region of CAV-1 were decreased. Moreover, the activity of NF-κB signaling pathway was inhibited by its regulator CAV-1. Taken together, we concluded that WTAP could methylate 3′-UTR of CAV-1 and downregulate CAV-1 expression to activate NF-κB signaling pathway in EC, which promoted EC progression.     

Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.