Chemokine receptor 4 gene silencing blocks neuroblastoma metastasis in vitro

This study investigated the effects of small interfering RNA （siRNA）-mediated silencing of chemokine receptor 4 （CXCR4） on the invasion capacity of human neuroblastoma cell line SH-SY5Y in vitro. Three siRNAs targeting CXCR4 were chemically synthesized and individually transfected into SH-SY5Y cells. Expression of CXCR4 mRNA and protein was signiifcantly sup-pressed in transfected cells by all three sequence-speciifc siRNAs compared with control groups. Furthermore, the invasion capacity of SH-SY5Y cells was signiifcantly decreased following trans-fection with CXCR4-speciifc siRNA compared with the control groups. These data demonstrate that down-regulation of CXCR4 can inhibit in vitro invasion of neuroblastoma.

C-X-C chemokine receptor type 7 antibody enhances neural plasticity after ischemic stroke

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4(CXCR4) have been shown to regulate neural regeneration after stroke.Howeve r,whether stromal cell-derived factor-1 receptor CXCR7,which is widely distributed in the develo ping and adult central nervous system,participates in neural regeneration remains poorly unde rstood.In this study,we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral co rtex and striatum.Starting on day 7 after injury,CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days.Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting co rticospinal tra ct fibers in rats with cerebral ischemia,increased the expression of vesicular glutamate transporter 1 and growth-related protein 43,marke rs of the denervated spinal cord synapses,and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum.In addition,CXCR7 antibody increased the expression of CXCR4 in the striatum,increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway,and im proved rat motor function.These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration,synaptogenesis,and myelin regeneration,which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

Overexpression of amplified in breast cancer 1 (AIB1) gene promotes lung adenocarcinoma aggressiveness in vitro and in vivo by upregulating C-X-C motif chemokine receptor 4

Background:We previously found that overexpression of the gene known as amplified in breast cancer 1(AIB1)was associated with lymph node metastasis and poor prognosis in patients with lung adenocarcinoma.However,the role of AIB1 in that malignancy remains unknown.The present study aimed to investigate the function of AIB1 in the process of lung adenocarcinoma cell metastasis.Methods:A series of in vivo and in vitro assays were performed to elucidate the function of AIB1,while real-time PCR and Western blotting were utilized to identify the potential downstream targets of AIB1 in the process of lung adenocarcinoma metastasis.Rescue experiments and in vitro assays were performed to investigate whether the invasive-ness of AIB1-induced lung adenocarcinoma was mediated by C-X-C motif chemokine receptor 4(CXCR4).Results:The ectopic overexpression of AIB1 in lung adenocarcinoma cells substantially enhanced cell migration and invasive abilities in vitro and tumor metastasis in vivo,whereas the depletion of AIB1 expression substantially inhibited lung adenocarcinoma cell migration and invasion.CXCR4 was identified as a potential downstream target of AIB1 in lung adenocarcinoma.The knockdown of AIB1 greatly reduced CXCR4 gene expression at both the transcription and protein levels,whereas the knockdown of CXCR4 in cells with AIB1 ectopic overexpression diminished AIB1-induced migration and invasion in vitro and tumor metastasis in vivo.Furthermore,we found a significant positive association between the expression of AIB1 and CXCR4 in lung adenocarcinoma patients(183 cases),and the co-overexpression of AIB1 and CXCR4 predicted the poorest prognosis.Conclusions:These findings suggest that AIB1 promotes the aggressiveness of lung adenocarcinoma in vitro and in vivo by upregulating CXCR4 and that it might be usable as a novel prognostic marker and/or therapeutic target for this disease.

Foodborne toxin Aflatoxin B_(1)induced glomerular podocyte inflammation through proteolysis of RelA,downregulation of miR-9 and CXCR4/TXNIP/NLRP3 pathway

Aflatoxin B_(1)(AFB_(1))is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin,particularly causing damages to kidney.Glomerular podocytes are terminally differentiated epithelial cells.AFB_(1)induces podocyte inflammation,proteinuria and renal dysfunction.Studying the mechanism of AFB_(1)-induced podocyte inflammation and murine kidney dysfunction,we detected that AFB_(1)increased ubiquitindependent degradation of the transcription factor RelA through enhanced interaction of RelA with E3 ubiquitin ligase tripartite motif containing 7(TRIM7)in mouse podocyte clone-5(MPC-5)and mouse glomeruli.Reduction of RelA resulted in decreasing microRNA-9(miR-9)and activating the chemokine receptor 4(CXCR4),thioredoxin interacting protein(TXNIP),and NOD-like receptor pyrin domain-containing 3(NLRP3)signaling axis(CXCR4/TXNIP/NLRP3 pathway),leading to podocyte inflammation.We also determined that downregulation of miR-9 led to CXCR4 expression and the downstream TXNIP/NLRP3 pathway activation.Overexpression of miR-9 or deletion of CXCR4 suppressed AFB_(1)-induced CXCR4/TXNIP/NLRP3 pathway,resulting in alleviating podocyte inflammation and kidney dysfunction.Our findings indicated that ubiquitin-dependent proteolysis of RelA,downregulation of miR-9,and activation of CXCR4/TXNIP/NLRP3 pathway played an essential role in AFB_(1)-induced glomerular podocyte inflammation.Our study revealed a novel mechanism,via RelA,for the control of AFB_(1)’s nephrotoxicity,leading to an effective protection of food safety and public health.

Functions and mechanisms of chemokine receptor 7 in tumors of the digestive system

Chemokine(C-X-C motif)receptor 7(CXCR7),recently termed ACKR3,belongs to the G protein-coupled cell surface receptor family,binds to stromal cellderived factor-1[SDF-1,or chemokine(C-X-C motif)ligand 12]or chemokine(CX-C motif)ligand 11,and is the most common chemokine receptor expressed in a variety of cancer cells.SDF-1 binds to its receptor chemokine(C-X-C motif)receptor 4(CXCR4)and regulates cell proliferation,survival,angiogenesis and migration.In recent years,another new receptor for SDF-1,CXCR7,has been discovered,and CXCR7 has also been found to be expressed in a variety of tumor cells and tumor-related vascular endothelial cells.Many studies have shown that CXCR7 can promote the growth and metastasis of a variety of malignant tumor cells.Unlike CXCR4,CXCR7 exhibits a slight modification in the DRYLAIV motif and does not induce intracellular Ca^2+release following ligand binding,which is essential for recruiting and activating G proteins.CXCR7 is generally thought to work in three ways:(1)Recruitingβ-arrestin 2;(2)Heterodimerizing with CXCR4;and(3)Acting as a“scavenger”of SDF-1,thus lowering the level of SDF-1 to weaken the activity of CXCR4.In the present review,the expression and role of CXCR7,as well as its prognosis in cancers of the digestive system,were investigated.

The biological role of the CXCL12/CXCR4 axis in esophageal squamous cell carcinoma

Esophageal cancer is the eighth most common malignant tumor and the sixth leading cause of cancer-related death worldwide.Esophageal squamous cell carcinoma(ESCC)is the main histological type of esophageal cancer,and accounts for 90%of all cancer cases.Despite the progress made in surgery,chemotherapy,and radiotherapy,the mortality rate from esophageal cancer remains high,and the overall 5-year survival rate is less than 20%,even in developed countries.The C-X-C motif chemokine ligand 12(CXCL12)is a member of the CXC chemokine subgroup,which is widely expressed in a variety of tissues and cells.CXCL12 participates in the regulation of many physiological and pathological processes by binding to its specific receptor,C-X-C motif chemokine receptor type 4(CXCR4),where it causes embryonic development,immune response,and angiogenesis.In addition,increasing evidence indicates that the CXCL12/CXCR4 axis plays an important role in the biological processes of tumor cells.Studies have shown that CXCL12 and its receptor,CXCR4,are highly expressed in ESCC.This abnormal expression contributes to tumor proliferation,lymph node and distant metastases,and worsening prognosis.At present,antagonists and imaging agents against CXCL12 or CXCR4 have been developed to interfere with the malignant process and monitor metastasis of tumors.This article summarizes the structure,function,and regulatory mechanism of CXCL12/CXCR4 and its role in the malignancy of ESCC.Current results from preclinical research targeting CXCL12/CXCR4 are also summarized to provide a reference for the clinical diagnosis and treatment of ESCC.

The Role of SDF-1/CXCR4 Axis in Ovarian Cancer Metastasis

This study was aimed to explore the role of stromal-derived factor 1 （SDF-1）/CXC chemokine receptor 4 （CXCR4） axis in mediating the metastasis of ovarian cancer cells through activation of extracellular signal-regulated kinase-1/2 （ERK-1/2） signaling pathway. A highly metastatic ovarian cancer cell line, SKOV3, was used in the study. Intracellular calcium mobilization was detected by using laser scanning confocal fluorescence microscopy. Western blotting was used to detect the phosphorylation of ERK1/2 in SDF-1α-treated SKOV3 cells. Adhesion capability and matrix metalloproteinase （MMP） activity of ovarian cancer cells after exposure to SDF-1 a were measured by adhesion assay and gelatin zymography. The results showed that SDF-1α induced rapid intracellular calcium mobilization in SKOV3 cells, as well as the phosphorylation of ERK-1/2. The adhesion of ovarian cancer cells to fibronectin and collagen Ⅳ was increased after SDF-1α treatment. An inhibitor of ERK-1/2 signaling, PD98059, could antagonize such effects of SDF-1α. SDF-1α could also increase the secretion of active MMP-2 and MMP-9. It was concluded that the SDF-1/CXCR4 axis played a critical role in the metastasis of human ovarian cancer by increasing the adhesion capability of cancer cells and the activity of MMP-2 and MMP-9 via ERK1/2 signaling pathway.

Transfection with CXCR4 potentiates homing of mesenchymal stem cells in vitro and therapy of diabetic retinopathy in vivo

AIM：To investigate the effect of the overexpression of C-X-C chemokine receptor type 4（CXCR4） on homing of mesenchymal stem cells（MSCs） in vitro and therapeutic effects of diabetic retinopathy（DR） in vivo.METHODS：MSCs were infected by lentivirus constructed with CXCR4.The expression of CXCR4 was examined by immunofluorescence,Western blot,and quantitative polymerase chain reaction.CXCR4-overexpressing MSCs were cultured in vitro to evaluate their chemotaxis,migration,and apoptotic activities.CXCR4-overexpressing MSCs were intravitreally injected to observe and compare their effects in a mouse model of DR.The histological structure of DR in rats was inspected by hematoxylin and eosin staining.The expression of rhodopsin,neuron-specific enolase（NSE）,and inflammatory cytokines interleukin（IL）-6 and tumor necrosis factor（TNF）-α was examined by Western blot and immunohistochemical analyses.RESULTS：The transduction of MSCs by lentivirus was effective,and the transduced MSCs had high expression levels of CXCR4 gene and protein.Improved migration activities were observed in CXCR4-overexpressing MSCs.Further,reduced retinal damage,upregulation of rhodopsin and NSE protein,and downregulation of inflammatory cytokines IL-6 and TNF-α were observed in CXCR4-overexpressing MSCs in vivo.CONCLUSION：The homing of MSCs can be enhanced by upregulating CXCR4 levels,possibly improving histological structures of DR.CXCR4-overexpressing MSCs can be a novel strategy for treating DR.

Stromal cell-derived factor-1α regulates chondrogenic differentiation via activation of the Wnt/β-catenin pathway in mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have been applied to treat degenerative articular diseases,and stromal cell-derived factor-1α(SDF-1α)may enhance their therapeutic efficacy.However,the regulatory effects of SDF-1αon cartilage differentiation remain largely unknown.Identifying the specific regulatory effects of SDF-1αon MSCs will provide a useful target for the treatment of degenerative articular diseases.AIM To explore the role and mechanism of SDF-1αin cartilage differentiation of MSCs and primary chondrocytes.METHODS The expression level of C-X-C chemokine receptor 4(CXCR4)in MSCs was assessed by immunofluorescence.MSCs treated with SDF-1αwere stained for alkaline phosphatase(ALP)and with Alcian blue to observe differentiation.Western blot analysis was used to examine the expression of SRY-box transcription factor 9,aggrecan,collagen II,runt-related transcription factor 2,collagen X,and matrix metalloproteinase(MMP)13 in untreated MSCs,of aggrecan,collagen II,collagen X,and MMP13 in SDF-1α-treated primary chondrocytes,of glycogen synthase kinase 3β(GSK3β)p-GSK3βandβ-catenin expression in SDF-1α-treated MSCs,and of aggrecan,collagen X,and MMP13 in SDF-1α-treated MSCs in the presence or absence of ICG-001(SDF-1αinhibitor).RESULTS Immunofluorescence showed CXCR4 expression in the membranes of MSCs.ALP stain was intensified in MSCs treated with SDF-1αfor 14 d.The SDF-1αtreatment promoted expression of collagen X and MMP13 during cartilage differentiation,whereas it had no effect on the expression of collagen II or aggrecan nor on the formation of cartilage matrix in MSCs.Further,those SDF-1α-mediated effects on MSCs were validated in primary chondrocytes.SDF-1αpromoted the expression of p-GSK3βandβ-catenin in MSCs.And,finally,inhibition of this pathway by ICG-001(5μmol/L)neutralized the SDF-1α-mediated up-regulation of collagen X and MMP13 expression in MSCs.CONCLUSION SDF-1αmay promote hypertrophic cartilage differentiation in MSCs by activating the Wnt/β-catenin pathway.These findings provide further evidence for the use of MSCs and SDF-1αin the treatment of cartilage degeneration and osteoarthritis.

Effect of CO2 pneumoperitoneum on the expression of the chemokine receptors CXCR4 and CCR7 in colorectal carcinoma cells in vitro

Background The ability of pneumoperitoneum in laparoscopic surgery to promote proliferation and metastasis of colorectal cancer has become a focus of research in the field of minimally invasive surgery. The aim of this research was to investigate the effect of CO~ pneumoperitoneum under different pressures and exposed times on the expression of chemokine receptors in colorectal carcinoma cells. Methods We constructed an in vitro pneumoperitoneum model. SW480 colon carcinoma cells were exposed to CO2 pneumoperitoneum under different pressures （6, 9, 12, and 15 mmHg） for 1, 2, and 4 hours. These cells were then cultivated under the same conditions as normal SW480 colon carcinoma cells without CO= pneumoperitoneum （control group）, treated at 37℃, and 5% CO2. The expression of the chemokine receptors CXC receptor 4 （CXCR4） and chemokine C receptor 7 （CCR7） was detected by immunocytochemistry and reverse transcriptase polymerase chain reaction after being cultivated for 0, 24, 48, and 72 hours. Results Immunocytochemistry showed that CXCR4 expression in SW480 cells was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups for the same exposure times compared with controls （P 〈0.05）. CCR7 expression in SW480 cells was significantly decreased in the 12 and 15 mmHg CO2 pneumoperitoneum- treated groups compared with controls （P 〈0.05）. CXCR4 and CCR7 expression increased up to the level of the control group after 24 and 48 hours （P 〉0.05）. If the CO2 pneumoperitoneum pressure increased, CXCR4 and CCR7 expression decreased at all exposure times. If the CO2 pneumoperitoneum exposure time prolonged, there were no significant differences in CXCR4 and CCR7 expression under the same pressure. Under all exposure times, CXCR4 and CCR7 mRNA expression was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups （P 〈0.05） compared with controls, and it increased up to the level of controls after being cultivated for 48 hours （P 〉0.05）. If the CO2 pneumoperitoneum pressure increased （with all exposure times） and exposure time prolonged （under the same pressure）, there were no significant differences in CXCR4 and CCR7 expression. Conclusions CXCR4 and CCR7 expression is temporarily affected after continuous CO2 pneumoperitoneum treatment. The high pressure of CO2 pneumoperitoneum plays an important role in suppressing the expression of these chemokine receptors. Different lengths of time of exposure to a CO2 pneumoperitoneum-like environment do not change CXCR4 and CCR7 expression.

Apoptosis in human germinal centre B cells by means of CC chemokine receptor 3 expression induced by interleukin-2 and interleukin-4

Background CC chemokine receptor 3 (CCR3), expressed on some inflammatory cells, is a member of the chemokine receptor family. Its ligand is eotaxin/CCL11. In this research, we studied the expression and function of CCR3 induced by interleukin-2 (IL-2) and interleukin-4 (IL-4) on human germinal centre (GC) B cells.Methods Cells isolated from human tonsils were stimulated with IL-2 or/and IL-4 followed by bonding with eotaxin/CCL11. Flow cytometry was used to detect expression of CCR3 on GC B cells and apoptosis of GC B cells. Real time quantitative reverse transcription polymerase chain reaction and Northern blot assays were used to analyse the CCR3 mRNA expressed in the GC B cells. Chemotaxis and adhesion assays were used to determine the effect of eotaxin/CCL11 ligand bonded to CCR3 on GC B cells.Results There was no CCR3 expression on human freshly isolated GC B cells. The combination IL-2 and IL-4 could upregulate CCR3 mRNA and protein expression on GC B cells. Eotaxin could not induce GC B cell chemotaxis and adhesion but triggered apoptosis of GC B cells.Conclusion IL-2 and IL-4 together induced expression of CCR3 on GC B cells, and the receptor acted as a death receptor.

Mobilization of hematopoietic progenitor cells in patients with liver cirrhosis

AIM:To test the hypothesis that liver cirrhosis is associated with mobilization of hematopoietic progenitor cells. METHODS:Peripheral blood samples from 72 patients with liver cirrhosis of varying etiology were analyzed by flow cytometry.Identified progenitor cell subsets were immunoselected and used for functional assays in vitro. Plasma levels of stromal cell-derived factor-1(SDF-1) were measured using an enzyme linked immunosorbent assay.RESULTS:Progenitor cells with a CD133 + /CD45 + CD14 + phenotype were observed in 61%of th patients.Between 1%and 26%of the peripheral bloo mononuclear cells(MNCs)displayed this phenotype Furthermore,a distinct population of c-kit + progenito cells(between 1%and 38%of the MNCs)could b detected in 91%of the patients.Additionally,18% of the patients showed a population of progenito cells(between 1%and 68%of the MNCs)that wa characterized by expression of breast cancer resistanc protein-1.Further phenotypic analysis disclosed tha the circulating precursors expressed CXC chemokin receptor 4,the receptor for SDF-1.In line with thi finding,elevated plasma levels of SDF-1 were presen in all patients and were found to correlate with th number of mobilized CD133 + progenitor cells.

Study on the Effect of Ligustrazine on Hematopoietic Reconstitution in Bone Marrow Transplantation Mice

To explore tile effects of ligustrazine on hematopoietic reconstitution and its mechanism after bone marrow transplantation (BMT), the allogenic BMT mice were given intra-abdominal injection of 2,mg ligustrazine twice a day. On the 1st, 7th, 14th, and 28th day after BMT, peripheral blood cells and bone marrow nuclear cells (BMNC) were counted, and the histological features were evaluated. On the 7th, 14th, 21st day after BMT, CXCR4 expression on the BMNC was assayed. The results showed that peripheral blood cell counts and BMNC counts in ligustrazine-treated group on the 7th, 14th, 28th day were higher than those in BMT group (P<0. 01 or P<O. 05). The percentage of hematopoietic tissue volume, fat tissue hyperplasia and congestion and dilation degree of microvessel in ligustrazine-treated group on the 7th, 14th, 21st, 28th day was higher than those in BMT group. The CXCR4 expression levels in ligustrazine-treated group were higher than in BMT group (P<0.01 or P<0. 05) on the 7th and 14th day, and were lower than in BMT group on the 21st day (P<0. 01 ). It is concluded that the ligustrazine can accelerate hematopoietic reconstruction, enhance growth of hematopoietic tissues and promote the repair of microvessels. The CXCR4 expres- sion levels on BMNC may be responsible for the effect of ligustrazine.

Berberine-Promoted CXCR4 Expression Accelerates Endothelial Repair Capacity of Early Endothelial Progenitor Cells in Persons with Prehypertension

Objective： To evaluate whether the berberine treatment can improve endothelial repair capacity of early endothelial progenitor cells （EPCs） from prehypertensive subjects through increasing CXC chemokine receptor 4 （CXCR4） signaling. Methods： EPCs were isolated from prehypertensive and healthy subjects and cultured. In vivo reendothelialization capacity of EPCs from prehypertensive patients with or without in vitro berberine treatment was examined in a nude mouse model of carotid artery injury. The protein expressions of CXCR4/Janus kinase-2 （JAK-2） signaling of in vitro EPCs were detected by Western blot analysis. Results： CXCR4 signaling and alteration in migration and adhesion functions of EPCs were evaluated. Basal CXCR4 expression was significantly reduced in EPCs from prehypertensive patients compared with normal subjects （P〈0.01）. Also, the phosphorylation of JAK-2 of EPCs, a CXCR4 downstream signaling, was significantly decreased （P〈0.01）. Berberine promoted CXCR4/JAK-2 signaling expression of in vitro EPCs （P〈0.01）. Transplantation of EPCs pretreated with berberine markedly accelerated in vivo reendothelialization （P〈0.01）. The increased in vitro function and in vivo reendothelialization capacity of EPCs were inhibited by CXCR4 neutralizing antibody or pretreatment with JAK-2 inhibitor AG490, respectively （P〈0.01）. Conclusion： Berberine- modified EPCs via up-regulation of CXCR4 signaling contributes to enhanced endothelial repair capacity in prehypertension, indicating that berberine may be used as a novel potential primary prevention means against prehypertension-related atherosclerotic cardiovascular disease.

CXCR4 Peptide Conjugated Au-Fe2O3 Nanoparticles for Tumor-targeting Magnetic Resonance Imaging

Peptide-functionalized Au-Fe2O3 nanoparticles（termed as anti-CXCR4-Au-Fe2O3 NPs） have been constructed through conjugation of dumbbell-like Au-Fe203 NPs with C-X-C motif chemokine receptor 4（CXCR4） binding cyclic peptide. One dumbbelMike Au-Fe2O3NP composes an Au NP[（3.3±0.3） nm in diameter] for conjugating CXCR4 binding cyclic peptide through Au-S covalent bond and a Fe2O3 NP[（8.7±0.8） nm in diameter] for using as T2-weighted magnetic resonance imaging（MRI） contrast agent. The anti-CXCR4-Au-Fe2O3 NPs have reasonable biocompatibility and integration of T2-weighted MRI contrast and tumor-targeting functionalities. The anti- CXCR4-Au-Fe2O3 NPs exhibit strong interactions with two kinds of breast tumor cells, MCF-7 cells and MDA-MB-231 cells, and high negative contrast in MRI of MDA-MB-231 tumor bearing mouse with 62% decreasing of MRI signal, indicating that the anti-CXCR4-Au-Fe2O3 NPs can recognize tumor with high efficacy and specificity.