CXC Chemokine Receptor 4 mRNA Expression Research Articles

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

CXCR4, regulated by HIF1A, promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45+ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.

Oxymatrine suppresses oral squamous cell carcinoma progression by suppressing CXC chemokine receptor 4 in an m6A modification decrease dependent manner.

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 Kit‑8, apoptosis and Transwell assays. The inhibitory effect of oxymatrine on tumor growth was evaluated invivo. The regulation of oxymatrine on the CXC chemokine receptor 4 (CXCR4) was analyzed using western blotting, reverse transcription‑quantitative 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 N6‑position of adenosine (m6A modification) of CXCR4 mRNA by decreasing the expression of the methyltransferase‑like 3 (METTL3) gene. In addition, oxymatrine inhibited tumor growth invivo. 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.

The Expression and Prognostic Significance of VEGF and CXCR4 in Gastric Cancer: Correlation with Angiogenesis, Lymphangiogenesis and Progression.

The cellular response to hypoxia includes the expression of hypoxia-inducible factor-1 (HIF-1) and its target genes: vascular endothelial growth factor (VEGF) and CXC chemokine receptor 4 (CXCR4). The aim of this study was to investigate the expression and prognostic significance of VEGF and CXCR4, which are responsible for angiogenesis and progression in gastric cancer. Twenty-eight gastric cancer patients were analyzed. The mRNA expression was examined in primary tumors and corresponding normal gastric mucosa by RT-PCR. The protein level was examined by immunohistochemistry staining. The high expression of VEGF and CXCR4 was found in 71.0 and 64.0% of tumors, respectively. The mean levels of VEGF and CXCR4 were upregulated in primary tumors compared to normal mucosa (p = 0.0007, p = 0.0052). A correlation between VEGF expression and tumor invasion (p = 0.0216) and stage (p = 0.0181) was found. CXCR4 expression correlated with lymph node metastases (p = 0.0237) and stage (p = 0.0054). The VEGF expression correlated with microvessel density (MVD) (p = 0.0491). The overall 3-year survival rate was 46.4% and correlated negatively with high CXCR4 mRNA expression (p = 0.0089). VEGF and CXCR4 play an important role in tumor progression. Their overexpression correlates with a bad prognosis and may improve high-risk patient selection, and these patients may obtain additional survival benefits if treated more aggressively.

CXCR4 overexpression: An indicator of poor survival and predictor of response to immunotherapy in patients with metastatic colorectal cancer.

3546 Background: CXC-chemokine receptor 4 (CXCR4) is a ubiquitous chemokine receptor activated by the CXCL12 ligand and is implicated in tumor invasion, metastasis, and immune cell (IC) trafficking. High CXCR4 expression is associated with poor prognosis in colorectal cancer (CRC). &lt; 10% of metastatic CRC cases harbor microsatellite instability (MSI-H) and demonstrate lower tumor mutation burden (TMB), decreased IC infiltration, and lack of response to current immunotherapy regimens. This study aims to interrogate the role of CXCR4 mRNA expression on the the tumor microenvironment (TME) and its prognostic and predictive value to tailor immunotherapeutic treatment strategies in CRC. Methods: A total of 15,026 CRC samples were analyzed using whole-exome sequencing, whole-transcriptome sequencing, and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Study cohort was stratified by CXCR4 mRNA expression levels in quartiles (Q1 (low) vs Q4 (high)). IC fraction was calculated by QuantiSeq, and real-world overall survival information was obtained from insurance claims data and calculated from tissue collection time to last day of contact. Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (q &lt; 0.05). Results: Samples obtained from metastatic sites showed higher CXCR4 mRNA expression than those from primary tumors (22.7 vs 18.6 median transcripts per million (TPM), p &lt; 0.001). CXCR4 mRNA expression was significantly lower in liver metastases than in non-liver metastases (21.2 vs 24.8 TPM, p &lt; 0.001). Median CXCR4 mRNA expression was highest in the consensus molecular subtypes 4 (33.3 TPM) and lowest in 3 (13.0 TPM, p &lt; 0.05). CXCR4 mRNA expression was positively associated with TMB-H, MSI-H/dMMR, and positive PD-L1 IHC status. In the TME, high CXCR4 mRNA expression was observed in tumors with a higher IC infiltration including B cells, M1/M2 macrophages, NK cells, CD8+ T cells and T-regs, regardless of MSI status. High CXCR4 mRNA expression in the primary tumor was associated with poor prognosis (HR 0.77, 95% CI 0.70-0.85; p &lt; 0.001), regardless of MSI-status. In metastatic tumors, low mRNA expression was correlated with improved survival (HR 0.89, 95% CI 0.80-0.99; p = 0.34); however, this did not reach statistical significance in the MSS cohort (HR 0.90, 95% CI 0.80-1.0; p = 0.06). Of note, high CXCR4 mRNA expression was associated with improved survival in all patients with CRC who received pembrolizumab (HR 2.12, 95% CI 1.16-3.91; p = 0.013). Conclusions: This is the largest clinical dataset to date demonstrating high CXCR4 expression as a predictor for poor survival in CRC. Furthermore, high CXCR4 expression was associated with improved outcome after checkpoint inhibition immunotherapy, indicating its strong potential as a predictive biomarker that could inform immunotherapeutic strategies in CRC.

Effects of miR-338 on morphine tolerance by targeting CXCR4 in a rat model of bone cancer pain.

The present study aimed to investigate the effects of miR-338 on morphine tolerance through the targeting of CXC chemokine receptor-4 (CXCR4) in a rat model of bone cancer pain (BCP). Sprague–Dawley (SD) rats were obtained and divided into model saline (n=10), model morphine (n=50), normal saline (n=10) and normal morphine (healthy rats, n=10) groups. After BCP rat model establishment, the remaining SD rats (n=40) in the model saline group were assigned into pLV-THM-miR-338, pLV-THM-anti-miR-338, CXCR4 shRNA, blank and PBS groups. Luciferase reporter gene assay was used for luciferase activity. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to detect the miR-338 and CXCR4 mRNA and protein expression. The model saline group showed increased mRNA and protein expressions of CXCR4 but decreased miR-338 compared with the model saline group, and the model morphine group had increased mRNA and protein expressions of CXCR4 but decreased miR-338 compared with the model saline group. The mRNA and protein expressions of miR-338 in the pLV-THM-miR-338 group increased remarkably while those of the pLV-THM-anti-miR-338 group decreased significantly compared with the CXCR4 shRNA, blank and PBS groups. The pLV-THM-miR-338, pLV-THM-anti-miR-338, CXCR4 shRNA and CXCR4 mRNA groups all had lower mRNA and protein expressions of CXCR4 than those in the blank and PBS groups. miR-338 exerts significant influence in the inhibition of morphine tolerance by suppressing CXCR4 in BCP.

In Vivo Tracking of Chemokine Receptor CXCR4-Engineered Mesenchymal Stem Cell Migration by Optical Molecular Imaging.

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells to injured and inflamed areas. Noninvasive cell tracking methods could be useful for monitoring cell fate. Therefore, in this study, we evaluated the efficacy of an intravenous infusion of genetically engineered mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4) to home to the tumor, by optical imaging. We constructed a retroviral vector containing CXCR with dual reporter genes, eGFP and Fluc2, under the control of an EF1α promoter (pBABE-EF1α-CXCR4-eGFP-IRES-Fluc2). We also developed an eGFP-Fluc2 construct in the Retro-X retroviral vector (Retro-X-eGFP-Fluc2). MSCs were transduced with retroviruses to generate CXCR4-overexpressing MSCs (MSC-CXCR4/Fluc2) and MSCs (MSC/Fluc2). CXCR4 mRNA and protein expression was confirmed by RT-PCR and Western blotting, respectively, and it was higher in MSC-CXCR4/Fluc2 than in naive MSCs. eGFP expression was confirmed by confocal microscopy. The transfected MSC-CXCR4/Fluc2 cells showed higher migratory capacity than naive MSCs observed in Transwell migration assay. The in vivo migration of CXCR4-overexpressing MSCs to MDAMB231/Rluc tumor model by BLI imaging was also confirmed. Intravenous delivery of genetically modified MSCs overexpressing CXCR4 with a Fluc2 reporter gene may be a useful, noninvasive BLI imaging tool for tracking cell fate.

Prognostic Significance of CXCR4 Overexpression in Pediatric Acute Myeloid Leukemia with Low-Risk: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Background:The outcome of acute myeloid leukemia (AML) has been improved by advanced risk-stratified chemotherapy based on prognostic factors. However, the long-term survival rate remains 60-70% and further investigation is needed. Core binding factor (CBF)-AML, characterized by t(8;21) or inv(16)/t(16;16), is the most frequent subtype in pediatric AML. Although CBF-AML is generally classified as a low-risk (LR) group, approximately 30% of the patients relapsed within 3 years in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. This finding suggests that the LR patients had other risk factors that could account for their adverse outcome. CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor that helps leukemia cells adhere to bone marrow stromal cells that affect the cell survival and proliferation. There are several reports that CXCR4 overexpression is an adverse prognostic factor in adult AML; however, few studies have examined their significance in pediatric AML. Here, we retrospectively examined whether CXCR4 overexpression can be an adverse prognostic factor in pediatric AML with LR.Patients and Methods:From 2006 to 2010, 485 de novo pediatric AML patients aged <18 years were registered in the AML-05 trial. Overall, 42 patients were excluded, mainly because of misdiagnosis. The risk groups were defined as follows: all of the CBF-AML patients with complete remission (CR) after the initial induction course without FLT3 -ITD were included in the LR group, while patients with -7, 5q-, t(16;21)(p11;q22), Ph1, or non-CR after the initial induction course or with FLT3-ITD were included in the high-risk (HR) group. Patients with no LR or HR features were in the intermediate-risk. CXCR4 mRNA expression was analyzed by quantitative RT-PCR using diagnostic bone marrow samples. CXCR4 expression levels were dichotomized based on the average expression level.Results and Discussion:A total of 248 samples were available for this study. No significant differences in the clinical outcomes were observed between these 248 patients and the 195 patients who did not have CXCR4 data (EFS, P =0.55; OS, P =0.87). CXCR4 overexpression (CXCR4 +) was present in 81 patients (32.7%). CXCR4 + was not correlated with age, sex, and WBC count. CBF-AML was similarly distributed between CXCR4 + and CXCR4 - patients. The frequency of FLT3 -ITD in CXCR4 + patients was significantly higher in CXCR4 - patients (P =0.049), while there were no significant differences in that of KIT and CEBPA mutation (P =0.08 and 0.71, respectively). In total (n=248), no significant differences were observed between CXCR4 + and CXCR4 - patients in 3-year OS (68.6% vs. 75.1%, P =0.42) and 3-year EFS (65.6% vs. 58.9%, P =0.27). However, in the LR group (n=93), a significant difference was observed between CXCR4 + and CXCR4 - patients in 3-year OS (79.2% vs. 98.3%, P =0.008), whereas no significant difference was observed in 3-year EFS (83.3% vs. 69.6%, P =0.33). We have revealed that KIT mutations are adverse prognostic factors in t(8;21) pediatric AML in our recent study (Leukemia, 2015). In t(8;21) AML without KIT mutations (n=44), a significant difference was observed between CXCR4 + and CXCR4 - patients in 3-year OS (76.1% vs. 100.0%, P =0.01), whereas no significant difference was observed in 3-year EFS (77.8% vs. 74.2%, P =0.85). Multivariate Cox regression analysis, including CXCR4 +, KIT mutation, and age identified CXCR4 + as the only prognostic factor predicting poor OS in the cohort of AML with LR (hazard ratio, 11.01; P =0.01), but not EFS (hazard ratio, 0.54; P =0.21). The results suggest that CXCR4 + is associated with an adverse prognosis in pediatric AML with LR. The adverse outcome was restricted in OS, not in EFS, suggesting that CXCR4 + may be associated with the poor prognosis after recurrence, although the underlying mechanism remains unknown. Furthermore, it is noteworthy that the OS of LR patients with CXCR4 - was quite high, which should be confirmed in larger studies.Conclusions:We identified the adverse prognostic significance of CXCR4 overexpression in pediatric AML with LR. The adverse prognosis may be restricted after recurrence. Further studies of the underlying biology are required. There are several promising CXCR4 antagonists including AMD3100, therefore, our findings will help to develop better therapeutic approaches in pediatric AML with LR. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3786: Gambogic acid inhibits chemokine receptor CXCR4 signaling pathways and osteoclastogenesis in multiple myeloma

Abstract Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation, as indicated by down-regulation of CXCR4 mRNA expression, inhibition of nuclear factor-kappa B (NF-κB) activity, and suppression of p65 binding at CXCR4 promoter analyzed by quantitative chromatin immunoprecipitation (qChIP) assay. Suppression of CXCR4 expression by GA correlated with both inhibition of SDF-1α-induced invasion of MM cells. GA also inhibited phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA suppressed the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. Finally, we found that MM cells, induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and thus has a strong potential to suppress osteoclastogenesis mediated by MM cells. Citation Format: Manoj K. Pandey, Vijay P. Kale, Chunhua Song, Shen-shu Sung, Arun K. Sharma, Sinisa Dovat, Shantu G. Amin. Gambogic acid inhibits chemokine receptor CXCR4 signaling pathways and osteoclastogenesis in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2014-3786

Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways

Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MMcells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-κB) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1α-induced chemotaxis of MMcells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MMcells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MMcells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MMcells.

Age-related CXC chemokine receptor-4-deficiency impairs osteogenic differentiation potency of mouse bone marrow mesenchymal stromal stem cells

Cysteine (C)-X-C chemokine receptor-4 (CXCR4) is the primary transmembrane receptor for stromal cell-derived factor-1 (SDF-1). We previously reported in mouse or human bone marrow-derived mesenchymal stromal stem cells (BMSCs) that deleting or antagonizing CXCR4 inhibits bone morphogenetic protein-2 (BMP2)-induced osteogenic differentiation. The goal of this study was to determine whether CXCR4-deficiency in BMSCs is an age-related effect in association with impaired osteogenic differentiation potency of aged BMSCs. Using BMSCs derived from C57BL/6J wild type mice at ages ranging from 3 to 23 months old, we detected decreased CXCR4 mRNA and protein expression as well as SDF-1 secretion with advancing aging. Moreover, CXCR4-deficient BMSCs from elderly vs. young mice exhibited impaired osteogenic differentiation in response to BMP2 stimulation or when cultured in dexamethasone (Dex)-containing osteogenic medium, evidenced by decreased alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition (markers for immature and mature osteoblasts). Mechanistically, impaired BMP2- or Dex-osteoinduction in BMSCs of elderly mice was mediated by inhibited phosphorylation of intracellular R-Smads and Erk1/2 or Erk1/2 and p38 proteins, and decreased Runx2 and Osx expression (osteogenesis “master” regulators) were also detected. Furthermore, adenovirus-mediated repair of CXCR4 expression in BMSCs of elderly mice restored their osteogenic differentiation potentials to both BMP2 treatment and osteogenic medium. Collectively, our results demonstrate for the first time that CXCR4 expression in mouse BMSCs declines with aging, and this CXCR4-deficiency impairs osteogenic differentiation potency of aged BMSCs. These findings provide novel insights into mechanisms underlying age-related changes in BMSC-osteogenesis, and will potentiate CXCR4 as a therapeutic target to improve BMSC-based bone repair and regeneration in broad orthopedic situations.

5-Aza-2'-deoxycytidine inhibiting metastasis to lungs of triple-negative breast cancer cells

Objective To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on metastasis to lungs of triple-negative breast cancer (TNBC) MDA-MB-231 cells and the mechanisms. Methods MDA-MB-231 cells were divided into two groups: control group and 5-Aza-CdR-treated group. The mRNA expression and promoter methylation status of breast cancer metastasis suppressor-1 (BRMS1 ) and CXC chemokine receptor-4 (CXCR4) in the MDA-MB-231 cells were evaluated by semi-quantitative reverse transcription-polymerase chain reaction ( SqRT-PCR) and methylation specific polymerase chain reaction (MSP) respectively. Then 2 x 105 cells were injected into each BALB/C nu/nu mouse (5 mice in each group) through lateral tail veins. Five weeks later, the metastasis of lungs was evaluated through the mRNA abundance of the target gene HPRT in the lungs tissue from the mice detected by fluorescent quantitative RT-PCR (FqRT-PCR). Results 5-Aza-CdR up-regulated the BRMS1 mRNA expression [0 versus 0. 390 ±0. 001 (relative integrated density value, IDV against GAPDH) ,P ＜0. 05] and demethylated the methylated CpG island B in promoter, while the CXCR4 mRNA expression (0. 580 ±0. 003 versus 0. 580 ±0. 010,P＞0. 05) and the status of unmethylated CXCR4 CpG island 1 in promoter had no significant change. The Ct values of HPRT and GAPDH in control and 5-Aza-CdR-treated groups were 24. 75 ± 1. 55,16. 19 ±0. 69 versus 27. 61 ± 1. 67, 17. 48 ±0. 96 respectively, 2-△△Ct =0. 34, and the inhibitory rate of metastasis was 66% . The mRNA abundance of the HPRT was lower and there were fewer metastases in the lungs of 5-Aza-CdR-treated group than in control group. Conclusion 5-Aza-CdR reactivated tumor metastasis suppressor gene BRMS1 and decreased the metastasis to lungs of TNBC MDA-MB-231 cells by demethylation mechanism. Key words: 5-Aza-CdR; Methylation; Breast carcinoma; Metastasis

Suppression of murine breast cancer metastasis by selective inhibition of CXCR4 by synthetic polypeptide derived from viral macrophage inflammatory protein II

Stromal cell-derived factor-1 and its receptor CXC chemokine receptor-4 (CXCR4) have been implicated in breast cancer metastasis. A significant association between HER2 and CXCR4 expression has been observed in human breast tumor tissues, and overexpression of CXCR4 is essential for HER2-mediated tumor metastasis. Moreover, CXCR4 expression is low in normal breast tissues and high in malignant tumors, suggesting that a blockade of CXCR4 may limit tumor metastasis. The present study investigated the action of a synthetic antagonist 21-mer peptide derived from viral macrophage inflammatory protein II against CXCR4 (NT21MP) in inhibiting metastasis in vitro and in vivo. The results showed that chemotaxis of SKBR3 cells toward SDF-1α was reduced by NT21MP in a dose-dependent manner (P < 0.05). NT21MP inhibited tumor growth at 500 μg/kg and in combination with Herceptin, the anti-HER2 antibody. The in vivo metastatic assay showed that NT21MP significantly inhibited pulmonary metastasis, and the number of metastatic tumor nodes on the surface of the lung was greatly decreased. Compared with the saline-treated control group, PCNA expression was dose-dependently decreased by NT21MP, the percentage of apoptotic cells was increased, and CXCR4 mRNA and protein expression were downregulated. In conclusion, NT21MP inhibits cellular proliferation, promotes apoptosis by downregulating CXCR4 expression, and suppresses the progression of primary and metastatic tumors. CXCR4 may be a useful therapeutic target for breast cancer, and NT21MP may serve as a potential target drug for the treatment of breast cancer metastasis.

Transforming growth factor-β1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells

To investigate whether rhTGF-beta1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-beta receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells. MCF-7 breast cancer cells were treated with rhTGF-beta1 or transfected with a recombinant vector, pIRES2-EGFP-TbetaRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1alpha. CXCR4 mRNA and protein expression were upregulated in TGF-beta1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1alpha. In MCF-7 cells transiently transfected with pIRES2-EGFP-TbetaRII-Fc, a fusion protein named TbetaRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1alpha-mediated chemotactic response. TGF-beta1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1alpha-induced chemotactic response. The results suggest a link between TGF-beta1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-beta1-mediated enhancement of metastatic potential in breast cancer cells.

Tonic Activation of CXC Chemokine Receptor 4 in Immature Granule Cells Supports Neurogenesis in the Adult Dentate Gyrus

Stromal-cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) play a well-established role during embryonic development of dentate gyrus granule cells. However, little is known about the regulation and function of CXCR4 in the postnatal dentate gyrus. Here, we identify a striking mismatch between intense CXCR4 mRNA and limited CXCR4 protein expression in adult rat subgranular layer (SGL) neurons. We demonstrate that CXCR4 protein expression in SGL neurons is progressively lost during postnatal day 15 (P15) to P21. This loss of CXCR4 protein expression was paralleled by a reduction in the number of SDF-1-responsive SGL neurons and a massive upregulation of SDF-1 mRNA in granule cells. Intraventricular infusion of the CXCR4-antagonist AMD3100 dramatically increased CXCR4 protein expression in SGL neurons, suggesting that CXCR4 is tonically activated and downregulated by endogenous SDF-1. Infusion of AMD3100 also facilitated detection of CXCR4 protein in bromodeoxyuridine-, nestin-, and doublecortin-labeled cells and showed that the vast majority of adult-born granule cells transiently expressed CXCR4. Chronic AMD3100 administration impaired formation of new granule cells as well as neurogenesis-dependent long-term recognition of novel objects. Therefore, our findings suggest that tonic activation of CXCR4 in newly formed granule cells by endogenous SDF-1 is essential for neurogenesis-dependent long-term memory in the adult hippocampus.

Epigenetic Up-regulation of C-C Chemokine Receptor 7 and C-X-C Chemokine Receptor 4 Expression in Melanoma Cells

Histone deacetylation and DNA methylation establish epigenetic modifications, which through chromatin remodeling may result in gene silencing. We hypothesized that chemokine receptors C-C chemokine receptor 7 (CCR7) and C-X-C chemokine receptor 4 (CXCR4) on melanoma cells undergo epigenetic regulation. We investigated whether a histone deacetylase inhibitor and a demethylating agent influence CCR7 and CXCR4 expression on melanoma cells. Initially, microarray analysis was done to screen changes in chemokine receptor expression on melanoma cells after treatment with trichostatin A (TSA) and 5-Aza-2-deoxycytidine (5-Aza). CCR7 and CXCR4 mRNA expression were uniformly altered and selected for further investigation. Quantitative real-time reverse transcription-PCR assay, immunohistochemistry, and Western blot analysis were used to assess changes in mRNA and protein expression induced by TSA and 5-Aza in melanoma lines. Cell migration assays were conducted to assess the effects of altered CCR7 and CXCR4 expression on cell function. Treatment with TSA or 5-Aza increased gene expression of both CCR7 and CXCR4 in melanoma lines. TSA was the strongest enhancer. With combined treatment, CCR7 and CXCR4 mRNA expression was also up-regulated. Immunohistochemistry after combined treatment showed enhanced staining of both CCR7 and CXCR4 compared with control cells. Melanoma cell migration in TSA- and 5-Aza-treated cells was 7- and 2-fold higher than control cells for CCR7 and CXCR4, respectively. In summary, a histone deacetylase inhibitor and a demethylating agent up-regulated CCR7 and CXCR4 expression on melanoma cells. This increase in chemokine receptor expression correlated with functional activity. Most importantly, we have identified an epigenetic mechanism that may endogenously regulate chemokine receptor expression on melanoma cells.

Redox regulation of chemokine receptor expression.

Cytokines and reactive oxygen intermediates (ROI) are frequent companions at sites of acute inflammation. We have shown previously that in human monocytes, bacterial lipopolysaccharide, IL-1, and tumor necrosis factor-alpha induce a rapid down-regulation of the monocyte chemotactic protein-1 receptor CCR2 (CC chemokine receptor-2). These stimuli also induce production of ROI. In this paper, we investigate the influence of antioxidants and/or ROI on chemokine-receptor expression. In human monocytes, the antioxidant pyrrolidine dithiocarbamate (PDTC) rapidly inhibited CCR2 (95-100% of inhibition) and CCR5 (77-100% of inhibition) mRNA expression by strongly decreasing transcript stability. CCR2 half-life was decreased from 1.5 h to 45 min; CCR5 half-life was decreased from 2 h to 70 min. This inhibitory activity also included CXCR4 (CXC chemokine receptor-4) but not CXCR2 receptor and, although to a lesser extent, was shared by the antioxidants N-acetyl-l-cysteine and 2-mercaptoethanol. In contrast, the ROI-generating system xanthine/xanthine oxidase increased CCR5 and CXCR4 mRNA expression and counteracted the inhibitory effect of PDTC. Accordingly, H(2)O(2) and the glutathione-depleting drug buthionine sulfoximine increased to different extents CCR2, CCR5, and CXCR4 mRNA expression. The PDTC-mediated inhibition of CCR5 and CXCR4 mRNA expression was associated with decreased chemotactic responsiveness (>90% inhibition) and with a marked inhibition of surface-receptor expression. In contrast, xanthine/xanthine oxidase opposed the bacterial lipopolysaccharide- and tumor necrosis factor-alpha-mediated inhibition of CCR5 and CXCR4 mRNA expression and increased both the CCR5 surface expression and the cell migration (3-fold) in response to macrophage inflammatory protein-1beta. These results suggest that the redox status of cells is a crucial determinant in the regulation of the chemokine system.

CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10.

We report that interleukin (IL)-4 and IL-10 can significantly up- or down-regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell-derived factor-1alpha (SDF-1alpha)-induced CD4+ T-lymphocyte chemotaxis was also correspondingly regulated by IL-4 and IL-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70,000 SDF-1alpha-binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 approximately 4.4 nM and Kd2 approximately 14.6 nM), and a total of approximately 130,000 SDF-1alpha-binding sites per cell, among IL-4-stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium-mobilization stimulation. These results indicate that the effects of IL-4 and IL-10 on the CXCR4-SDF-1 receptor-ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection.