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1.
Int J Mol Sci ; 23(17)2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36077241

RESUMO

CXCR7 and CXCR4 are G protein-coupled receptors (GPCRs) that can be stimulated by CXCL12 in various human cancers. CXCR7/4-CXCL12 binding can initiate activation of multiple pathways including JAK/STAT and manganese superoxide dismutase (MnSOD) signaling, and initiate epithelial-mesenchymal transition (EMT) process. It is established that cancer cell invasion and migration are caused because of these events. In particular, the EMT process is an important process that can determine the prognosis for cancer. Since the antitumor effect of leelamine (LEE) has been reported in various previous studies, here, we have evaluated the influence of LEE on the CXCR7/4 signaling axis and EMT processes. We first found that LEE suppressed expression of CXCR7 and CXCR4 both at the protein and mRNA levels, and showed inhibitory effects on these chemokines even after stimulation by CXCL12 ligand. In addition, LEE also reduced the level of MnSOD and inhibited the EMT process to attenuate the invasion and migration of breast cancer cells. In addition, phosphorylation of the JAK/STAT pathway, which acts down-stream of these chemokines, was also abrogated by LEE. It was also confirmed that LEE can induce an imbalance of GSH/GSSG and increases ROS, thereby resulting in antitumor activity. Thus, we establish that targeting CXCR7/4 in breast cancer cells can not only inhibit the invasion and migration of cancer cells but also can affect JAK/STAT, EMT process, and production of ROS. Overall, the findings suggest that LEE can function as a novel agent affecting the breast cancer.


Assuntos
Neoplasias da Mama , Receptores CXCR , Abietanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacologia , Transição Epitelial-Mesenquimal , Feminino , Humanos , Janus Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais
2.
Oxid Med Cell Longev ; 2022: 4455183, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35982734

RESUMO

Stem cell-based therapeutic strategies have obtained a significant breakthrough in the treatment of cardiovascular diseases, particularly in myocardial infarction (MI). Nevertheless, limited retention and poor migration of stem cells are still problems for stem cell therapeutic development. Hence, there is an urgent need to develop new strategies that can mobilize stem cells to infarcted myocardial tissues effectively. Electroacupuncture (EA) intervention can improve cardiac function and alleviate myocardial injury after MI, but its molecular mechanism is still unclear. This study is aimed at observing the effects of EA treatment on the stem cell mobilization and revealing possible mechanisms in the MI model of mice. EA treatment at Neiguan (PC6) and Xinshu (BL15) acupoints was conducted on the second day after the ligation surgery. Then, the number of stem cells in peripheral blood after EA in MI mice and their cardiac function, infarct size, and collagen deposition was observed. We found that the number of CD34-, CD117-, Sca-1-, and CD90-positive cells increased at 6 h and declined at 24 h after EA intervention in the blood of MI mice. The expression of CXC chemokine receptor-4 (CXCR4) protein was upregulated at 6 h after EA treatment, while the ratio of LC3B II/I or p-ERK/ERK showed a reverse trend. In addition, there was obvious difference in EF and FS between wild-type mice and CXCR4+/- mice. The infarct size, collagen deposition, and apoptosis of the injured myocardium in CXCR4+/- mice increased but could be ameliorated by EA. In a word, our study demonstrates that EA alleviates myocardial injury via stem cell mobilization which may be regulated by the SDF-1/CXCR4 axis.


Assuntos
Quimiocina CXCL12 , Eletroacupuntura , Infarto do Miocárdio , Receptores CXCR4 , Animais , Quimiocina CXCL12/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Camundongos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Receptores CXCR4/metabolismo
3.
Oncol Rep ; 48(4)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36004481

RESUMO

Oxymatrine has been revealed to exert antitumor activity; however, its role in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, the effects and underlying molecular mechanisms of oxymatrine in OSCC were explored. The antineoplastic effects of oxymatrine were measured using Cell Counting Kit­8, apoptosis and Transwell assays. The inhibitory effect of oxymatrine on tumor growth was evaluated in vivo. The regulation of oxymatrine on the CXC chemokine receptor 4 (CXCR4) was analyzed using western blotting, reverse transcription­quantitative PCR, RNA stability and methylated RNA immunoprecipitation assays. The present results revealed that oxymatrine inhibited the proliferation and migration of OSCC cells and promoted cell apoptosis. Furthermore, oxymatrine reduced CXCR4 mRNA and protein expression levels by promoting CXCR4 mRNA degradation. Mechanistically, oxymatrine inhibited the methylation at the N6­position of adenosine (m6A modification) of CXCR4 mRNA by decreasing the expression of the methyltransferase­like 3 (METTL3) gene. In addition, oxymatrine inhibited tumor growth in vivo. Taken together, our findings demonstrated the antitumor effect of oxymatrine on OSCC. Mechanistically, oxymatrine inhibited the progression of OSCC by downregulating METTL3 and degrading CXCR4 mRNA by decreasing the level of m6A modification.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Alcaloides , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Metiltransferases/genética , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Quinolizinas , RNA Mensageiro/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética
4.
Biochem Biophys Res Commun ; 625: 16-22, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35944359

RESUMO

Esophageal cancer is one of the most common malignant tumors worldwide and half of the patients present tumor metastasis at initial diagnosis. CXCR4 has been reported to be upregulated in esophageal cancer tissues and associated with tumor metastasis. However, the upstream transcriptional regulator of CXCR4 in esophageal cancer is still unclear. In this study, we found that transcription factor FOXA2 directly bound on the CXCR4 promoter region and activated its expression in esophageal cancer cells. The expression of FOXA2 was upregulated in esophageal cancer tissues and positively correlated with CXCR4. Moreover, knockout of FOXA2 significantly inhibited esophageal cancer migration and metastasis, which can be rescued by ectopically expressed CXCR4. Taken together, we reveal a novel transcriptional activator of CXCR4 in esophageal cancer, which might provide new strategies for esophageal cancer treatment.


Assuntos
Neoplasias Esofágicas , Receptores CXCR4 , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Esofágicas/patologia , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
5.
Mol Med Rep ; 26(4)2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35946444

RESUMO

The purpose of this study is to address the effect and mechanism of stromal cell­derived factor­1 (SDF­1)α/chemokine (C­X­C motif) receptor 4 (CXCR4) signaling on capillary tube formation of human retinal vascular endothelial cells (HRECs). The expression of CXCR4 in HRECs was quantified by reverse transcription (RT­PCR) and western blotting. The effects of SDF­1α/CXCR4 signaling in capillary tube formation and migration of HRECs was examined using three­dimensional Matrigel assay and wound scratching assay respectively in vitro. Cell proliferation of HRECs was examined using cell counting kit (CCK)­8 assay in the presence of different concentrations of SDF­1α protein. The effect of SDF­1α/CXCR4 signaling in HREC expression of VEGF, basic fibroblast growth factor (bFGF), IL­8 and intercellular cell adhesion molecule (ICAM)­1 was examined using RT­PCR and western blotting. RT­PCR and western blot analysis revealed CXCR4 was expressed in HRECs. The number of intact capillary tubes formed by HRECs in the presence of SDF­1α was markedly more compared with a PBS treated control group. However, it was reduced with treatment with an CXCR4 antagonist. Wound scratching assay showed a significant increase in the number of migrated HRECs under SDF­1α stimulation and the number was reduced with treatment with an CXCR4 antagonist. RT­PCR and western blotting showed that SDF­1α significantly promoted VEGF, bFGF, IL­8 and ICAM­1 expression in HRECs. The proliferation of HRECs in the presence of SDF­1α was promoted in a dosage­dependent manner. SDF­1α/CXCR4 signaling can increase HREC capillary tube formation through promoting HREC migration, proliferation and expression of VEGF, bFGF, IL­8 and ICAM­1.


Assuntos
Quimiocina CXCL12 , Células Endoteliais , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12/metabolismo , Células Endoteliais/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CXCR4/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Sci Adv ; 8(28): eabn8063, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35857509

RESUMO

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein-coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward ß-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs.


Assuntos
Receptores CXCR4 , Transdução de Sinais , Arrestina , Ligação Proteica , Receptores CXCR4/metabolismo , beta-Arrestinas/metabolismo
7.
Mol Pharmacol ; 102(3): 128-138, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35809897

RESUMO

Chemokines such as stromal derived factor 1 and their G protein coupled receptors are well-known regulators of the development and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C chemokine motif receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3). ACKR3 has been described as an atypical "biased" receptor because it does not appear to signal through G proteins and, instead, signals solely through the ß-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mammalian G α isoforms and have generated a comprehensive map of the G α activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective agonists for ACKR3 as assessed by their ability to recruit ß-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the transmembrane domains of ACKR3. The development of more selective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signaling axis and better understand the structural determinants for ACKR3 activation. SIGNIFICANCE STATEMENT: We are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions between these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.


Assuntos
Quimiocina CXCL12 , Receptores CXCR4 , Animais , Quimiocina CXCL12/metabolismo , Ligantes , Mamíferos/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo
8.
Front Immunol ; 13: 925559, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35903108

RESUMO

Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-particle tracking analysis of diffusion in the plasma membrane to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with bacterial sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.


Assuntos
Receptores CXCR4 , Esfingomielinas , Movimento Celular , Ceramidas/metabolismo , Quimiocina CXCL12/antagonistas & inibidores , Quimiocina CXCL12/metabolismo , Humanos , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo
9.
Dis Markers ; 2022: 3276925, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35774848

RESUMO

Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous malignant lymphoma with distinct characteristics. Patients with treatment failure after the standard immunochemotherapy have worse prognosis, which implies the necessity to uncover novel targets. The C-X-C chemokine receptor 4 (CXCR4) overexpression has been identified in several hematopoietic malignancies. However, the expression signatures and prognostic significance of CXCR4 in DLBCL associated with clinicopathological features remain unclear. Methods: Gene expression profiles of DLBCL were obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Then, a meta-analysis with an integrated bioinformatic analysis was performed to assess the relationship between CXCR4 expression and clinicopathological features of DLBCL. Finally, experimental verification including immunohistochemical (IHC) staining and real-time quantitative PCR (qPCR) was carried out using patient samples. In vitro cell line viability tests were conducted using CXCR4 inhibitor WZ811. Results: DLBCL patients with activated B-cell-like (ABC) subtype have higher expression level of CXCR4 with worse survival. Differential expressed genes in the CXCR4-upregulation group were enriched in canonical pathways associated with oncogenesis. DLBCL with CXCR4 upregulation had lower degree of CD8+ T cell infiltration. TIMER analysis demonstrated that the CXCR4 expression was positively correlated with the expression of CD5, MYC, NOTCH1, PDCD1, CD274, mTOR, FOXO1, and hnRNPA2B1 in DLBCL. IHC study in patient samples showed the positive correlation between CXCR4 and nongerminal center B-cell (non-GCB) subtype and mTOR expression. Meanwhile, quantitative polymerase chain reaction results revealed that high CXCR4 mRNA level was correlated to double-hit DLBCL. Finally, cell viability test showed that WZ811 exerted antiproliferation effect in DLBCL cell lines in a dose-dependent manner. Conclusion: CXCR4 was upregulated in ABC-DLBCL associated with worse prognosis. Our analysis predicted CXCR4 as a potential target for DLBCL treatment, which may serve as an inhibitor both on BCR signaling and nuclear export warranting further investigation in clinical trials.


Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Prognóstico , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Regulação para Cima
10.
Cell Mol Biol (Noisy-le-grand) ; 67(5): 88-95, 2022 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-35818267

RESUMO

With the changes in people's dietary life, the incidence and mortality of colon cancer have risen sharply. Invasive metastasis of colon cancer is the main reason affecting the prognosis. Therefore, it is very important to study the molecular mechanism of SDF-1/CXCR4 and integrin αvß6 in liver metastasis of colon cancer. Floating cells were used to detect the appearance of ß6, and the relationship between SDF-1/CXCR4 and the molecular mechanism of colon cancer redirection was analyzed. Use immunohistochemistry to detect the appearance of SDF-1/CXCR4 and αvß6 protein, and combine the data with clinical-pathological data for statistical analysis. Experimental data showed that the positive expression rates of αvß6 protein in well-differentiated and poorly differentiated colon cancer tissues were 21.4% and 30.6%, respectively, and the difference was statistically significant (P<0.01). The results show that the appearance of SDF-1/CXCR4 in colon cancer cells (CCC) has nothing to do with the type of cancer cells, and increases with the decrease of cell differentiation. This has a great relationship with the classification of the clinical TNM disease stage. The later the disease stage, the higher the expression level. The αvß6 has a strong correlation with the invasion and metastasis of colon cancer and can be used as a criterion for judging liver metastasis and prognosis.


Assuntos
Antígenos de Neoplasias , Quimiocina CXCL12 , Neoplasias do Colo , Integrinas , Neoplasias Hepáticas , Receptores CXCR4 , Antígenos de Neoplasias/metabolismo , Quimiocina CXCL12/metabolismo , Neoplasias do Colo/patologia , Humanos , Integrinas/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo
11.
BMC Cancer ; 22(1): 740, 2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35799158

RESUMO

BACKGROUND: Papillary and follicular thyroid carcinomas can be treated surgically and with radioiodine therapy, whereas therapeutic options for advanced stage IV medullary and for anaplastic tumours are limited. Recently, somatostatin receptors (SSTs) and the chemokine receptor CXCR4 have been evaluated for the treatment of thyroid carcinomas, however, with contradictory results. METHODS: The expression of the five SSTs and of CXCR4 was assessed in 90 samples from 56 patients with follicular, papillary, medullary, or anaplastic thyroid carcinoma by means of immunohistochemistry using well-characterised monoclonal antibodies. The stainings were evaluated using the Immunoreactivity Score (IRS) and correlated to clinical data. In order to further substantiate the immunohistochemistry results, in serial sections of a subset of the samples receptor expression was additionally examined at the mRNA level using qRT-PCR. RESULTS: Overall, SST and CXCR4 protein expression was low in all four entities. In single cases, however, very high IRS values for SST2 and CXCR4 were observed. SST2 was the most frequently expressed receptor, found in 38% of cases, followed by SST5 and SST4, found in 14 and 9% of tumours, respectively. SST1 and SST3 could not be detected to any significant extent. CXCR4 was present in 12.5% of medullary and 25% of anaplastic carcinomas. Expression SST3, SST4, SST5 and CXCR4 was positively correlated with expression of the proliferation marker Ki-67. Additionally, a negative interrelationship between SST4 or SST5 expression and patient survival and a positive association between SST3 expression and tumour diameter were observed. qRT-PCR revealed a similar receptor expression pattern to that seen at the protein level. However, probably due to the low overall expression, no correlation was found for the SSTs or the CXCR4 between the IRS and the mRNA values. CONCLUSIONS: SST- or CXCR4-based diagnostics or therapy in thyroid carcinomas should not be considered in general but may be feasible in single cases with high levels of expression of these receptors.


Assuntos
Receptores CXCR4 , Receptores de Somatostatina , Neoplasias da Glândula Tireoide , Anticorpos Monoclonais , Humanos , Radioisótopos do Iodo , RNA Mensageiro/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Neoplasias da Glândula Tireoide/genética
12.
Oxid Med Cell Longev ; 2022: 8547563, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35799894

RESUMO

Growing evidence suggests that the interactions between astrocytes and neurons exert important functions in the central sensitization of the spinal cord dorsal horn in rodents with diabetes and neuropathic pain (DNP). However, it still remains unclear how signal transmission occurs in the spinal cord dorsal horn between astrocytes and neurons, especially in subjects with DNP. Chemokine CXC receptor 4 (CXCR4) plays critical roles in DNP, and connexin 43 (CX43), which is also primarily expressed by astrocytes, contributes to the development of neuropathy. We thus postulated that astrocytic and neuronal CXCR4 induces and produces inflammatory factors under persistent peripheral noxious stimulation in DNP, while intercellular CX43 can transmit inflammatory stimulation signals. The results showed that streptozotocin-induced type 1 diabetic rats developed heat hyperalgesia and mechanical allodynia. Diabetes led to persistent neuropathic pain. Diabetic rats developed peripheral sensitization at the early phase (2 weeks) and central sensitization at the late phase (5 weeks) after diabetes induction. Both CXCR4 and CX43, which are localized and coexpressed in neurons and astrocytes, were enhanced significantly in the dorsal horn of spinal cord in rats undergoing DNP during late phase of diabetes, and the CXCR4 antagonist AMD3100 reduced the expression of CX43. The nociceptive behavior was reversed, respectively, by AMD3100 at the early phase and by the antioxidant N-acetyl-L-cysteine (NAC) at the late phase. Furthermore, rats with DNP demonstrated downregulation of glial fibrillary acidic protein (GFAP) as well as upregulation of c-fos in the spinal cord dorsal horn at the late phase compared to the controls, and upregulation of GFAP and downregulation of c-fos were observed upon treatment with NAC. Given that GFAP and c-fos are, respectively, makers of astrocyte and neuronal activation, our findings suggest that CXCR4 as an inflammatory stimulation protein and CX43 as an intercellular signal transmission protein both may induce neurons excitability and astrocytes dysfunction in developing DNP.


Assuntos
Conexina 43 , Diabetes Mellitus Experimental , Neuropatias Diabéticas , Neuralgia , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Animais , Antioxidantes/metabolismo , Astrócitos/metabolismo , Conexina 43/metabolismo , Diabetes Mellitus Experimental/metabolismo , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Medula Espinal/metabolismo
13.
Life Sci ; 303: 120661, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35643380

RESUMO

AIM: The C-X-C chemokine-receptor type 4 (CXCR4) is an emerging target for cancer drug discovery due to its high expression in cancer cells. The present study aimed to produce CXCR4 overexpressing HEK293T cells for a non-radioactive binding assay as a platform to identify drug candidates targeting CXCR4. MAIN METHODS: HEK293T cells stably expressing human CXCR4 were constructed by transfection of CXCR4 plasmids from the human CXCR4 gene. The CXCR4 overexpressing HEK293T cells were obtained by fluorescence-activated sorting and verified by conducting the competition binding assay of a known CXCR4 inhibitor, AMD3100 (plerixafor), to determine the IC50 value against monoclonal anti-human CD184 (hCD184) antibody tagged with fluorescence probe, phycoerythrin (PE). The non-radioactive binding assay using CXCR4 overexpressing HEK293T cells and PE-anti hCD184 was applied as a platform for identifying the target of natural compounds that exhibited cytotoxicity against cancer cell lines. KEY FINDINGS: The CXCR4 overexpressing HEK293T cells were produced with high expression (99.8%). The IC50 value of plerixafor determined by fluorescence tagged antibody-based competition assay using our developed cells agree with previously reported values using a radioligand binding assay. We observed no significant displacement of bound PE-anti-hCD184 by the test natural compounds which could be due to non-specific binding to other functional targets or organelles, low potency of the natural compounds, or binding to CXCR4 at deeper pockets. SIGNIFICANCE: The verified non-radioactive binding assay can serve as an alternative screening tool for anticancer lead compounds targeting CXCR4 and an essential tool for proof of mechanism study in the drug discovery.


Assuntos
Ciclamos , Compostos Heterocíclicos , Quimiocina CXCL12/metabolismo , Células HEK293 , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos/farmacologia , Humanos , Rim/metabolismo , Receptores CXCR4/metabolismo
14.
Arch Virol ; 167(10): 1947-1959, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35752684

RESUMO

Epstein-Barr virus (EBV) belongs to the subfamily Gammaherpesvirinae and was the first human tumor virus to be discovered. The global rate of EBV infection in adults exceeds 90%. EBV can participate in the regulation of multiple genes and signal pathways through its latency genes. Many studies have shown that CXCR4 is involved in the development of gastric cancer, but there have been few studies on the specific mechanisms involved in EBV-associated gastric cancer (EBVaGC). In this study, we explored the mechanism by which EBV-encoded products maintain latent EBV infection through interaction with CXCR4 and investigated the role of CXCR4 in EBV-positive cells. The results show that there is a positive feedback between the EBV-encoded products and CXCR4, and LMP2A can activate CXCR4 through the NF-κB pathway. In addition, CXCR4 can be fed back to LMP2A and EBNA1 through the ERK signaling pathway. At the same time, CXCR4 can promote the proliferation and migration of EBV-positive cells, reduce the expression of the immediate early protein BZLF1, the late protein EBV gp350, and the viral capsid antigen, and play an important role in maintaining the incubation period of EBV infection. These findings are applicable to the further targeted therapy of EBVaGC.


Assuntos
Infecções por Vírus Epstein-Barr , Receptores CXCR4 , Proteínas da Matriz Viral , Latência Viral , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Neoplasias Gástricas/virologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
15.
Pharmacol Res ; 182: 106328, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35772647

RESUMO

Atherosclerosis (AS) is an essential pathological changes of ischemic cardio-cerebrovascular disease, and monocyte migration and adhesion to endothelial cells are the critical pathological process in AS. Our previous studies demonstrated a beneficial effect of zedoarondiol in AS, but whether the mechanism is associated with monocyte migration and adhesion to endothelial cells remains unclear. In this study, we investigated whether the anti-atherosclerotic effects of zedoarondiol were associated with decreasing migration and adhesion of monocytes. The oil red O staining demonstrated that zedoarondiol ameliorated AS plaques in en face aorta and aortic root of apolipoprotein E gene knocked (apoE-/-) mice. In vitro, zedoarondiol decreased human monocytic macrophage-like cell line (THP-1) monocytes migration and adhesion to endothelial cells. Single-cell RNA sequencing analysis (scRNA-seq) in mice indicated that zedoarondiol decreased monocytes adhesion to endothelial cells by regulating CXC chemokine ligand 12/CXC chemokine receptor 4 (CXCL12/CXCR4) pathway, which was verified by Western blot of THP-1 monocytes;zedoarondiol also decreased the expressions of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor-kappa B (NF/κB), the downstream proteins of CXCL12/CXCR4 pathway. In conclusion, zedoarondiol ameliorated AS plaque and inhibited monocyte migration and adhesion to endothelial cells via regulating CXCL12/CXCR4 pathway, suggesting that zedoarondiol might be a new promising drug for AS.


Assuntos
Aterosclerose , Placa Aterosclerótica , Animais , Aterosclerose/metabolismo , Adesão Celular , Quimiocina CXCL12/metabolismo , Células Endoteliais/metabolismo , Humanos , Lactonas , Camundongos , Monócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/metabolismo , Receptores CXCR4/metabolismo , Sesquiterpenos
16.
Br J Radiol ; 95(1136): 20211272, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35731811

RESUMO

OBJECTIVES: 68Ga-Pentixafor positron emission tomography (PET) imaging targets CXCR4 expression which is overexpressed in multiple myeloma (MM). In this study, we evaluated the diagnostic utility of 68Ga-Pentixafor PET/CT for imaging CXCR4 expression in MM and compared results with 18F-fluorodeoxyglucose (18F-FDG) PET/CT. METHODS: 34 (21M; 13F; median age = 57.5 years) treatment naive multiple myeloma patients were recruited. All the patients underwent 18F-FDG PET/CT and 68Ga-Pentixafor PET/CT imaging. Freshly prepared 68Ga-Pentixafor (148-185 MBq) was injected intravenously and whole-body PET/CT (low-dose CT) was acquired at 1 h post-injection. The pattern of uptake (diffuse, focal or mixed) and the mean SUVmax value of all the lesions (when lesions were ≤5) or of the five most tracer avid lesions (when lesions was >5) were evaluated. Tumor to background ratio (TBRmax) was calculated for both the tracers. Durie Salmon plus staging (DSPS) was used for disease staging on PET and the results were compared with International staging system (ISS). RESULTS: 68Ga-Pentixafor PET/CT showed higher disease extent than seen on 18F-FDG PET/CT in 23/34 patients (68.0%), lesser disease extent in 2/34 (6%) and similar disease extent in 9/34 (26%) patients. Significantly (p < 0.001) higher TBRmax values (5.7; IQR 8.8) were observed on 68Ga-Pentixafor PET/CT as compared to 18F-FDG PET/CT values (2.9; IQR = 4.0). Both the techniques detected extramedullary lesions in six patients. On the other hand, 68Ga-Pentixafor detected medullary lesions in five, whereas, 18F-FDG PET in three patients. Further, only 68Ga-Pentixafor TBRmax correlated significantly (ρ = 0.421; 0.013) with bone marrow plasma cell percentage. 68Ga-Pentixafor PET upstaged more number (9/29) of patients as compared to (4/29) 18F-FDG PET imaging. On the other hand, 18F-FDG PET down-staged 9/29, whereas 68Ga-Pentixafor PET downstaged only 3/29 patients. CONCLUSION: 68Ga-Pentixafor PET/CT evaluated the whole-body disease burden of CXCR4 receptors non-invasively which is not possible by tissue sampling methods. This novel PET tracer has also implication for disease staging. Dual 68Ga-Pentixafor/18F-FDG PET/CT imaging may help in determining the tumor heterogeneity in MM. ADVANCES IN KNOWLEDGE: This CXCR4 targeting PET tracer has a promising role in the development of CXCR4 targeting theranostics and also for response assessment to these therapies including the conventional treatment.


Assuntos
Fluordesoxiglucose F18 , Mieloma Múltiplo , Complexos de Coordenação , Radioisótopos de Gálio , Humanos , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico por imagem , Mieloma Múltiplo/patologia , Peptídeos Cíclicos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Tomografia por Emissão de Pósitrons/métodos , Receptores CXCR4/metabolismo
17.
Arterioscler Thromb Vasc Biol ; 42(7): e217-e227, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35652755

RESUMO

BACKGROUND: An activated, proinflammatory endothelium is a key feature in the development of complications of obesity and type 2 diabetes and can be caused by insulin resistance in endothelial cells. METHODS: We analyzed primary human endothelial cells by RNA sequencing to discover novel insulin-regulated genes and used endothelial cell culture and animal models to characterize signaling through CXCR4 (C-X-C motif chemokine receptor 4) in endothelial cells. RESULTS: CXCR4 was one of the genes most potently regulated by insulin, and this was mediated by PI3K (phosphatidylinositol 3-kinase), likely through FoxO1, which bound to the CXCR4 promoter. CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared with lean controls and 37% higher in db/db mice than db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. SDF-1 (stromal cell-derived factor-1)-the ligand for CXCR4-increased leukocyte adhesion to cultured endothelial cells. This effect was lost after deletion of CXCR4 by gene editing while 80% of the increase was prevented by treatment of endothelial cells with insulin. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling after intravenous injection of SDF-1, but most of this response was prevented in transgenic mice with endothelial overexpression of IRS-1 (insulin receptor substrate-1). CONCLUSIONS: Endothelial cell insulin signaling limits leukocyte/endothelial cell interaction induced by SDF-1 through downregulation of CXCR4. Improving insulin signaling in endothelial cells or inhibiting endothelial CXCR4 may reduce immune cell recruitment to the vascular wall or tissue parenchyma in insulin resistance and thereby help prevent several vascular complications.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Receptores CXCR4/metabolismo , Animais , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Insulina , Leucócitos/metabolismo , Camundongos , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CXCR4/genética
18.
Cell Biol Int ; 46(9): 1519-1529, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35731168

RESUMO

Glioma initiating cells (GICs), also known as glioma stem cells, display the capacity to recapitulate the functional diversity within the tumor. Despite the great progress achieved over the last decades, defining the key molecular regulators of GICs has represented a major obstacle in this field. In our study, data from The Cancer Genome Atlas database illustrated a relationship between C-X-C motif chemokine receptor 4 (CXCR4) expression and the survival of glioma patients. Mechanistically, we further indicated that CXCR4 mediated the upregulation of Kruppel like factor 5 (KLF5), a zinc-finger-containing transcription factor, to facilitate the proliferation of GICs. What's more, CXCR4 also enhanced the chemoresistance through KLF5/Bcl2-like 12 (BCl2L12) in glioma. The elevated expression of KLF5 and BCL2L12 induced by CXCR4 was dependent on phosphoinositide 3-kinases (PI3K)/serine/threonine kinase (AKT) signaling. Importantly, combined application of temozolomide and a CXCR4 inhibitor efficiently reversed CXCR4 mediated drugs resistance and improved anticancer effects in vivo. Collectively, our findings confirmed that CXCR4 promoted GICs proliferation via the KLF5/BCL2L12 dependent pathway, which may enrich the understanding of GICs and help drive the design of efficacious therapeutic strategies.


Assuntos
Neoplasias Encefálicas , Glioma , Receptores CXCR4 , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Proteínas Musculares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Temozolomida/metabolismo , Temozolomida/farmacologia
19.
J Exp Med ; 219(7)2022 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-35670812

RESUMO

Regulatory T (Treg) cells represent a specialized lineage of suppressive CD4+ T cells whose functionality is critically dependent on their ability to migrate to and dwell in the proximity of cells they control. Here we show that continuous expression of the chemokine receptor CXCR4 in Treg cells is required for their ability to accumulate in the bone marrow (BM). Induced CXCR4 ablation in Treg cells led to their rapid depletion and consequent increase in mature B cells, foremost the B-1 subset, observed exclusively in the BM without detectable changes in plasma cells or hematopoietic stem cells or any signs of systemic or local immune activation elsewhere. Dysregulation of BM B-1 B cells was associated with a highly specific increase in IgM autoantibodies and total serum IgM levels. Thus, Treg cells control autoreactive B-1 B cells in a CXCR4-dependent manner. These findings have significant implications for understanding the regulation of B cell autoreactivity and malignancies.


Assuntos
Subpopulações de Linfócitos B , Linfócitos T Reguladores , Subpopulações de Linfócitos B/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Imunoglobulina M/metabolismo , Receptores CXCR4/metabolismo
20.
J Transl Med ; 20(1): 263, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35672854

RESUMO

BACKGROUND: Metabolic disorders are associated with increased incidence, aggressive phenotype and poor outcome of breast cancer (BC) patients. For instance, hyperinsulinemia is an independent risk factor for BC and the insulin/insulin receptor (IR) axis is involved in BC growth and metastasis. Of note, the anti-diabetic metformin may be considered in comprehensive therapeutic approaches in BC on the basis of its antiproliferative effects obtained in diverse pre-clinical and clinical studies. METHODS: Bioinformatics analysis were performed using the information provided by The Invasive Breast Cancer Cohort of The Cancer Genome Atlas (TCGA) project. The naturally immortalized BC cell line, named BCAHC-1, as well as cancer-associated fibroblasts (CAFs) derived from BC patients were used as model systems. In order to identify further mechanisms that characterize the anticancer action of metformin in BC, we performed gene expression and promoter studies as well as western blotting experiments. Moreover, cell cycle analysis, colony and spheroid formation, actin cytoskeleton reorganization, cell migration and matrigel drops evasion assays were carried out to provide novel insights on the anticancer properties of metformin. RESULTS: We first assessed that elevated expression and activation of IR correlate with a worse prognostic outcome in estrogen receptor (ER)-positive BC. Thereafter, we established that metformin inhibits the insulin/IR-mediated activation of transduction pathways, gene changes and proliferative responses in BCAHC-1 cells. Then, we found that metformin interferes with the insulin-induced expression of the metastatic gene CXC chemokine receptor 4 (CXCR4), which we found to be associated with poor disease-free survival in BC patients exhibiting high levels of IR. Next, we ascertained that metformin prevents a motile phenotype of BCAHC-1 cells triggered by the paracrine liaison between tumor cells and CAFs upon insulin activated CXCL12/CXCR4 axis. CONCLUSIONS: Our findings provide novel mechanistic insights regarding the anti-proliferative and anti-migratory effects of metformin in both BC cells and important components of the tumor microenvironment like CAFs. Further investigations are warranted to corroborate the anticancer action of metformin on the tumor mass toward the assessment of more comprehensive strategies halting BC progression, in particular in patients exhibiting metabolic disorders and altered insulin/IR functions.


Assuntos
Neoplasias da Mama , Metformina , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Insulina/farmacologia , Insulina/uso terapêutico , Metformina/farmacologia , Metformina/uso terapêutico , Receptores CXCR4/metabolismo , Transdução de Sinais , Microambiente Tumoral
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