C-C Motif) Ligand 2 Expression Research Articles

Abstract 2072: Ormeloxifene attenuates wnt/β-catenin signaling in colon cancer cells by modulation of PKD1 and glycolytic pathways

Abstract Background: Colorectal cancer is the third most commonly diagnosed cancer and third leading cause of cancer-related deaths. Aberrant Wnt/β-catenin signaling is implicated in development and progression of colon cancers. Therefore, modulation of this master regulatory signaling is essential for the prevention and treatment of colon cancer. Protein Kinase D1 (PKD1) which is downregulated in colon cancer, regulates β-catenin transcriptional activity by its phosphorylation. Therefore, drug mediated upregulation of PKD1 may have high clinical significance for the prevention/treatment of Wnt/β-catenin signaling induced cancers. We have identified, ormeloxifene, a synthetic non-steroidal selective estrogen receptor modulator (SERM), as a novel PKD1 modulator. Herein, we have investigated the effect of ormeloxifene on tumorigenic phenotypes of colon cancer cells and its effect on PKD1 and glycolytic pathways mediated oncogenic β-catenin signaling. Materials and Methods: We have used SW480-PKD1-GFP and SW480-GFP colon cancer cells (SW480 has a mutated APC gene leading to stabilization of β-catenin due to lack of degradation) to investigate the effect of PKD1 on β-catenin activity and xenograft mouse model. SW480, SW48, SW620 and Lovo cell lines have been used to observe the effect of ormeloxifene (5-20 μM) on various cellular characteristics, including cell proliferation, clonogenic potential, motility and aggregation. β-catenin/T-cell factor 4 (TCF4) transcription activity was measured by Dual-Glo luciferase reporter assays. The expression and subcellular localization of proteins was determined by immunoblotting and confocal microscopy. Chemokine (C-C) motif ligand 2 (CCL2) and glucose assay were performed using ELISA technique. Results: Ormeloxifene treatment restores PKD1 expression, thereby, attenuates β-catenin transcriptional activity. We have also observed that ormeloxifene treatment effectively inhibits CCL2 expression, glucose uptake, glucose metabolism and Glut1 receptor expression in cancer cells. Ormeloxifene (5-20μM) inhibits cell proliferation and motility of colon cancer cells. Additionally, ormeloxifene effectively inhibited CCL2 mediated cellular migration of colon cancer cells. Furthermore, Western blot results depict that PKD1 inhibits TCF4 activity in colon cancer cells. SW480 PKD1 overexpressing cells clearly delayed tumor formation in nude mice, compared to control cells. In addition, SW480-PKD1-GFP cells showed less hypoxia (Glut1) and increased Vasculature (CD31) in tumors. Conclusion: Our findings demonstrate that ormeloxifene treatment effectively attenuates β-catenin mediated oncogenic signaling and tumorigenic phenotypes of colon cancer cells via upregulation of PKD1 and suppression of glycolytic pathways. Thus it can be used as a potential new preventive/treatment modality for colon cancer. Citation Format: Aditya Ganju, Rishi Gara, Sonam Kumari, Man Mohan Singh, Subhash Chauhan, Meena Jaggi. Ormeloxifene attenuates wnt/β-catenin signaling in colon cancer cells by modulation of PKD1 and glycolytic pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2072. doi:10.1158/1538-7445.AM2015-2072

Local production of the chemokines CCL5 and CXCL10 attracts CD8+ T lymphocytes into esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a very common malignant tumor with poor prognosis in China. Chemokines secreted by tumors are pivotal for the accumulation of CD8(+) T lymphocytes within malignant lesions in several types of cancers, but the exact mechanism underlying CD8(+) T lymphocyte homing is still unknown in ESCC. In this study, we revealed that, compared with marginal tissues, the expression of both chemokine (C-C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) was upregulated in ESCC tissues. CCL5 expression was positively associated with the overall survival of patients. Meanwhile, RT-PCR data showed that the expression of CCL5 and CXCL10 was positively correlated with the local expressions of the CD8(+) T lymphocyte markers (CD8 and Granzyme B) in tumor tissues. Correspondingly, CD8(+) T lymphocytes were more frequently CCR5- and CXCR3-positive in tumor than in peripheral blood. Transwell analysis showed both CCL5 and CXCL10 were important for the chemotactic movement of CD8(+) T lymphocytes. Our data indicate that CCL5 and CXCL10 serve as the key chemokines to recruit CD8(+) T lymphocytes into ESCC tissue and may play a role in patient survival.

Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage infiltration into the human corpus luteum during luteolysis.

Intense macrophage infiltration is observed during luteolysis in various animals including women; however, we still do not know how macrophage infiltration into the human corpus luteum (CL) during luteolysis is regulated. In this study, we examined the expression, localization and regulation of an important chemokine for the recruitment of monocyte/macrophage lineages, C-C motif ligand 2 (CCL2), in the human CL across the luteal phase and in cultured human luteinized granulosa cells (LGCs), with special reference to the number of infiltrating macrophages and luteal cell function. CCL2 mRNA increased in the non-functional regressing CL during menstruation (P < 0.01), corresponding to an elevated mRNA expression of a macrophage-derived cytokine, tumor necrosis factor (TNF), and an increased number of infiltrating macrophages positively stained with a macrophage marker, CD68. CCL2 protein was immunohistochemically localized to the cytoplasm of granulosa-lutein and theca-lutein cells, and CCL2 mRNA was significantly reduced by hCG both in vivo (P < 0.05) and in vitro (P < 0.01). CCL2 was also down-regulated by luteotrophic prostaglandin (PG) E (P < 0.0001), but up-regulated by luteolytic PGF (P < 0.05) in vitro. Administration of TNF significantly enhanced the CCL2 mRNA expression in cultured LGCs (P < 0.01). A greater abundance of infiltrating macrophages were found around granulosa-lutein cells lacking 3β-HSD or PGE synthase (PGES) immunostaining. CCL2 mRNA expression was negatively correlated with both HSD3B1 and PGES, suggesting that locally produced progesterone and PGE suppress macrophage infiltration into the CL. Taken together, the infiltration of macrophages in the human CL is regulated by endocrine and paracrine molecules via regulation of the CCL2 expression in luteal cells.

TLR4 activation by lipopolysaccharide confers survival advantage to growth factor deprived prostate cancer cells.

Prostate cancer (PCa) cells express Toll-like receptor-4 (TLR4), a known pro-tumorigenic molecule for different cancer cells. The cancer cells residing in the avascular region of the tumor confront various metabolic stresses and continuously adapt mechanisms to overcome them. We hypothesized that TLR4 activation might provide direct survival advantage to metabolically stressed PCa cells. We first investigated the effect of LPS on survival of serum deprived PCa cells. To understand the molecular mechanisms involved in TLR4 mediated PCa survival, we next investigated change in expression of markers for apoptosis, senescence and autophagy. Ultimately, the effect of LPS on established prostate tumors was confirmed in vivo using a syngeneic rat model for PCa. Lipopolysaccharide (LPS)-mediated TLR4 activation significantly enhanced survival of serum deprived (SD) PC3, DU145 and MAT-LyLu PCa cells. TLR4 inhibition by a specific inhibitor resulted in rapid death of SD-PC3 cells, which was significantly suppressed by LPS. Interestingly, LPS treatment suppressed macroautophagy in SD-PC3 cells and increased expression of CCL2 (C-C motif ligand-2), a known autophagy inhibitor and pro-survival factor. Intra-tumor LPS injection resulted in increased tumor mass, induced TLR4 activation, suppressed autophagy, and increased the macrophage population in MAT-LyLu-tumors. Our study reveals that bacterial LPS enhance survival of PCa cells under conditions of nutrient stress through TLR4 activation. Moreover, LPS induces overexpression of CCL2 involved in the suppression of starvation-induced macroautophagy in PCa cells, and enhanced macrophage population in prostate tumors in vivo. Taken together, the current study suggests the importance of bacterial infection or TLR4-activation in prostate cancer pathogenesis.

Carcinoembryonic Antigen Cell Adhesion Molecule 1 long isoform modulates malignancy of poorly differentiated colon cancer cells

ObjectiveNearly 20%–29% of patients with colorectal cancer (CRC) succumb to liver or lung metastasis and there is a dire need for novel targets to improve the survival of patients with...

Spinal NF-κB and chemokine ligand 5 expression during spinal glial cell activation in a neuropathic pain model.

BackgroundThe NF-κB pathway and chemokine (C-C motif) ligand 5 (CCL5) are involved in pain modulation; however, the precise mechanisms of their interactions in chronic neuropathic pain have yet to be established.MethodsThe present study examined the roles of spinal NF-κB and CCL5 in a neuropathic pain model after chronic constriction injury (CCI) surgery. CCI-induced pain facilitation was evaluated using the Plantar and von Frey tests. The changes in NF-κB and CCL5 expression were analyzed by immunohistochemistry and Western blot analyses.ResultsSpinal NF-κB and CCL5 expression increased after CCI surgery. Repeated intrathecal infusions of pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) decreased CCL5 expression, inhibited the activation of microglia and astrocytes, and attenuated CCI-induced allodynia and hyperalgesia. Intrathecal injection of a CCL5-neutralizing antibody attenuated CCI-induced pain facilitation and also suppressed spinal glial cell activation after CCI surgery. However, the CCL5-neutralizing antibody did not affect NF-κB expression. Furthermore, selective glial inhibitors, minocycline and fluorocitrate, attenuated the hyperalgesia induced by intrathecal CCL5.ConclusionsThe inhibition of spinal CCL5 expression may provide a new method to prevent and treat nerve injury-induced neuropathic pain.

Monocyte-Induced Prostate Cancer Cell Invasion is Mediated by Chemokine ligand 2 and Nuclear Factor-κB Activity.

Study BackgroundThe tumor microenvironment contains inflammatory cells which can influence cancer growth and progression; however the mediators of these effects vary with different cancer types. The mechanisms by which prostate cancer cells communicate with monocytes to promote cancer progression are incompletely understood. This study tested prostate cancer cell and monocyte interactions that lead to increased prostate cancer cell invasion.MethodsWe analyzed the prostate cancer cell invasion and NF-κB activity and cytokine expression during interaction with monocyte-lineage cells in co-cultures. The roles of monocyte chemotactic factor (MCP-1/CCL2) and NF-κB activity for co-culture induced prostate cancer invasion were tested. Clinical prostate cancer NF-κB expression was analyzed by immunohistochemistry.ResultsIn co-cultures of prostate cancer cell lines with monocyte-lineage cells, (C-C motif) ligand 2 (CCL2) levels were significantly increased when compared with monocytes or cancer cells cultured alone. Prostate cancer cell invasion was induced by recombinant CCL2 in a dose dependent manner, similar to co-cultures with monocytes. The monocyte-induced prostate cancer cell invasion was inhibited by CCL2 neutralizing antibodies and by the CCR2 inhibitor, RS102895. Prostate cancer cell invasion and CCL2 expression induced in the co-cultures was inhibited by Lactacystin and Bay11-7082 NF-κB inhibitors. Prostate cancer cell NF-κB DNA binding activity depended on CCL2 dose and was inhibited by CCL2 neutralizing antibodies. Clinical prostate cancer NF-κB expression correlated with tumor grade.ConclusionsCo-cultures with monocyte-lineage cell lines stimulated increased prostate cancer cell invasion through increased CCL2 expression and increased prostate cancer cell NF-κB activity. CCL2 and NF-κB may be useful therapeutic targets to interfere with inflammation-induced prostate cancer invasion.

The role of chemokine (C-C motif) ligand 2 in morphine tolerance to analgesia in spinal cord of rats

Objective To investigate the role of spinal chemokine (C-C motif) ligand 2 (CCL2) in morphine tolerance to analgesia.Methods Male Sprague-Dawley (SD)rats with successful intrathecal catheter were randomly divided into ten groups by means of random number table (n=6):IgG plus morphine group (IgG+MOR),CCL2 neutralizine antibody plus morphine group (CCL2 neutralizine antibody +MOR),IgG plus saline group (IgG +NS),CCL2 neutralizine antibody plus saline group (CCL2 neutralizine antibody +NS),IgG4 plus morphine group (IgG4 +MOR),CCL2 neutralizine antibody4 plus morphine group (CCL2 neutralizine antibody4+MOR),IgG4 plus saline group (IgG4+NS),IgG8 plus morphine group (IgG8+MOR),CCL2 neutralizine antibody8 plus morphine group (CCL2 neutralizine antibody8+MOR),IgG8 plus saline group (IgG8+NS).A rat model of morphine tolerance to analgesia was induced by intrathecal injection of morphine 15 μg once daily for 7 consecutive days.The role of CCL2 in morphine antinociceptive tolerance is explored by tail flick latency (TFL) and mechanical withdrawal threshold (MWT).ELISA was used to evaluate the change in spinal CCL2 immunoreactivity.Results On days 3,5 and 7 after chronic morphine,percent of maximal possible potential effect by TFL (％MPETFL) and percent of maximal possible potential effect by MWT (％MPEMT) were significantly increased in the CCL2 neutralizing antibody plus morphine group [％MPETFL:3 d (65±6)％,5 d (47±4)％,7 d (42± 4)％.％MPEMWT:3 d (65±5)％,5 d (46±4)％,7 d (41±4)％] versus the IgG plus morphine group [％ MPETFL:3 d (55±6)％,5 d (27±4)％,7d (17±3)％.％MPEMT:3d (54±6)％,5d (27±4)％,7d (17±4)％](P＜0.05,P＜0.01),％MPEFL and ％MPEMWT were significantly up-regulated in the CCL2 neutralizine antibody4 plus morphine group [％MPETFL:5 d (46±4)％,7 d (43±4)％,％ MPEMWT:5 d (45±4)％,7 d (44±4)％]versus the IgG4 plus morphine group [％MPETFL:5 d (27±4)％,7 d (17±3)％,％MPEMT:5 d (27±4)％,7 d (17±3)％](P＜0.01).On days 9 and 11 after chronic morphine,％MPETFL and ％MPEMT were significantly up regulated in the CCL2 neutralizine antibody 8 plus morphine group [％MPETFL:9 d (26±4)％,11 d (36±4)％,％MPEMWT:9 d (26± 4)％,11 d (35±4)％] versus the IgG8 plus morphine group [％ MPETFL:9 d (16±3)％,1 1 d (15±4)％,％MPEMWT:9 d (16±3)％,11 d (14±3)％] (P＜0.05,P＜0.01).On day 7 after chronic morphine,CCL2 expression in the IgG plus morphine group compared with the IgG plus saline group was significantly up-regulated,while CCL2 expression in the CCL2 neutralizine antibody plus morphine group compared with IgG plus MOR group was significantly down-regulated.Conclusions CCL2 in the spinal cord is involved in the development and maintenance of morphine tolerance to analgesia.Inhibition of CCL2 may provide a new therapy for morphine tolerance to analgesia. Key words: Morphine antinociceptive tolerance; Hemokine (C-Cmotif) ligand 2; Spinal cord

Understanding the role of circulating chemokine (C-C motif) ligand 2 in patients with chronic ischemia threatening the lower extremities

The role of chemokine (C-C motif) ligand 2 (CCL2) in peripheral artery disease is unclear. We measured the difference between serum and plasma levels of CCL2 in patients with chronic ischemia threatening the lower extremities following the observation that atypical chemokine receptors in blood and tissue cells may prevent CCL2 from entering the circulation and consequently modulate its function of attracting monocytes to the site of lesion. To identify the influence of CCL2, we compared the patients' values to those in bio-banked samples from a control population. Further, we explored the association with the Asp42Gly polymorphism (rs12075) in Duffy antigen chemokine receptor; one of these atypical chemokine receptors. When possible, we evaluated in surgically excised normal and affected arteries the calcium burden as well as the expression of CCL2 and related receptors reflecting the inflammatory status. Our findings indicate that circulating CCL2 was significantly associated with the severity and presence of the disease (OR 0.966, 95% CI 0.944 to 0.988, p = 0.003). Circulating CCL2 was dependent on the rs12075 genotype (AA>AG>GG), which, probably, indicates a higher expression of chemokine receptor in the arteries of AA subjects. The associations with genetic variants and the over-expression of atypical chemokine receptors in diseased arteries may have potential implications and our data indicate that CCL2 may represent a previously unrecognized factor that needs to be considered in the screening of patients with risk factors for peripheral artery disease.

Chronic pelvic allodynia is mediated by CCL2 through mast cells in an experimental autoimmune cystitis model.

The cause of chronic pelvic pain in interstitial cystitis/painful bladder syndrome (IC/PBS) remains unclear; autoimmunity is a possible etiology. We have recently shown that injection of a single immunogenic peptide of uroplakin 3A (UPK3A 65-84) induces experimental autoimmune cystitis (EAC) in female BALB/cJ mice that is unique among experimental models in accurately reflecting both the urinary symptoms and pelvic pain of IC/PBS. The aim of this project was to identify the roles of mast cells and mast cell chemoattractant/activator monocyte chemoattractant protein-1 [chemokine (C-C motif) ligand 2 (CCL2)] in the allodynia in this model. We immunized 6- to 8-wk-old female BALB/cJ mice with UPK3A 65-84 peptide and, 5-40 days later, observed increased responses to stimulation of the suprapubic abdominal and hindpaw surfaces with von Frey monofilaments compared with mice injected with adjuvant alone. Suprapubic and hindpaw tactile allodynia responses by EAC mice were blocked by instillation of lidocaine into the bladder but not by lidocaine in the uterus, confirming the bladder as the source of the hypersensitivity. Markedly increased numbers of activated mast cells and expression of CCL2 were found in the bladder after immunization with UPK3A 65-84. Hypersensitive responses were inhibited by mast cell stabilizer cromolyn sodium and antagonists of histamine receptors 1 and 2. Furthermore, BALB/cJ mice with deletion of the Ccl2 or chemokine (C-C motif) receptor 2 gene exhibited markedly reduced allodynia and accumulation of mast cells after UPK3A 65-84 immunization. These results show that UPK3A 65-84 immunization causes chronic visceral allodynia and suggest that it is mediated by CCL2-driven mast cell accumulation in the bladder.

Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress

Objective To observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress.Methods The third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier.The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10％ normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro.hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group.Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours.Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment.TER was measured to evaluate the barrier function of hRPE.The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2),together with complement bioactive fragments (C3a,C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay.Results Stable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert.Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51％ compared with normal hRPE barrier.CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48％ compared with normal hRPE barrier(t =21.60,P＜0.05).Compared with model group,CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48％ and 23.47 ％ secreted by hRPE under complement-activated oxidative stress (t =3.26,2.43; P＜0.05).Compared with model group,CR1 treatment could also decreased the concentration of C3a,C5a and MAC by 24.00 ％,27.87 ％,22.44 ％.The difference were statistically significant (t =9.86,2.63,6.94 ; P＜0.05).Conclusions CR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress.The underlying mechanism may involve inhibiting complement activation and downregulating the expression of VEGF and CCL2. Key words: Retinal pigment epithelium; Receptors, complement 3b/antagonists & inhibitors; Complement activation; Animal experimentation

Selective inhibition of sphingosine kinase-1 protects adipose tissue against LPS-induced inflammatory response in Zucker diabetic fatty rats.

Obesity is associated with a state of chronic inflammation. The chemokine (C-C motif) ligand 5 (CCL5) has been proposed to modulate the inflammatory response in adipose tissue (AT). However, the mechanisms underlying CCL5 upregulation in AT remain undefined. The objective of the present study was to evaluate whether the enzyme sphingosine kinase-1 (SK1) would modulate the expression of CCL5 and other inflammatory biomarkers in primary adipocytes and its potential role in lipopolysaccharide (LPS)-induced AT inflammation in a rat model of diabetes. To address this, LPS-stimulated primary adipocytes and 3T3-L1 cells were treated with a SK inhibitor, and the expression of Ccl5 and other CC chemokines were studied. Moreover, the effect of SK1 knockdown on cytokine production was analyzed in 3T3-L1 cells by transfection of SK1-specific small-interfering RNA (siRNA). The anti-inflammatory effects of SK inhibitor in AT were also investigated in vivo using the Zucker lean normoglycemic control (ZLC) rats. LPS treatment stimulated Ccl5, IL-6, pentraxin 3 (Ptx3), and Tnfα mRNA expression in primary adipocytes and 3T3-L1 cells, whereas pharmacologically and siRNA-mediated SK1 inhibition strongly reduced mRNA levels of proinflammatory cytokines in these cells. Similarly, administration of SK inhibitor to ZLC rats prevented the LPS-induced inflammatory response in AT. Our data demonstrate a role for SK1 in endotoxin-induced cytokine expression in adipocytes and suggest that inhibition of SK1 may be a potential therapeutic tool in the prevention and treatment of chronic and common metabolic disorders, including obesity, insulin-resistance, and type 2 diabetes.

Association Between Ligands and Receptors Related to the Progression of Early Breast Cancer in Tumor Epithelial and Stromal Cells

BackgroundDespite advances in the study of breast cancer (BC), it remains the second leading cause of mortality among women. BC is a heterogeneous system, mainly composed of tumor epithelial cells (TEpCs) and stromal cells (SCs); the interaction through the ligands and their receptors (Rs) plays a major role in BC progression. The aim of the present study was to evaluate the association between ligands, such as osteoprotegerin, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand (RANKL), stromal cell–derived factor (SDF)-1, interleukin (IL)-6, macrophage colony stimulating factor, chemokine (C-C motif) ligand-2 (CCL-2), and their Rs in TEpC and spindle-shaped SCs not closely associated with the vasculature. Patients and MethodsWe studied the expression of all those factors in 63 primary tumors of untreated patients with BC with infiltrative ductal carcinoma (I/II stage) and 10 non-neoplastic tissues. The percentage of positive cells and the staining intensity were analyzed by immunohistochemistry. Mann–Whitney test and Spearman's rank correlation coefficient were used (P ≤ .05). ResultsWe found a significant association between the expression of RANKL, IL-6, SDF-1, and CCL-2 in TEpC and the receptor activator of nuclear factor kappa B (RANK), IL-6R, C-X-C chemokine R type 4, and chemokine (C-C motif) R-2 (CCR-2) in spindle-shaped SC. The expression of TRAIL, RANKL, and CCL-2 in spindle-shaped SC also was associated with the expression of TRAIL-receptor 1, TRAIL-receptor 4, RANK, and CCR-2 in TEpC. ConclusionsBecause the described ligands and Rs are implicated in BC progression, our results suggest that these factors could be involved in the crosstalk between TEpC and SC in the early stages of BC.

Induction of chemokine (C-C motif) ligand 2 by sphingosine-1-phosphate signaling in neuroblastoma

Background/purposeNeuroblastoma (NB) is the most common extracranial solid tumor of childhood. Preliminary data derived from a human angiogenesis array in NB showed that the bioactive lipid sphingosine-1-phosphate (S1P) induced the secretion of several angiogenesis-related proteins including the important inflammatory factor chemokine (C-C motif) ligand 2 (CCL2). In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB. MethodsQuantitative real-time PCR and CCL2 ELISA were conducted to detect the mRNA expression and protein secretion of CCL2 in NB cells. Gain and loss of function studies were performed by using specific S1PR antagonists, adenoviral transduction and siRNA transfection. Macrophage F4/80 receptor in NB xenografts was detected by quantitative real-time PCR and immunohistochemistry staining. ResultsS1P induced CCL2 mRNA expression and protein secretion in a time- and concentration-dependent manner in NB cells. Blockade of S1P2 signaling using the selective S1P2 antagonist JTE-013 inhibited S1P-induced CCL2 expression. Overexpression of S1P2 by adenoviral transduction increased CCL2 secretion while knockdown of S1P2 by siRNA transfection decreased S1P-induced CCL2 secretion in NB cells. Macrophage infiltration, as detected by F4/80 staining, was significantly decreased in JTE-013-treated NB xenografts. ConclusionsTaken together, our data for the first time demonstrate that S1P induced the macrophage-recruiting factor CCL2 expression in NB cells via S1P2, providing new insights into the complicated functions of S1P2 in cancer.

Gene expression in subcutaneous adipose tissue differs in women with polycystic ovary syndrome and controls matched pair-wise for age, body weight, and body mass index

Adipose tissue dysfunction may be a central factor in the pathogenesis of insulin resistance in women with polycystic ovary syndrome (PCOS). Gene expression in subcutaneous adipose tissue in PCOS and its relation to metabolic and endocrine features of the syndrome have been fragmentarily investigated. The aim was to assess in subcutaneous adipose tissue the expression of genes potentially associated with adipose tissue dysfunction and to explore their relation to features of the syndrome. Twenty-one women with PCOS (body mass index [BMI] 18.2–33.4 kg/m2) and 21 controls (BMI 19.2–31.7 kg/m2) were matched pair-wise for age, body weight, and BMI. Tissue biopsies were obtained to measure mRNA expression of 44 genes (TaqMan Low Density Array). Differential expression levels were correlated with BMI, glucose infusion rate (GIR), sex hormone binding globulin (SHBG), and sex steroids. In PCOS, expression of adiponectin receptor 2 (ADIPOR2), LPL, and twist-related protein 1 (TWIST1) was decreased, while expression of chemokine (C-C motif) ligand 2 (CCL2) and heme oxygenase (decycling 1) (HMOX1) was increased. TWIST1 and HMOX1, both novel adipokines, correlated with BMI and GIR. After BMI adjustment, LPL and ADIPOR2 expression correlated with plasma estradiol, and CCL2 expression correlated with GIR, in all women. We conclude that adipose tissue mRNA expression differed in PCOS women and controls and that two novel adipokines, TWIST1 and HMOX1, together with adiponectin, LPL, and CCL2, and their downstream pathways merit further investigation.

A CCL2/ROS autoregulation loop is critical for cancer-associated fibroblasts-enhanced tumor growth of oral squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) have been described to play critical roles in initiation, progression and metastasis of various cancers. However, the involvement of CAFs in oral cancer (OC) has not been well addressed. In this study, we demonstrate that CAFs, when cocultured with OC cells (OCCs), produce high levels of chemokine (C-C motif) ligand 2 (CCL2) and, subsequently, enhance endogenous reactive oxygen species production in cells. Oxidative stress stimulates expression of cell cycle progression proteins in OCCs, leading to promotion of OCC proliferation, migration, invasion and, OC tumor growth. On the other hand, oxidative stress triggered the activation of nuclear factor-kappaB (NF-κB) and STAT3 in CAFs, resulting in accelerating CCL2 expression. In this way, CAFs-OCCs coculture creates a favorable cytokine-rich microenvironment, beneficial for both CAFs and OCCs. In addition, upregulation of CCL2 expression has been observed in oral squamous cell carcinoma tumors and patient plasma. We also showed that inhibition of CCL2 reduced OC tumor burden in mice. Therefore, our data suggested that CCL2 represents a potential therapeutic target for treatment of OC.

Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis

BackgroundBone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.MethodsBleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.ResultsPirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.ConclusionsPirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.

Forkhead box Q1 promotes hepatocellular carcinoma metastasis by transactivating ZEB2 and VersicanV1 expression

Forkhead box Q1 (FoxQ1) is a master regulator of tumor metastasis. However, the molecular mechanism of FoxQ1 in regulating hepatocellular carcinoma (HCC) metastasis remains unknown. Here we report a novel function for FoxQ1 in modifying the tumor microenvironment to promote HCC metastasis. FoxQ1 expression was an independent and significant risk factor for the recurrence and survival in two independent cohorts totaling 1,002 HCC patients. FoxQ1 induced epithelial-mesenchymal transition (EMT) through the transactivation of ZEB2 expression by directly binding to the ZEB2 promoter. Knockdown of ZEB2 decreased FoxQ1-enhanced HCC metastasis, whereas up-regulation of ZEB2 rescued the decreased metastasis induced by FoxQ1 knocking down. Additionally, serial deletion, site-directed mutagenesis, and a chromatin immunoprecipitation assays showed that VersicanV1, which promoted HCC metastasis and macrophage attraction, was a direct transcriptional target of FoxQ1. FoxQ1-induced VersicanV1 expression promoted the secretion of chemokine (C-C motif) ligand 2 (CCL2) from HCC cells. Chemotaxis assay showed that the culture media from FoxQ1-overexpressing HCC cells increased the migratory activity of the macrophages. Inhibition of VersicanV1 and CCL2 expression significantly inhibited FoxQ1-mediated macrophage migration. In animal studies, the up-regulation of FoxQ1 in HCC cells promoted HCC metastasis and intratumoral tumor associated macrophage (TAM) infiltration, whereas knockdown of VersicanV1 reduced FoxQ1-mediated HCC metastasis and intratumoral TAM infiltration. Depletion of macrophages using clodronate liposomes dramatically decreased FoxQ1-enhanced HCC metastasis. In human HCC tissues, FoxQ1 expression was positively correlated with ZEB2 and VersicanV1 expression and intratumoral TAM infiltration. Patients with positive coexpression of FoxQ1 and ZEB2, FoxQ1, and VersicanV1, or FoxQ1 and intratumoral TAMs were associated with poorer prognosis. FoxQ1 promotes HCC metastasis by transactivating ZEB2 and VersicanV1 expression, resulting in the induction of EMT and the recruitment of macrophage infiltration.

Clock gene Per1 regulates the production of CCL2 and interleukin-6 through p38, JNK1 and NF-κB activation in spinal astrocytes

It has been previously reported that spinal clock genes controlled under circadian rhythm contribute to the regulation of astrocytic function, which in turn is involved in diverse processes such as nociceptive transduction and the induction of inflammation. However, how clock genes expressed in spinal cord astrocytes are associated with the modulation of the inflammatory response is poorly understood. In the current study, the role of Period1 (Per1), one of clock genes, in the expression of chemokine (C-C motif) ligand 2 (CCL2) and interleukin-6 (IL-6), which are typical pro-inflammatory mediators produced by spinal astrocytes, was investigated. It was found that the knockdown of Per1 by using RNA interference led to a significant increase of the expression of CCL2 and IL-6 in cultured rat spinal astrocytes. Moreover, the silencing of the Per1 gene also increased the phosphorylation of p38, c-Jun N-terminal kinase (JNK) 1 and IκBα, and led to the translocation of p65 from the cytosol to the nucleus. The induction of CCL2 and IL-6 was significantly inhibited by treatment with the inhibitors of p38, JNK, and NF-κB. By contrast, the overexpression of PER1 by transfection vector significantly blocked the expression of CCL2 and IL-6, and the activation of p38, JNK, and NF-κB. Together, these results suggest that down-regulation of Per1 induced the phosphorylation of p38 and JNK1 and the subsequent activation of NF-κB, and that these events contribute to neuroinflammatory state in the spinal cord via the induction of the release of inflammatory mediators.

CCL5/RANTES is important for inducing osteogenesis of human mesenchymal stem cells and is regulated by dexamethasone.

In this study, we examine the effect of chemokine (C-C motif) ligand 5 (CCL5)/Regulated on Activation Normal T cell Expressed and Secreted (RANTES), a pro-inflammatory cytokine on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We found CCL5 expression was increased during osteogenic differentiation of hMSCs and CCL5 expression is dependent on the presence of dexamethasone. Knocking down endogenous CCL5 expression blocked osteogenesis, as revealed by decreasing alkaline phosphatase (ALP) activity and a reduction in the expression levels of ALP, bone sialoprotein (BSP), and osteopontin (OPN). Of note, the overexpression of CCL5 was sufficient to increase ALP expression and activity. Moreover, the down-regulation of chemokine (C-C motif) receptor 1 (CCR1), one of the CCL5 receptors, significantly decreased the osteogenesis of hMSCs. Interestingly, the down-regulation of CCR1, but not CCL5, was sufficient to affect the cell numbers during the process of osteogenesis. Our findings reveal that both CCL5 and CCR1 are required for osteogenesis of human MSCs, CCL5 is sufficient for the osteogenesis, and provide a novel link between dexamethasone and CCL5 in human osteogenesis.