Retracted: Role of CXCL12–CXCR4 axis in ovarian cancer metastasis and CXCL12–CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro

Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12–CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis. OC (nonmetastatic and metastatic) and normal ovarian tissues were collected to determine the expressions of CXCL12 and CXCR4. A series of AMD3100, shRNA against CXCR4, and pCNS-CXCR4 were introduced to treat CAOV3 cells with the highest CXCR4 was assessed. Cell viability, apoptosis, migration, and invasion were all evaluated. The microarray analysis screened out the differential expression of CXCL12–CXCR4 in OC. CXCL12 and CXCR4 expressions were increased in OC tissues, particularly in the metastatic OC tissues. Downregulation of CXCR4 by AMD3100 or shRNA was observed to have a critical role in inhibiting cell proliferation, migration, and invasion of the CAOV3 OC cell line while promoting cell apoptosis. Overexpressed CXCR4 brought significantly promoting effects on the proliferation and invasiveness of OC cells. These results reinforce that the blockade of CXCL12–CXCR4 axis with AMD3100 inhibits the growth of OC cells. The antitumor role of the inhibition of CXCL12–CXCR4 axis offers a preclinical validation of CXCL12–CXCR4 axis as a therapeutic target in OC.     

The chemokine SDF-1/CXCL12 regulates the migration of melanocyte progenitors in mouse hair follicles

Mouse skin melanocytes originate from the neural crest and subsequently invade the epidermis and migrate into the hair follicles (HF) where they proliferate and differentiate. Here we demonstrate a role for the chemokine SDF-1/CXCL12 and its receptor CXCR4 in regulating the migration and positioning of melanoblasts during HF formation and cycling. CXCR4 expression by melanoblasts was upregulated during the anagen phase of the HF cycle. CXCR4-expressing cells in the HF also expressed the stem cell markers nestin and LEX, the neural crest marker SOX10 and the cell proliferation marker PCNA. SDF-1 was widely expressed along the path taken by migrating CXCR4-expressing cells in the outer root sheath (ORS), suggesting that SDF-1-mediated signaling might be required for the migration of CXCR4 cells. Skin sections from CXCR4-deficient mice, and skin explants treated with the CXCR4 antagonist AMD3100, contained melanoblasts abnormally concentrated in the epidermis, consistent with a defect in their migration. SDF-1 acted as a chemoattractant for FACS-sorted cells isolated from the anagen skin of CXCR4–EGFP transgenic mice in vitro, and AMD3100 inhibited the SDF-1-induced migratory response. Together, these data demonstrate an important role for SDF-1/CXCR4 signaling in directing the migration and positioning of melanoblasts in the HF.     

Imaging CXCL12-CXCR4 Signaling in Ovarian Cancer Therapy

Chemokine CXCL12 and receptor CXCR4 have emerged as promising therapeutic targets for ovarian cancer, a disease that continues to have a dismal prognosis. CXCL12-CXCR4 signaling drives proliferation, survival, and invasion of ovarian cancer cells, leading to tumor growth and metastasis. Pleiotropic effects of CXCR4 in multiple key steps in ovarian cancer suggest that blocking this pathway will improve outcomes for patients with this disease. To quantify CXCL12-CXCR4 signaling in cell-based assays and living mouse models of ovarian cancer, we developed a click beetle red luciferase complementation reporter that detects activation of CXCR4 based on recruitment of the cytosolic adapter protein β-arrestin 2. Both in two-dimensional and three-dimensional cell cultures, we established that bioluminescence from this reporter measures CXCL12-dependent activation of CXCR4 and inhibition of this pathway with AMD3100, a clinically-approved small molecule that blocks CXCL12-CXCR4 binding. We used this imaging system to quantify CXCL12-CXCR4 signaling in a mouse model of metastatic ovarian cancer and showed that treatment with AMD3100 interrupted this pathway in vivo. Combination therapy with AMD3100 and cisplatin significantly decreased tumor burden in mice, although differences in overall survival were not significantly greater than treatment with either agent as monotherapy. These studies establish a molecular imaging reporter system for analyzing CXCL12-CXCR4 signaling in ovarian cancer, which can be used to investigate biology and therapeutic targeting of this pathway in cell-based assays and living mice.     

A point mutation (G574A) in the chemokine receptor CXCR4 detected in human cancer cells enhances migration

The chemokine receptor CXCR4 is widely expressed in human cancers and regulates cell invasion, proliferation and survival. Because mutations in the CXCR4 gene could regulate its function we sequenced the coding region of the CXCR4 gene in 18 human melanoma and 3 human colon carcinoma cell lines. The same somatic point mutation (G574A; V160I) in the fourth trans-membrane region of CXCR4 was detected in one colon cancer cell line (PD) and one melanoma cell line (LB). CXCR4 was expressed and functional in both PD and LB cells, PD and LB cells migrated specifically toward the receptor ligand, CXCL12 and P-Erk was specifically induced by CXCL12. To give insight into the function of the mutant CXCR4 receptor, human A431, epidermoid carcinoma cells, were stably transfected with both mutant and wild type CXCR4. In vitro, A431 cells harboring CXCR4^G574A migrated specifically toward CXCL12 and CXCL12 induced ERK phosphorylation. Interestingly, in vivo studies showed that the growth of A431 tumors harboring CXCR4G574A was delayed compared to those harboring WT CXCR4. As expected, treatment with AMD3100, a specific CXCR4 inhibitor, reduced the in vivo growth of CXCR4_^G574A tumor b^G574A rprisingly, increased the growth of CXCR4^G574A A431 cells. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells. While the mutation impairs cell growth in vivo, the CXCR4 inhibitor, AMD3100, stimulated the growth of cells harboring CXCR4^G574A.     

AMD3100对apoE-/-小鼠骨髓源性内皮祖细胞增殖、迁移和黏附的影响

探讨AMD3100对apoE-/-小鼠骨髓内皮祖细胞的动员作用及其增殖、迁移和黏附的影响.12只8周龄雄性apoE-/-小鼠随机分为AMD3100组(2.5 mg/(kg·2d))和对照组(PBS 0.1 ml/2d),高脂高胆固醇饲料喂养12周后,差速贴壁法结合微孔法分离培养小鼠骨髓细胞,免疫荧光鉴定CD133/VEGFR-2双阳性细胞为内皮祖细胞;MTT比色法、Transwell、黏附试验分别检测细胞的增殖、迁移和黏附能力;通过计数典型内皮祖细胞克隆形成单位,观察次级集落单位的大小及细胞密度,检测各组内皮祖细胞的克隆形成能力;RT-PCR和Western blot检测内皮祖细胞上CXCR4 mRNA和蛋白质表达水平.与对照组比较,AMD3100组骨髓源性内皮祖细胞的增殖、迁移、黏附和克隆形成能力均显著低于对照组,其CXCR4mRNA和蛋白质表达均显著低于对照组.结果表明:持续注射AMD3100可抑制骨髓源内皮祖细胞的增殖、迁移、黏附和克隆形成能力,并下调CXCR4的表达.     

64Cu-AMD3100—A novel imaging agent for targeting chemokine receptor CXCR4

CXCR4 is a chemokine receptor which has been shown to be exploited by various tumors for increased survival, invasion, and homing to target organs. We developed a one step radiosynthesis for labeling the CXCR4-specific antagonist AMD3100 with Cu-64 to produce ⁶⁴Cu-AMD3100 with a specific activity of 11.28 Ci/μmol (417 GBq/μmol) at the end of radiosynthesis. Incorporation of Cu(II) ion into AMD3100 did not change its ability to inhibit cellular migration in response to the (only) CXCR4 ligand, SDF-1/CXCL12. ⁶⁴Cu-AMD3100 binding affinity to CXCR4 was found to be 62.7 μM. Biodistribution of ⁶⁴Cu-AMD3100 showed accumulation in CXCR4-expressing organs and tissues, a renal clearance pathway, and an anomalous specific accumulation in the liver. We conclude that ⁶⁴Cu-AMD3100 exhibits promise as a potential PET imaging agent for visualization of CXCR4-positive tumors and metastases and might be used to guide and monitor anti-CXCR4 tumor therapy.     

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4⁺ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.     

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In β-arrestin recruitment assays, ubiquitin did not sufficiently recruit β-arrestin2 to CXCR4 (EC₅₀ > 10 μM), whereas the EC₅₀ for CXCL12 was 4.6 nM (95% confidence interval 3.1–6.1 nM). Both agonists induced similar chemical shift changes in the ¹³C-¹H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the ¹³C-¹H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.     

CXCL12/CXCR4 Axis Improves Migration of Neuroblasts Along Corpus Callosum by Stimulating MMP-2 Secretion After Traumatic Brain Injury in Rats

To investigate the effect of CXCL12 on migration of neural precursor cells after traumatic brain injury (TBI). We randomly divided 48 rats into four groups: (1) the sham group, rats were performed craniotomy only, (2) the control group, saline were injected into the ipsilateral cortex after TBI, (3) the CXCL12 group, CXCL12 were injected into the ipsilateral cortex after TBI, and (4) the CXCL12 + AMD3100 group, CXCL12 and AMD3100 were mixed together and injected into the ipsilateral cortex after TBI. At 7 days after TBI, the brain tissues were subjected to immunofluorescent double-labeled staining with the antibodies of CXCR4/DCX, MMP-2/DCX, MMP-2/GFAP, MMP-2/NeuN. Western blot assay was used to measure the protein levels of MMP-2. Compared with the control group, the number of CXCR4/DCX and MMP-2 positive cells around the injured corpus callosum area were significantly increased in the CXCL12 treatment group. The area occupied by these cells expanded and the shape changed from chain distribution to radial. CXCL12 + AMD3100 treatment significantly decreased the number and distribution area of CXCR4/DCX and MMP-2 positive cells compared with the CXCL12 treatment and control group. The DCX positive cells could not form chain or radial distribution. The protein expressions of MMP-2 had the similar change trends as the results of immunofluorescent staining. MMP-2 could be secreted by DCX, GFAP and NeuN positive cells. CXCL12/CXCR4 axis can improve the migration of the neuroblasts along the corpus callosum by stimulating the MMP-2 secretion of different types of cells.     

NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.     

Insights into the mechanism of enhanced mobilization of hematopoietic progenitor cells and release of CXCL12 by a combination of AMD3100 and aminoglycoside-polyarginine conjugates

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow to the peripheral blood is utilized in clinical HSPC transplantation protocols. Retention of HSPCs in the bone marrow is determined by relationships between the chemokine chemokine (C-X-C motif) ligand 12 (CXCL12) and its major receptor C-X-C chemokine receptor type 4 (CXCR4), and disruption of this retention by CXCR4 antagonists such as AMD3100 induces rapid HSPC mobilization. Here, we report that aminoglycoside-polyarginine conjugates (APACs) and N-α-acetyl-nona-D-arginine (r9) induce mobilization of white blood cells and, preferentially, immature hematopoietic progenitor cells (HPCs) in mice, similarly to AMD3100. Remarkably, administration of AMD3100 with each one of the APACs or r9 caused additional HPC mobilization. The mobilizing activity of APACs and r9 was accompanied by a significant elevation in plasma CXCL12 levels. To further understand how APACs, r9 and their combinations with AMD3100 compete with CXCL12 binding to CXCR4, as well with antibody against CXCR4 for CXCR4 binding, we have undertaken an approach combining experimental validation and docking to determine plausible binding modes for these ligands. On the basis of our biological and docking findings, and recently published NMR data, we suggest that combination of pairs of compounds such as APACs (or r9) with AMD3100 induces more efficient disruption of the CXCL12-CXCR4 interaction than AMD3100 alone, resulting in enhanced HPC mobilization.     

Synthesis and evaluation of 2,5 and 2,6 pyridine-based CXCR4 inhibitors

Targeting the interaction between G-Protein Coupled Receptor, CXCR4, and its natural ligand CXCL12 is a leading strategy to mitigate cancer metastasis and reduce inflammation. Several pyridine-based compounds modeled after known small molecule CXCR4 antagonists, AMD3100 and WZ811, were synthesized. Nine hit compounds were identified. These compounds showed lower binding concentrations than AMD3100 (1000 nM) and six of the nine compounds had an effective concentration (EC) less than or equal to WZ811 (10 nM). Two of the hit compounds (2g and 2w) inhibited invasion of metastatic cells at a higher rate than AMD3100 (62%). Compounds 2g and 2w also inhibit inflammation in the same range as WZ811 in the paw edema test at 40% reduction in inflammation. These preliminary results are the promising foundation of a new class of pyridine-based CXCR4 antagonists.     

肠道病毒71型感染小鼠心肌损害与CXCL12/CXCR4通路激活的关系

重症手足口病患儿中心肌损害常见,肠道病毒71型(Enterovirus 71,EV71)是引起手足口病的主要病原体之一,EV71感染小鼠可以出现心肌炎的病理改变,但EV71引起心肌损害的机制尚不明确。为了阐明EV71引起心肌损害的机制,本实验观察了EV71感染小鼠心肌损害与CXC趋化因子配体12(CXC chemokine ligand 12,CXCL12)/CXC趋化因子受体4(CXC chemokine receptor 4,CXCR4)通路激活的关系。BALB/c乳鼠随机分为对照组、EV71组、EV71+AMD3100组、AMD3100组,对照组、AMD3100组给予生理盐水腹腔注射,EV71组、EV71+AMD3100组给予EV71病毒液腹腔注射;而后EV71+AMD3100组、AMD3100组给予CXCR4抑制剂AMD3100腹腔注射、连续7d。比较四组间血清中CXCL12、磷酸肌酸激酶同工酶(CreatineKinase-MB,CK-MB)、乳酸脱氢酶(Lactate dehydrogenase,LDH)含量、心肌病理改变及细胞凋亡率、心肌中CXCR4、含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase-3)表达及肿瘤坏死因子-α(Tumor necrosis factor,TNF-α)、白介素-6(Interleukin-6,IL-6)含量的差异。与对照组比较,EV71组血清中CXCL12、CK-MB、LDH的含量明显增加,心肌出现了典型的心肌炎病理改变且细胞凋亡率、CXCR4及caspase-3表达水平、TNF-α及IL-6含量明显增加;与EV71组比较,EV71+AMD3100组血清中CXCL12、CK-MB、LDH的含量明显降低,心肌的病理改变改善且细胞凋亡率、CXCR4及caspase-3表达水平、TNF-α及IL-6含量明显降低。以上结果表明EV71感染小鼠心肌中CXCL12/CXCR4通路过度激活,该通路的激活介导了心肌细胞凋亡及炎症反应。本研究创新点为阐明了CXCL12/CXCR4通路在EV71病毒感染引起心肌损害中的作用,CXCL12/CXCR4通路激活能够激活EV71感染小鼠心肌的炎症反应及细胞凋亡,这为今后研究手足口病发病过程中心肌损害的机制提供了依据。     

CXCL12 Induces Connective Tissue Growth Factor Expression in Human Lung Fibroblasts through the Rac1/ERK,JNK, and AP-1 Pathways

CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4⁺ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.     

Long-term high-fat diet induces pancreatic injuries via pancreatic microcirculatory disturbances and oxidative stress in rats with hyperlipidemia

Stromal cell-derived factor 1 (CXCL12) is an angiogenic chemokine that is believed to act solely via its cognate receptor CXCR4. Evidence is now provided for the existence of a different CXCL12 binding and signaling receptor on endothelial cells. Bovine aortic endothelial cells (BAECs) strongly expressed CXCR4 and exhibited high binding capacity for fluorescently labeled CXCL12. However, CXCL12 binding was not correlated with the CXCR4 expression level and was virtually unaffected by the specific CXCR4 antagonists AMD3100 or T22. Similar observations were made in endothelial cells of mouse and human origin. Also, AMD3100 failed to block CXCL12 internalization and CXCL12-induced intracellular signal transduction via extracellular signal-regulated kinases 1/2 in BAECs. In contrast, CXCL12 binding and signaling were almost completely inhibited by the CXCR4 antagonist in T-lymphoid SupT1 cells. Together, our data point to the existence of an additional receptor through which CXCL12 exerts its biological effects in endothelial cells.     

The peptidomimetic CXCR4 antagonist TC14012 recruits beta-arrestin to CXCR7: roles of receptor domains

CXCR7 is an atypical chemokine receptor that signals through β-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of β-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC(50) 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 μM for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7.     

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell “dormancy.” Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from “sanctuary” niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda“protective niche” for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.     

Inhibition of the CXCL12/CXCR4-Axis as Preventive Therapy for Radiation-Induced Pulmonary Fibrosis

Background

A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process.

Methodology/Principal Findings

The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process.

Conclusions/Significance

CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.     

AMD3100 inhibits epithelial–mesenchymal transition,cell invasion,and metastasis in the liver and the lung through blocking the SDF-1α/CXCR4 signaling pathway in prostate cancer

Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have been found to be tightly correlated with the progression of prostate cancer (PC). In this study, we investigated the effects of an SDF-1α/CXCR4 inhibitor, AMD3100, on cell progression and metastasis potential of human PC cells. Human PC cell lines (LNCaP, PC3, and DU145) were cultured to detect SDF-1α/CXCR4, which showed higher SDF-1α and CXCR4 expression than the normal human prostate epithelial cell line, RWPE-1. AMD3100 was confirmed to be an inhibitor of SDF-1α, and to detect the effect of SDF-1α/CXCR4 inhibition on PC, PC cells were treated with AMD3100 or/and CXCR4 siRNA. The results suggested that inhibition of the SDF-1α/CXCR4 pathway could promote the E-cadherin level but inhibit the levels of invasion and migration of vimentin, N-cadherin and α5β1 integrin. Finally, tumor formation in nude mice was conducted, and the cell experiment results were verfied. These data show that AMD3100 suppresses epithelial–mesenchymal transition and migration of PC cells by inhibiting the SDF-1α/CXCR4 signaling pathway, which provides a clinical target in the treatment of PC.     

Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice

Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl₄)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl₄-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis.