CXC Chemokine Receptor 4 Gene Research Articles

Construction of an Inducible CRISPR/Cas9 System for CXCR4 Gene and Demonstration of its Effects on MKN-45 Cells.

The CRISPR/Cas9 system is an effective tool for gene editing. However, this conventional expression system cannot control the timing of gene editing and does not utilize resistance screening markers. Therefore, carrying out CRISPR/Cas9 experiments is extremely inconvenient. Our aim is to develop an inducible lentiviral vector-based gene-editing system for C-X-C chemokine receptor 4 (CXCR4) by CRISPR/Cas9, and to demonstrate its function in MKN-45 cell. The DNA fragments of Blasticidin and T2A-GFP were produced using the lenti-Cas9-BLAST and PX458 plasmids as templates. The PCR products were harvested and cloned into the lenti-guide-puro plasmid to yield the lenti-guide-BLAST-GFP plasmid. Three double-stranded guide RNA (gRNA) sequences targeting the exon 2 of CXCR4 gene were designed online (http://crispr.mit.edu), synthesized, and recombined into the lenti-guide-BLAST-GFP plasmid, to yield the lenti-guide-BLAST-GFP-gRNA plasmid. The pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA plasmids were packaged with lentiviral vectors, which were then transfected into MKN-45 cells, to identify the CXCR4 gene-editing effects using the T7 endonuclease 1 (T7E1) and Western blot assays. The lenti-guide-BLAST-GFP and lenti-guide-BLAST-GFP-gRNA plasmids were successfully constructed and packaged, to yield lentiviral particles. Transfection of the pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA viral vectors could decrease the expression of CXCR4 protein, and lead to gene editing in MKN-45 cells. The efficiencies of gRNA-1, gRNA-2, and gRNA-3 were 45.6%, 53.6%, and 56.7%, respectively. Furthermore, the chemotactic efficiency of the dual viral vector-infected MKN-45 cells was significantly decreased in response to SDF-1. The numbers of migratory cells in the lower chamber of the transwell system were 30.0 ± 0.23, 29.7 ± 1.55, 28.2 ± 1.11 and 36.1 ± 2.00 cells per field (400×) for gRNA-1, gRNA-2, gRNA-3 and the control, respectively (P < 0.05). We constructed an inducible CXCR4 gene-editing, dual-vector CRISPR/Cas9 system, which could induce CXCR4 gene editing in MKN-45 cells in a doxycycline-dependent manner and thus reduce the migration of MKN-45 cells.

MiR‑494.3p expression in synovial sarcoma: Role of CXCR4 as a potential target gene.

Synovial sarcoma (SS) is a rare tumour, with dismal survival when metastasis occurs. SS contains a characteristic translocation (X;18)(p11;q11) and the fusion genes appear to be mutually exclusive and concordant in primary and metastatic tumours. Novel prognostic and predictive factors are required. The C‑X‑C motif chemokine ligand12 (CXCL12)/C‑X‑C chemokine receptor4 (CXCR4) axis is involved in tumour development and metastatic spread in many types of cancer and previous data have demonstrated a pivotal role of CXCR4 in SS cell migration and invasion. Bioinformatics and biological data indicated CXCR4 is a possible candidate target of miR‑494.3p, known to be involved in tumour progression. In this study, we analysed the expression of miR‑494.3p and its potential target, CXCR4, in a series of SS specimens. A significantly lower miR‑494.3p expression was found in the tumour compared to normal tissue associated with higher levels of CXCR4 both at the gene and protein level. The role of CXCR4 as a potential target of miR‑494.3p was assessed in two SS cell lines (SW982 and SYO‑I). Transfection with miR‑494.3p expression plasmid led to a marked decrease in CXCR4 gene and protein expression, concomitant with a transitory decrease in cell proliferation and migration. The SYO‑I cells also responded with an increased apoptotic fraction. The data of this study also demonstrate that the downregulation of miR‑494.3p in SS surgical specimens, concomitant with an increased expression of its potential target, CXCR4, was more evident in the metastatic subset. In vitro experiments confirmed that miR‑494.3p functioned as a tumour suppressor through the involvement of CXCR4 and ongoing studies are directed to better clarify its role in SS therapeutic strategies.

Effect of Lentiviral Vector-Mediated CXCR4 Gene Overexpression on Mesenchymal Stem Cell Homing Capacity

To explore the effects of lentiviral-mediated CXC chemokine receptor-4（CXCR-4）gene over-expression on the homing capacity of mesenchymal stem cells（MSC）in vivo. The MSC overexpressing CXCR-4 were constructed by using the lentiviral vector-mediated mouse MSC overexpressing the CXCR-4 gene. The BALB/c mice were divided into 3 group: simple radiation group（TBI）in which mice exposed to total body irradiation, then were infused with normal saline; EGFP-MSC group in which mice were infused with MSC（5×105）transducted by EGFP via tail vein after TBI; and CXCR-4-MSC group in which mice were infused with MSC (5×105) simultaneously carraying EGFP and CXCR-4 gene via tail vein after TBI. The mice were sacrified at 24 hours after infusion, the frozen sections were prepared to detect the distribution of infused MSC. Furthermore, the numbers of MSC homing into spleen and bone marrow was detected by flow cytometry, and the level of stromal cell-derived factor-1(SDF-1) was detected by ELISA. The frozen section showed that the CXCR-4 over-expression could significantly enhance the efficacy of MSC homing into lung, liver and spleen; the flow cytonetry detection slowed that the number of over-expressed CXCR-4 MSC homing into spleen and bone matrow was sigmificantly higher than that in EGFP-MSC group（P<0.05）, the ELISA showed that the SDF-1 level in peripheral blood and bone marrow after 24 hours of irradiation significantly incrtaoed （P<0.05）, moreover, the SDF-1 level increase was associcted with horming efficacy of MSC with CXCR-4 overexpression. overexpression CXCR-4 gene mediated by lentiviral vector can prmote the efficacy of MSC homing into spleen and bone marrow.

Influence of Lead Exposure in the Expression of Calmodulin – Related Genes: A Preliminary Study on Workers Working in Industry of Batteries, In Iraq

Most pathological effects of lead on the body are due to ability of lead to bind with important cellular molecules of various tissues and organs leading to formation abnormal molecules and thus to emergence of pathological conditions. To evaluation the risk to the health status of Iraqi workers who work in the batteries industry, expression of three types of calmodulin related genes were examined. Blood samples were collected from worker working in Iraqi industry of batteries (located in Al-Waziriya), then RNAs extraction were done thereby gene expression for Calcium/Calmodulin- dependent protein kinase2 (CaMKK2), C-X-C Chemokine receptor 4 (CXCR4) and mitogen activated protein kinase kinase 6 (MAP2K6) was done for each sample by using RT-qPCR. The study showed that the expression of CXCR4 gene was significantly decrement in the lead exposed workers meanwhile the MAP2K6 gene insignificantly increment in those workers, but no effect appeared on their CaMKK2 gene expression. Conclusion obtained from this study is that lead has ability to impact on calmodulin related genes in the workers working in the batteries manufacture, but this ability is variable according to the type of gene.

Mesenchymal stem cells and CXC chemokine receptor 4 overexpression improved the therapeutic effect on colitis via mucosa repair.

The present study intended to observe the homing capability and therapeutic effect of CXC chemokine receptor 4 (CXCR-4) gene overexpressed bone marrow mesenchymal stem cells (BMSCs) on colitis, and to study the possible mechanisms involved. BMSCs were derived from male BALB/c mice and CXCR-4 gene was transfected into BMSCs by the utilization of the lentiviral vector. The expression of CXCR-4 gene was analyzed and the biological characteristics, and vitality of BMSCs and CXCR-4 gene overexpressed BMSCs (CXCR-BMSCs) were detected. The chemotaxis assay was performed to investigate migration in vitro. Colitis was induced by TNBS in female BALB/c mice. BMSCs and CXCR-BMSCs were injected into experimental models intravenously. The homing of cells was confirmed by fluorescence observation and Sry gene detection. Clinical manifestation and histological changes were also evaluated. The expression levels of occludin and vascular endothelial growth factor (VEGF) were detected to measure mucosal repair. Furthermore, CXCR-4 gene was successfully transfected into BMSCs by the utilization of lentiviral vector. Results indicated that overexpression of CXCR-4 gene did not influence the biological characteristics and vitality of BMSCs, but enhanced the capability of migration and homing of BMSCs in vitro and in vivo. Notably, CXCR-BMSCs had an improved effect on treating colitis, and the expression levels of Occludin and VEGF were higher. The results suggested that overexpression of CXCR-4 gene may enhance BMSC homing to damaged intestinal mucosa and their curative effect on colitis. The present findings indicated that using lentiviral vector to transfect CXCR-4 gene into BMSCs may be a potential method to improve the outcomes of BMSCs treatment on inflammatory bowel disease.

Dual expression of CXCR4 and IL-35 enhances the therapeutic effects of BMSCs on TNBS-induced colitis in rats through expansion of Tregs and suppression of Th17 cells

Bone marrow-derived mesenchymal stem cells (BMSCs) hold great promise for the treatment of inflammatory bowel disease owing to their immunosuppressive property and tissue healing potential. The balance between regulatory T cells (Tregs) and T helper (Th)17 cells plays a crucial role in BMSC-mediated immunosuppression. Interleukin (IL)-35 is a newly identified anti-inflammatory cytokine required for the expansion of Tregs and suppression of Th17 cell differentiation. IL-35 can amplify the immunosuppressive property of BMSCs when overexpressed in these cells. However, the reparative capability of BMSCs in vivo is limited, partly due to the poor homing efficiency of BMSCs to inflamed colons. Up-regulation of CXC chemokine receptor 4 (CXCR4) expression in BMSCs may affect the directional homing of implanted BMSCs via stromal-derived factor-1. In this study, by lentivirus-mediated introduction of CXCR4 and IL-35 genes to modify rat BMSCs, we observed enhanced migration and strengthened immunomodulatory activities of the genetically engineering BMSCs. These results suggest that modification of BMSCs by dual expression of CXCR4 and IL-35 may provide an effective therapeutic strategy for inflammatory bowel disease.

Deferoxamine promotes mesenchymal stem cell homing in noise-induced injured cochlea through PI3K/AKT pathway.

Over 5% of the world's population suffers from disabling hearing loss. Stem cell homing in target tissue is an important aspect of cell-based therapy, which its augmentation increases cell therapy efficiency. Deferoxamine (DFO) can induce the Akt activation, and phosphorylation status of AKT (p-AKT) upregulates CXC chemokine receptor-4 (CXCR4) expression. We examined whether DFO can enhance mesenchymal stem cells (MSCs) homing in noise-induced damaged cochlea by PI3K/AKT dependent mechanism. Mesenchymal stem cells were treated with DFO. AKT, p-AKT protein and hypoxia inducible factor 1- α (HIF-1α) and CXCR4 gene and protein expression was evaluated by RT- PCR and Western blot analysis. For in vivo assay, rats were assigned to control, sham, noise exposure groups without any treatment or receiving normal, DFO-treated and DFO +LY294002 (The PI3K inhibitor)-treated MSCs. Following chronic exposure to 115 dB white noise, MSCs were injected into the rat cochlea through the round window. Number of Hoechst- labelled cells was determined in the endolymph after 24hours. Deferoxamine increased P-AKT, HIF-1α and CXCR4 expression in MSCs compared to non-treated cells. DFO pre-conditioning significantly increased the homing ability of MSCs into injured ear compared to normal MSCs. These effects of DFO were blocked by LY294002. Pre-conditioning of MSCs by DFO before transplantation can improve stem cell homing in the damaged cochlea through PI3K/AKT pathway activation.

Influence of Lead Exposure in the Expression of Calmodulin – Related Genes: A Preliminary Study on Workers Working in Industry of Batteries, In Iraq

Most pathological effects of lead on the body are due to ability of lead to bind with important cellular molecules of various tissues and organs leading to formation abnormal molecules and thus to emergence of pathological conditions. To evaluation the risk to the health status of Iraqi workers who work in the batteries industry, expression of three types of calmodulin related genes were examined. Blood samples were collected from worker working in Iraqi industry of batteries (located in Al-Waziriya), then RNAs extraction were done thereby gene expression for Calcium/Calmodulin- dependent protein kinase2 (CaMKK2), C-X-C Chemokine receptor 4 (CXCR4) and mitogen activated protein kinase kinase 6 (MAP2K6) was done for each sample by using RT-qPCR. The study showed that the expression of CXCR4 gene was significantly decrement in the lead exposed workers meanwhile the MAP2K6 gene insignificantly increment in those workers, but no effect appeared on their CaMKK2 gene expression. Conclusion obtained from this study is that lead has ability to impact on calmodulin related genes in the workers working in the batteries manufacture, but this ability is variable according to the type of gene.

Canonical and alternative transcript expression of PAX6 and CXCR4 in pancreatic cancer.

Pancreatic cancer is a lethal disease with a propensity for invading and metastasizing into the surrounding tissues, including the liver and intestines. A number of factors are aberrantly overexpressed in this tumor type and actively promote cancer progression and metastasis. The present study demonstrates that paired box transcription factor 6 (PAX6) and C-X-C chemokine receptor 4 (CXCR4) are frequently co-expressed in primary pancreatic adenocarcinoma tumors and established cell lines. Expression analysis methods used in the present study included evaluation of protein expression by western blot analysis and immunofluorescence, transcript expression levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and luciferase assays utilizing regulatory elements from the CXCR4 gene locus. Canonical PAX6 and alternative splice variant PAX6(5a) proteins are expressed in pancreatic cancer and can drive gene expression through a conserved enhancer element within the first intron of the CXCR4 gene. As demonstrated by the introduction of an exogenous reporter construct with or without the intronic enhancer, loss of this element inhibited gene expression within numerous pancreatic cancer cell lines including Panc1, MIA-PaCa2 and BxPC3. All of the pancreatic cancer cell lines expressed the canonical CXCR4B transcript in addition to the alternatively spliced variant CXCR4A as determined by RT-qPCR experiments. The discovery of variant transcripts in pancreatic cancer cells may provide new candidates for future targeted therapies.

Expression of chemokine receptor CXCR4 is closely correlated with clinical outcome in human nasopharyngeal carcinoma.

The CXC chemokine receptor 4 (CXCR4) has been reported to be involved in the development and progression of nasopharyngeal carcinoma (NPC). However, the role of CXCR4 in clinical outcome and prognosis of NPC patients remains controversial. In the present study, we investigated and reviewed the expression of CXCR4 in NPC tissues and then analyzed the definitive role of CXCR4 in clinical outcome and prognosis. Here, we found that the expression level of CXCR4 was significantly higher in NPC cancer specimens (61/98) than that in paired non-tumor tissues (p < 0.001). Together with our pathological analysis, statistic analysis revealed that CXCR4 expression was indeed closely correlated with UICC stage (p = 0.000), N stage (p = 0.019), and metastasis (p = 0.000). Most importantly, the systematic review combined with our survival and multivariate analysis that revealed high expression of CXCR4 was obviously associated with poor overall survival (OS) (p = 0.000) and progression-free survival (PFS) (p = 0.000) and can act as an independent prognostic factor in NPC patients. In conclusion, this study suggests that CXCR4 is highly activated and expressed in the development of NPC and may be recommended as an indicator in the diagnosis of NPC. Thus, targeting of CXCR4 gene or protein could be used as a potential therapy for NPC.

Abstract 16620: Enhancing transplanted Cardiac Progenitor Cells homing and survival in Ischemic Heart By CRISPR-dcas9/sgRNA-based Endogenous CXCR4 Promoter Activation

Cardiac progenitor cell (CPC) therapy is promising for repairing the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow-derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Strategies to overexpress CXCR4 genes in CPC have been shown to improve the survival of donor cells within the ischemic myocardium. The CRISPR/dCas9-VP64/sgRNA activation system (CAS), was recently developed as a simple and efficient method in activation of endogenous loci by targeting their promoter region in mammalian cells. With single guide chimeric RNA's (sgRNA) targeting proximal promoter region of CXCR4, we found that the CAS activation system can upregulate protein level of endogenous CXCR4 gene expression about 140 fold by qRT-PCR. The CAS -CXCR4 improves CPC response to SDF-1 induced migration and survival against H2O2 induced oxidative stress. Half million CAS-Control or CAS-CXCR4 CPC were intramyocardially injected into the peri-infarction zone after myocardial infarction (MI) induced by left anterior descending coronary artery (LAD) ligation in mice. The CAS- CXCR4 gene modification improved CPC survival by bioluminescence imaging (BLI), and CAS-CXCR4 CPC also exhibited improved left ventricular (LV) ejection fraction (EF) at 3 wk after MI (n=6, P&lt;0.05). Our results demonstrate that activation of endogenous CXCR4 expression by CRISPR/dCas9-VP64 activation system using sgRNA is very effective in improving homing and survival of donor CPC, and attenuate cardiomyopathy after MI.

SDF-1/CXCR4 axis in Tie2-lineage cells including endothelial progenitor cells contributes to bone fracture healing.

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal-derived-factor 1 (SDF-1). SDF-1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF-1/CXCR4 pathway in Tie2-lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2-Cre(ER) CXCR4 knockout mice (CXCR4(-/-) ) and wild-type mice (WT). We report here that in vitro, EPCs derived from of CXCR4(-/-) mouse bone marrow demonstrated severe reduction of migration activity and EPC colony-forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4(-/-) group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4(-/-) group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4(-/-) group. Especially, CXCR4(-/-) group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real-time RT-PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE-cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4(-/-) group. In the gain-of-function study, the fracture in the SDF-1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF-1 injected CXCR4(-/-) group. We demonstrated that an EPC SDF-1/CXCR4 axis plays an important role in bone fracture healing using Tie2-Cre(ER) CXCR4 conditional knockout mice.

Construction of lentiviral vector for mouse CXC chemokine receptor 4 gene and its expression in eukaryotic cells

This study was aimed to clone the gene coding mouse CXC chemokine receptor 4 (CXCR4), to construct the recombinant lentiviral vector carrying enhanced green fluorescence protein (EGFP) and to explore its expression in eukaryotic cells (293FT cells). The full length CXCR4 gene was cloned by RT-PCR using bone marrow cells from C57BL/6 mouse as template and inserted into PCR-Blunt vector. CXCR4 fragment was generated by digestion with restriction endonuclease and subcloned into a lentiviral vector to generate recombinant lentiviral vector LV-IRES-EGFP-CXCR4, which was co-transfected into 293FT cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM). And the expression of CXCR4 protein was detected by Western blot. The results demonstrated that mouse CXCR4 gene was cloned and the lentiviral vector was successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in the transfected 293FT cells, and the transfection efficacy > 95% was determined by FCM. Expression of CXCR4 protein detected by FCM and Western blot was significantly higher than that in control group. It is concluded that the CXCR4 gene along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.

BreastDefend™ prevents breast-to-lung cancer metastases in an orthotopic animal model of triple-negative human breast cancer

We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of MDA-MB-231 invasive human breast cancer cells in vitro. In the present study, we evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. Oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastric gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67% (control) to 20% (BD) (p<0.05) and the number of metastases from 2.8 (0.0, 48.0) in the control group to 0.0 (0.0, 14.2) in the BD treatment group (p<0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the downregulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers.

Abstract 5633: BreastDefend suppresses growth and breast-to-lung cancer metastasis in an orthotopic model of human breast cancer

Abstract We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of invasive human breast cancer cells MDA-MB-231 in vitro. In the present study we have evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. An oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastrical gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time when compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67 % (control) to 20% (BD) (p&amp;lt;0.05) and number of metastasis from 2.8 [0.0, 48.0] in the control group to 0.0 [0.0, 14.2] in the BD treatment group (p&amp;lt;0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the down-regulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5633. doi:1538-7445.AM2012-5633

Cationic Liposome-MediatedCXCR4Gene Delivery into Hematopoietic Stem/Progenitor Cells: Implications for Clinical Transplantation and Gene Therapy

The chemokine stromal cell-derived factor (SDF)-1α/CXCL12 and its receptor CXC chemokine receptor 4 (CXCR4) play a crucial role in the homing/engraftment and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. It has been shown using the viral gene transfer technique that CXCR4 overexpression on human CD34(+) HSPC significantly improves their engraftment in murine models. However, clinical trials with gene therapy have revealed safety concerns related to the immunogenicity of the viral carriers, due to the random integration of viral genes into the host genome. Therefore, a method for CXCR4 gene delivery into HSPC that is safe, nonviral, and highly efficient is needed to improve clinical transplantation and gene therapies. In this work, we investigated the nonviral CXCR4 gene delivery into HSPC using the cationic liposome agent IBAfect. We used CD34(+) cells from cord blood and the models of immature hematopoietic cells expressing CD34 antigen, namely, leukemic cell lines KG-1a and KG-1. Transfection efficiency was determined by flow cytometric analysis 12, 24, 48, and 72 h after transfection, and the viability of cells analyzed by trypan blue exclusion and MTS assays. The functional response of CXCR4-transfected HSPC toward an SDF-1α gradient was determined by chemotaxis assay. We found that ~25% transfection is achieved for KG-1a and KG-1 cells and 20% for HSPC, and that the viability of CXCR4-transfected HSPC is not significantly altered. More importantly, overexpression of CXCR4 using IBAfect significantly increased the chemotaxis of KG-1 cells and HSPC toward SDF-1α. However, we tested 2 other commercially available cationic liposomes (Lipofectamine 2000 and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) in parallel, and we found that they failed to deliver the CXCR4 gene into cells under the same conditions. These results suggest that IBAfect-mediated in vitro gene delivery to overexpress CXCR4 on HSPC is a safe and efficient technique with great potential for improving the efficacy of HSPC transplantation and gene therapy protocols.

Plumbagin inhibits invasion and migration of breast and gastric cancer cells by downregulating the expression of chemokine receptor CXCR4

BackgroundIncreasing evidence indicates that the interaction between the CXC chemokine receptor-4 (CXCR4) and its ligand CXCL12 is critical in the process of metastasis that accounts for more than 90% of cancer-related deaths. Thus, novel agents that can downregulate the CXCR4/CXCL12 axis have therapeutic potential in inhibiting cancer metastasis.MethodsIn this report, we investigated the potential of an agent, plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), for its ability to modulate CXCR4 expression and function in various tumor cells using Western blot analysis, DNA binding assay, transient transfection, real time PCR analysis, chromatin immunoprecipitation, and cellular migration and invasion assays.ResultsWe found that plumbagin downregulated the expression of CXCR4 in breast cancer cells irrespective of their HER2 status. The decrease in CXCR4 expression induced by plumbagin was not cell type-specific as the inhibition also occurred in gastric, lung, renal, oral, and hepatocellular tumor cell lines. Neither proteasome inhibition nor lysosomal stabilization had any effect on plumbagin-induced decrease in CXCR4 expression. Detailed study of the underlying molecular mechanism(s) revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, inhibition of NF-κB activation, and suppression of chromatin immunoprecipitation activity. In addition, using a virtual, predictive, functional proteomics-based tumor pathway platform, we tested the hypothesis that NF-κB inhibition by plumbagin causes the decrease in CXCR4 and other metastatic genes. Suppression of CXCR4 expression by plumbagin was found to correlate with the inhibition of CXCL12-induced migration and invasion of both breast and gastric cancer cells.ConclusionsOverall, our results indicate, for the first time, that plumbagin is a novel blocker of CXCR4 expression and thus has the potential to suppress metastasis of cancer.

MiR-1 Is a Tumor Suppressor in Thyroid Carcinogenesis Targeting CCND2, CXCR4, and SDF-1α

Micro-RNA have emerged as an important class of short endogenous RNA that act as posttranscriptional regulators of gene expression and are constantly deregulated in human cancer. MiR-1 has been found down-regulated in lung, colon, and prostate cancer. In this study, we investigated the possible role of miR-1 in thyroid carcinogenesis. We have analyzed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions and searched for miR-1 targets. Our results show that miR-1 expression is drastically down-regulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 down-regulation was also found in thyroid hyperproliferative nonneoplastic lesions such as goiters. We identified the CCND2, coding for the cyclin D2 (CCND2) protein that favors the G1/S transition, CXCR4, and SDF-1α genes, coding for the receptor for the stromal cell derived factor-1 (SDF-1)/CXCL12 chemokine and its ligand SDF-1/CXCL12, respectively, as miR-1 targets. An inverse correlation was found between miR-1 expression and CXC chemokine receptor 4 (CXCR4) and SDF-1α protein levels in papillary and anaplastic thyroid carcinomas. Consistent with a role of the CCND2 protein in cell proliferation and CXCR4 and SDF-1α proteins in cell invasion and metastasis, functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration. These results indicate the involvement of miR-1 in thyroid cell proliferation and migration, validating a role of miR-1 down-regulation in thyroid carcinogenesis.

The Mood Stabilizers Valproic Acid and Lithium Enhance Mesenchymal Stem Cell Migration via Distinct Mechanisms

Mesenchymal stem cells (MSCs) show high potential for the therapy of several human diseases; however, the effectiveness of MSC transplantation has been hampered by the relatively poor migratory capacity of these cells toward disease target sites. This study investigated whether treatment of MSCs with two mood stabilizers-valproic acid (VPA) and lithium-would enhance cell migration and, if so, to explore the mechanisms underlying their effects. Short-term (3 h) exposure of MSCs to a relatively high concentration (2.5 mM) of VPA markedly increased the transcript and protein levels of CXC chemokine receptor 4 (CXCR4). VPA-induced CXCR4 expression required inhibition of histone deacetylases (HDACs), including the HDAC1 isoform, and involved histone hyperacetylation at the promoter region of the CXCR4 gene. Notably, VPA treatment enhanced stromal cell-derived factor-1α (SDF-1α)-mediated MSC migration, which was completely blocked by AMD3100, a CXCR4 antagonist. Treatment of MSCs with lithium (2.5 mM for 1 day) selectively elevated the transcript and protein levels of matrix metalloproteinase-9 (MMP-9) and its enzymatic activity; these effects were mimicked by inhibition or gene silencing of glycogen synthase kinase-3β (GSK-3β). Lithium treatment also potentiated SDF-1α-dependent MSC migration across the extracellular matrix, which was suppressed by two MMP-9 inhibitors, doxycycline and GM6001. Combining VPA and lithium treatment further increased MSC migration. Overall, VPA and lithium stimulated MSC migration through distinct targets and mediators: HDAC-CXCR4 and GSK-3β-MMP-9, respectively.

A case of WHIM syndrome associated with diabetes and hypothyroidism

The WHIM syndrome is a rare immunological disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. We hypothesized that immunological or genetic mechanisms may link WHIM syndrome and type 1 diabetes. We report that the young girl with WHIM syndrome developed diabetes and transient hypothyroidism. A nonsense mutation (C-->T) truncating the CXC chemokine receptor 4 (CXCR4) C-terminal cytoplasmic tail domain occurred at nucleotide position 1000(R334X) of the CXCR4 gene in one allele of the patient was identified, and the person was diagnosed as having WHIM syndrome. Recent observation suggested that the CXCR4, a G-protein-coupled receptor with a unique ligand, CXCL12, might be involved in the pathogenesis for type 1 diabetes. Taken into consideration the concurrent prevalence of the two disorders and the speculated common pathogenesis associated with the CXCR4, our patient may enable us to understand the genetic damage related to accelerated apoptosis.