Rational design,synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor

(?)-Dehydroxymethylepoxyquinomicin ((?)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-β-salicyloylamino-α-exo-methylene-?-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (?)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.     

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.     

LPS-induced CXCR4-dependent migratory properties and a mesenchymal-like phenotype of colorectal cancer cells

Colorectal cancer (CRC) is the most commonly diagnosed cancer worldwide, and over 50% of patients will develop hepatic metastasis during the course of their disease. CXCR4 and its ligand, stromal cell-derived factor 1α (SDF-1α)/chemokine (C-X-C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we have shown that lipopolysaccharides (LPS) promoted the migratory capacity of colon cancer cells in vivo and in vitro, which correlated with the activation of SDF-1α/CXCR4 axis and epithelial-mesenchymal transition (EMT) occurrence. Additionally, we found that LPS-induced CXCR4 expression and EMT through NF-κB signaling pathway activation. And inhibition of NF-κB pathway, which recovered the epithelial phenotype and attenuated CXCR4 expression, inhibited cell migratory capacity. Clinically, high levels of CXCR4 always correlated with metastasis and poor prognosis of CRC patients. In conclusion, LPS participate in the whole process of hepatic metastasis of CRC, not only causing liver damage resulting in the production of SDF-1α, but also enhancing the invasive potential of CRC cells by promoting CXCR4 expression and EMT occurrence, which would contribute to the enhancement of cell migration and invasion.     

趋化因子SD-1及其受体CXCR4在卵巢癌中的研究进展

基质细胞衍生因子-1(Stromal cell derived factor-1,SDF-1)是CXC趋化因子家族的重要成员,系统命名为CXCL12,能与它的唯一受体CXC趋化因子受体-4(CXC chemokine receptor-4,CXCR4)形成CXCL12-CXCR4生物学轴,CXCL12-CXCR4生物学轴在肿瘤生长、侵袭、转移过程中发生重要作用。到目前为止,已发现CXCL12-CXCR4在卵巢癌、胰腺癌、肝癌等多种肿瘤组织中表达。然而,国内目前还没有关于CXCL12-CXCR4与卵巢癌关系的相关综述,本文将从趋化因子CXCL12及其受体CXCR4,CXCL12/CXCR4轴与卵巢癌细胞系实验研究,CXCL12-CXCR4轴与卵巢癌的临床研究,CXCL12/CXCR4与卵巢癌预后,CXCL12/CXCR4与卵巢癌治疗展望等五个方面对CXCL12-CXCR4生物轴与卵巢癌的关系,及其在卵巢癌治疗中的应用展开综述。     

Imaging CXCL12-CXCR4 Signaling in Ovarian Cancer Therapy

Chemokine CXCL12 and receptor CXCR4 have emerged as promising therapeutic targets for ovarian cancer, a disease that continues to have a dismal prognosis. CXCL12-CXCR4 signaling drives proliferation, survival, and invasion of ovarian cancer cells, leading to tumor growth and metastasis. Pleiotropic effects of CXCR4 in multiple key steps in ovarian cancer suggest that blocking this pathway will improve outcomes for patients with this disease. To quantify CXCL12-CXCR4 signaling in cell-based assays and living mouse models of ovarian cancer, we developed a click beetle red luciferase complementation reporter that detects activation of CXCR4 based on recruitment of the cytosolic adapter protein β-arrestin 2. Both in two-dimensional and three-dimensional cell cultures, we established that bioluminescence from this reporter measures CXCL12-dependent activation of CXCR4 and inhibition of this pathway with AMD3100, a clinically-approved small molecule that blocks CXCL12-CXCR4 binding. We used this imaging system to quantify CXCL12-CXCR4 signaling in a mouse model of metastatic ovarian cancer and showed that treatment with AMD3100 interrupted this pathway in vivo. Combination therapy with AMD3100 and cisplatin significantly decreased tumor burden in mice, although differences in overall survival were not significantly greater than treatment with either agent as monotherapy. These studies establish a molecular imaging reporter system for analyzing CXCL12-CXCR4 signaling in ovarian cancer, which can be used to investigate biology and therapeutic targeting of this pathway in cell-based assays and living mice.     

Ursolic acid inhibits nuclear factor-κB signaling in intestinal epithelial cells and macrophages,and attenuates experimental colitis in mice

Aims

Ursolic acid (UA), a natural pentacyclic triterpenoid acid, has been reported to show immunomodulatory activity. This study investigated the effects of UA on nuclear factor-kappa B (NF-κB) signaling in cells and experimental murine colitis.

Main methods

Human intestinal epithelial cells (IECs) COLO 205 and peritoneal macrophages from IL-10-deficient (IL-10^−/−) mice were pretreated with UA and then stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), respectively. The expression of pro-inflammatory cytokines was determined by real-time RT-PCR and ELISA. The effect of UA on NF-κB signaling was examined by immunoblot analysis to detect IκBα phosphorylation/degradation and electrophoretic mobility shift assay to assess the DNA binding activity of NF-κB. For in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6 wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without UA. Colitis was quantified by histopathologic evaluation. Immunohistochemical staining for phosphorylated IκBα was performed in the colonic tissue.

Key findings

UA significantly inhibited the production of pro-inflammatory cytokines, IκBα phosphorylation/degradation and NF-κB DNA binding activity in both IEC and IL-10^−/− peritoneal macrophages stimulated with TNF-α and LPS, respectively. UA significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histopathology. UA also significantly ameliorated the severity of colitis in IL-10^−/− mice. Furthermore, UA suppressed IκBα phosphorylation in the colonic tissue.

Significance

UA inhibits NF-κB activation in both IECs and macrophages, and attenuates experimental murine colitis. These results suggest that UA is a potential therapeutic agent for inflammatory bowel disease.     

Retracted: Role of CXCL12–CXCR4 axis in ovarian cancer metastasis and CXCL12–CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro

Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12–CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis. OC (nonmetastatic and metastatic) and normal ovarian tissues were collected to determine the expressions of CXCL12 and CXCR4. A series of AMD3100, shRNA against CXCR4, and pCNS-CXCR4 were introduced to treat CAOV3 cells with the highest CXCR4 was assessed. Cell viability, apoptosis, migration, and invasion were all evaluated. The microarray analysis screened out the differential expression of CXCL12–CXCR4 in OC. CXCL12 and CXCR4 expressions were increased in OC tissues, particularly in the metastatic OC tissues. Downregulation of CXCR4 by AMD3100 or shRNA was observed to have a critical role in inhibiting cell proliferation, migration, and invasion of the CAOV3 OC cell line while promoting cell apoptosis. Overexpressed CXCR4 brought significantly promoting effects on the proliferation and invasiveness of OC cells. These results reinforce that the blockade of CXCL12–CXCR4 axis with AMD3100 inhibits the growth of OC cells. The antitumor role of the inhibition of CXCL12–CXCR4 axis offers a preclinical validation of CXCL12–CXCR4 axis as a therapeutic target in OC.     

Classical NF-kappaB activation negatively regulates noncanonical NF-kappaB-dependent CXCL12 expression

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.     

Na+/Ca2+ exchanger-1 protects against systolic failure in the Akitains2 model of diabetic cardiomyopathy via a CXCR4/NF-κB pathway

Diabetic cardiomyopathy is characterized, in part, by calcium handling imbalances associated with ventricular dysfunction. The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) has been implicated as a compensatory mechanism in response to reduced contractility in the heart; however, its role in diabetic cardiomyopathy remains unknown. We aimed to fully characterize the Akita(ins2) murine model of type 1 diabetes through assessing cardiac function and NCX1 regulation. The CXCL12/CXCR4 chemokine axis is well described in its cardioprotective effects via progenitor cell recruitment postacute myocardial infarction; however, it also functions in regulating calcium dependent processes in the cardiac myocyte. We therefore investigated the potential impact of CXCR4 in diabetic cardiomyopathy. Cardiac performance in the Akita(ins2) mouse was monitored using echocardiography and in vivo pressure-volume analysis. The Akita(ins2) mouse is protected against ventricular systolic failure evident at both 5 and 12 mo of age. However, the preserved contractility was associated with a decreased sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a)/phospholamban ratio and increased NCX1 content. Direct myocardial injection of adenovirus encoding anti-sense NCX1 significantly decreased NCX1 expression and induced systolic failure in the Akita(ins2) mouse. CXCL12 and CXCR4 were both upregulated in the Akita(ins2) heart, along with an increase in IκB-α and NF-κB p65 phosphorylation. We demonstrated that CXCR4 activation upregulates NCX1 expression through a NF-κB-dependent signaling pathway in the cardiac myocyte. In conclusion, the Akita(ins2) type 1 diabetic model is protected against systolic failure due to increased NCX1 expression. In addition, our studies reveal a novel role of CXCR4 in the diabetic heart by regulating NCX1 expression via a NF-κB-dependent mechanism.     

The effect of intestinal alkaline phosphatase on intestinal epithelial cells,macrophages and chronic colitis in mice

Aims

Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme that is shown to function as a gut mucosal defense factor, but its defensive mechanism remains unclear. The aims of this study were to evaluate the effect of IAP on intestinal epithelial cells and macrophages, and on chronic colitis in interleukin-10-deficient (IL-10^−/−) mice.

Main methods

Human intestinal epithelial cells COLO 205 and peritoneal macrophages from IL-10^−/− mice were pretreated with IAP and then stimulated with lipopolysaccharide (LPS). IL-8 secretion from COLO205 cells and TNF-α, IL-6, IL-12 from peritoneal macrophages were measured by ELISA. Electrophoretic mobility shift assay was used to assess the DNA binding activity of NF-κB and IκBα phosphorylation/degradation was evaluated by immunoblot assay in COLO 205. For the in vivo study, colitis was induced in IL-10^−/− mice with piroxicam, the mice were then treated with 100 or 300 units of IAP by oral gavage for 2 weeks. Colitis was quantified by histopathologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

IAP significantly inhibited LPS-induced inflammatory cytokine production in both IECs and peritoneal macrophages. IAP also attenuated LPS-induced NF-κB binding activity and IκBα phosphorylation/degradation in IECs. Oral administration of IAP significantly reduced the severity of colitis and down-regulated colitis-induced IκBα phosphorylation in IL-10^−/− mice.

Significance

IAP may inhibit the activation of intestinal epithelial cells and peritoneal macrophages, and may attenuate chronic murine colitis. This finding suggests that IAP supplementation is a potential therapeutic option for inflammatory bowel disease.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

趋化因子CXCL12及其受体CXCR4在胶质瘤中的研究进展

CXCL12是趋化因子家族成员之一,是能够特异性结合其受体CXCR4发挥趋化性作用的细胞因子。最初,CXCL12及CXCR4被发现于炎症细胞,参与机体炎症、免疫等病理反应。接下来的几年中发现,它在机体发育、成熟过程中也有重要作用。如今,大量研究表明它与肿瘤的生长、侵袭及转移密切相关。据报道,在乳腺癌、肺癌、卵巢癌等二十余种肿瘤组织中发现CXCL12及CXCR4的表达,其中也包括中枢系统肿瘤-胶质瘤。CXCL12/CXCR4参与胶质瘤生长过程的多个步骤,包括肿瘤增殖、侵袭、转移等。有实验指出,转移灶的CXCR4表达水平较原发灶高,CXCR4有可能成为抑制胶质瘤生长、转移的重要靶目标。     

Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.