The Exploration of Chemokines Importance in the Pathogenesis and Development of Endometrial Cancer

Endometrial cancer (EC) is one of the most frequent female malignancies. Because of a characteristic symptom, vaginal bleeding, EC is often diagnosed in an early stage. Despite that, some EC cases present an atypical course with rapid progression and poor prognosis. There have been multiple studies conducted on molecular profiling of EC in order to improve diagnostics and introduce personalized treatment. Chemokines—a protein family that contributes to inflammatory processes that may promote carcinogenesis—constitute an area of interest. Some chemokines and their receptors present alterations in expression in tumor microenvironment. CXCL12, which binds the receptors CXCR4 and CXCR7, is known for its impact on neoplastic cell proliferation, neovascularization and promotion of epidermal–mesenchymal transition. The CCL2–CCR2 axis additionally plays a pivotal role in EC with mutations in the LKB1 gene and activates tumor-associated macrophages. CCL20 and CCR6 are influenced by the RANK/RANKL pathway and alter the function of lymphocytes and dendritic cells. Another axis, CXCL10–CXCR3, affects the function of NK-cells and, interestingly, presents different roles in various types of tumors. This review article consists of analysis of studies that included the roles of the aforementioned chemokines in EC pathogenesis. Alterations in chemokine expression are described, and possible applications of drugs targeting chemokines are reviewed.     

SMM-chemokines: a class of unnatural synthetic molecules as chemical probes of chemokine receptor biology and leads for therapeutic development

Chemokines and their receptors play important roles in numerous physiological and pathological processes. To develop natural chemokines into receptor probes and inhibitors of pathological processes, the lack of chemokine-receptor selectivity must be overcome. Here, we apply chemical synthesis and the concept of modular modifications to generate unnatural synthetically and modularly modified (SMM)-chemokines that have high receptor selectivity and affinity, and reduced toxicity. A proof of the concept was shown by transforming the nonselective viral macrophage inflammatory protein-II into new analogs with enhanced selectivity and potency for CXCR4 or CCR5, two principal coreceptors for human immunodeficiency virus (HIV)-1 entry. These new analogs provided insights into receptor binding and signaling mechanisms and acted as potent HIV-1 inhibitors. These results support the concept of SMM-chemokines for studying and controlling the function of other chemokine receptors.     

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3⁺CCR4^-CD4⁺ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-γ, TNF-α, and IL-2 in CXCR3⁺CCR4^-CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4⁺ or CXCR3⁺CCR4^-CD4⁺ T cells with DC (CD4⁺/DC or CXCR3⁺CD4⁺/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-α and LPS (mDC). Although there was no significant difference between the effects of the CXCR3⁺CCR4^-CD4⁺ and CD4⁺ T cells on DC phenotype expression, CXCR3⁺CD4⁺/DC in CTL culture were able to expand number of CD8⁺ T cells and increased frequencies of IFN-γ secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3⁺CCR4^-CD4⁺ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.     

CXCR4 inhibition suppresses Cd-induced renal oxidative stress,apoptosis, and fibrosis by inhibiting the TGF-β1/Smad pathway

This study aimed to investigate the role and mechanism of CXC chemokine receptor 4 (CXCR4) in cadmium (Cd)-induced renal injury. CXCR4 and TGF-β1/Smad pathway protein levels were detected by western blotting. Indicators related to renal function and oxidative stress factors were assessed and reactive oxygen species (ROS) level was evaluated by staining. TUNEL was used to measure apoptosis rate. PAS and Masson's trichrome staining were used to detect the level of renal fibrosis. The expression of Bcl-2, Bax, Cleaved-caspase 3, fibronectin, and collagen I proteins were detected by western blotting, immunohistochemistry, or immunofluorescence. The expression of CXCR4 was increased in a Cd-induced chronic renal injury model in rats. Si-CXCR4 decreased levels of TGF-β1, TGF-βR1, p-Smad2/Smad2, p-Smad3/Smad3, the renal weight index, urine protein, blood urea nitrogen, blood creatinine, and levels of MDA but raised the levels of SOD and GSH-Px. In addition, si-CXCR4 inhibited apoptosis in Cd-treated rats. CXCR4 inhibition alleviated fibrosis levels in Cd-treated rats. In Cd-treated cells, TGF-β attenuated the suppressive effect of CXCR4 inhibition on the TGF-β1/Smad pathway. TGF-β intervention increased MDA and ROS, and downregulated SOD and GSH-Px. TGF-β attenuated the inhibitory effect of CXCR4 on apoptosis and fibrosis. CXCR4 inhibition decreased levels of Cd-induced renal oxidative stress, apoptosis, and fibrosis by inhibiting the TGF-β1/Smad pathway.     

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.     

An integrated stochastic model of "inside-out" integrin activation and selective T-lymphocyte recruitment

The pattern of T-lymphocyte homing is hypothesized to be controlled by combinations of chemokine receptors and complementary chemokines. Here, we use numerical simulation to explore the relationship among chemokine potency and concentration, signal transduction, and adhesion. We have developed a form of adhesive dynamics-a mechanically accurate stochastic simulation of adhesion-that incorporates stochastic signal transduction using the next subvolume method. We show that using measurable parameter estimates derived from a variety of sources, including signaling measurements that allow us to test parameter values, we can readily simulate approximate time scales for T-lymphocyte arrest. We find that adhesion correlates with total chemokine receptor occupancy, not the frequency of occupation, when multiple chemokine receptors feed through a single G-protein. A general strategy for selective T-lymphocyte recruitment appears to require low affinity chemokine receptors. For a single chemokine receptor, increases in multiple cross-reactive chemokines can lead to an overwhelming increase in adhesion. Overall, the methods presented here provide a predictive framework for understanding chemokine control of T-lymphocyte recruitment.     

An Ultra‐High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4

Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high‐throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip‐coating poly(styrene‐co‐N‐isopropylacrylamide) spheres on the glass substrate. The self‐ assembled spheres form three‐dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF‐1α activation in various cancer cells, even for those with extremely low expression level.     

Discovery of rare sulfated N-unsubstituted glucosamine based heparan sulfate analogs selectively activating chemokines

Achieving selective inhibition of chemokines with structurally well-defined heparan sulfate (HS) oligosaccharides can provide important insights into cancer cell migration and metastasis. However, HS is highly heterogeneous in chemical composition, which limits its therapeutic use. Here, we report the rational design and synthesis of N-unsubstituted (NU) and N-acetylated (NA) heparan sulfate tetrasaccharides that selectively inhibit structurally homologous chemokines. HS analogs were produced by divergent synthesis, where fully protected HS tetrasaccharide precursor was subjected to selective deprotection and regioselectively O-sulfated, and O-phosphorylated to obtain 13 novel HS tetrasaccharides. HS microarray and SPR analysis with a wide range of chemokines revealed the structural significance of sulfation patterns and NU domain in chemokine activities for the first time. Particularly, HT-3,6S-NH revealed selective recognition by CCL2 chemokine. Further systematic interrogation of the role of HT-3,6S-NH in cancer demonstrated an effective blockade of CCL2 and its receptor CCR2 interactions, thereby impairing cancer cell proliferation, migration and invasion, a step towards designing novel drug molecules.     

Disulfide Bond Replacement with 1,4- and 1,5-Disubstituted [1,2,3]-Triazole on C-X-C Chemokine Receptor Type 4 (CXCR4) Peptide Ligands: Small Changes that Make Big Differences

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.     

The synergistic effects of CXCR4 and EGFR on promoting EGF-mediated metastasis in ovarian cancer cells

CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been reported to mediate the metastasis of many solid tumors including ovarian, breast, lung and prostate. The over-expression of the epidermal growth factor receptor (EGFR) is associated with the majority of ovarian cancer and has been implicated in the process of malignant transformation by promoting cell proliferation, survival, and motility. In this research, the result first showed that epidermis growth factor (EGF) enhanced the expression of CXCR4 and the migration of ovarian cancer cells, moreover, both stromal cell derived factor-1alpha (SDF-1alpha) and EGF-induced high matrix metallopeptidase 9 (MMP9) expressions. Molecular analysis indicated that augmented CXCR4 and MMP9 expression was regulated by phosphatidylinositol-3-kinase(PI3K)/Akt signal transduction pathway. These results suggested a possible important "cross-talk" between CXCR4 and EGFR intracellular pathways that might link signals of tumor deteriorated and provided a plausible explanation for the poor overall survival rate of patients whose co-expression of CXCR4 and EGFR was detected in their tissue sections. It enlightened that, compared to the respective inhibition of the EGFR or CXCR4 signaling, the simultaneous inhibition of them might be a more useful therapeutic strategy of cancer.     

T lymphocyte migration to lymph nodes is maintained during homeostatic proliferation.

The immune system maintains appropriate cell numbers through regulation of cell proliferation and death. Normal tissue distribution of lymphocytes is maintained through expression of specific adhesion molecules and chemokine receptors such as L-selectin and CCR7, respectively. Lymphocyte insufficiency or lymphopenia induces homeostatic proliferation of existing lymphocytes to increase cell numbers. Interestingly, homeostatic proliferation of T lymphocytes induces a phenotypic change from na?ve- to memory-type cell. Na?ve T cells recirculate between blood and lymphoid tissues whereas memory T cells migrate to nonlymphoid sites such as skin and gut. To assess effects of homeostatic proliferation on migratory ability of T cells, a murine model of lymphopenia-induced homeostatic proliferation was used. Carboxyfluorescein diacetate, succinimidyl ester-labeled wild-type splenocytes were adoptively transferred into recombination activation gene-1-deficient mice and analyzed by flow cytometry, in vitro chemotactic and in vivo migration assays, and immunofluorescence microscopy. Homeostatically proliferated T cells acquired a mixed memory-type CD44high L-selectinhigh CCR7low phenotype. Consistent with this, chemotaxis to secondary lymphoid tissue chemokine in vitro was reduced by 22%-34%. By contrast, no differences were found for migration or entry into lymph nodes during in vivo migration assays. Therefore, T lymphocytes that have undergone homeostatic proliferation recirculate using mechanisms similar to na?ve T cells.     

Assessment of Growth Factors,Cytokines, and Cellular Markers in Saliva of Patients with Trigeminal Neuralgia

We proposed to perform a comparative analysis of growth factors, cytokines, and chemokine receptors on the salivary cells in the saliva obtained from trigeminal neuralgia (TN) and normal subjects. Saliva was collected from TN and healthy subjects. Salivary cells were isolated by centrifugation. The expression of the cell surface marker was analyzed by flow cytometry. A cytometric bead array was done to measure the levels of cytokines and growth factors on the flow cytometer. Saliva from TN subjects showed lower growth factor levels of Angiopoietin-2, bFGF, HGF, SCF, TGF-α, and VEGF and higher cytokine levels of IL-1β, TNF-α, CCL2, IL-17A, IL-6, and CXCL8, as well as higher expression levels of chemokine receptors CCR1 (CD191), CR3 (CD11b), CCR2 (CD192), CXCR5 (CD185), and CCR5 (CD196) in the cells from TN saliva. A certain set of cytokines and growth factors in the saliva, as well as chemokine receptors on salivary cells, could be a useful tool in the diagnostics and prognostics of trigeminal neuralgia. Trigeminal neuralgia is one of the significant pathological conditions in the class of chronic diseases around the world. Many targeted approaches are being tried by various research groups to utilize the information of the inflammatory microenvironment to resolve the pathology of chronic TN.     

Inhibition of Human Immunodeficiency Type 1 Virus (HIV-1) Life Cycle by Different Egg White Lysozymes

Lysozyme is a relatively small enzyme with different biological activities, which is found in tears, saliva, egg white, and human milk. In the study, the anti-HIV-1 activity of lysozymes purified from quail, Meleagris, and hen egg white has been determined. For this end, a time-of-drug-addition assay was performed to identify the target of anti-HIV-1 agents and for determination of probable anti HIV-1 mechanism of the studied lysozyme, the binding affinity of the lysozymes to the human CD4 receptor was studied by molecular docking method. To define structural differences between studied lysozymes, structural motifs of them were predicted by MEME tool. Quail, hen, and Meleagris lysozymes showed potent anti-HIV-1 activity with EC₅₀ of 7.5, 10, and 55 nM, respectively. The time-of-drug-addition study demonstrated that the inhibitory effect of all purified lysozymes is before HIV-1 infection. The frequency and intensity of CD4 expression in PBMCs decreased in the presence of all mentioned lysozymes. Also, the expression level of C-C chemokine receptor type 5 (CCR5) and chemokine receptor type 4 (CXCR4) on CD4+ T cells was not changed in cells treated with these lysozymes. The results of in silico study confirmed that the binding energy of quail lysozyme with CD4 was more than that of other studied lysozymes. The results revealed that these lysozymes restrict HIV-1 attachment to host cell CD4.     

Peptide and peptidomimetic ligands for CXC chemokine receptor 4 (CXCR4)

The development of novel peptide and peptidomimetic ligands for the CXC chemokine receptor 4 (CXCR4) as therapeutic agents for HIV-1 infection, cancer, and immune system diseases has grown over the last decade. In this perspective article, the design of CXCR4 agonists and antagonists from endogenous stromal cell-derived factor-1 (SDF-1)/CXCL12 and horseshoe crab-derived antimicrobial peptides and their therapeutic and diagnostic applications are described.     

Sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.     

Synthesis of peptides mimicking chemokine receptor CCR5 and their inhibitory effects against HIV-1 infection

Peptides mimicking chemokine receptor CCR5 were synthesized and their anti-HIV-1 activities evaluated. Prepared compounds, especially a sulfated derivatives, showed significant anti-HIV-1 activities. Furthermore, a hybrid molecule linked to an N-carbomethoxycarbonyl-prolyl-phenylalanine (CPF) moiety had a greater effect.     

Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.     

Alcoholic monoterpenes found in essential oil of aromatic spices reduce allergic inflammation by the modulation of inflammatory cytokines

Allergic inflammation is a response of the body against pathogens by cytokine release and leucocyte recruitment. Recently, there was an increase in morbimortality associated with allergic inflammation, especially asthma. The treatment has many adverse effects, requiring the search for new therapies. Monoterpenes are natural products with anti-inflammatory activity demonstrated in several studies and can be an option to inflammation management. Thus, we investigated the effects of citronellol, α-terpineol and carvacrol on allergic inflammation. The model of asthma was established by OVA induction in male Swiss mice. The monoterpenes were administered (25, 50 or 100 mg/kg, i.p.) 1 h before induction. After 24hs, the animals were sacrificed to leucocytes and TNF-α quantification. Monoterpenes significantly decrease leucocyte migration and TNF-α levels, possibly by modulation of COX, PGE₂ and H1 receptor, as demonstrated by molecular docking. These findings indicate that alcoholic monoterpenes can be an alternative for treatment of allergic inflammation and asthma.