Macrophage-conditioned Medium Research Articles

Anti-cancer effect of danshen and dihydroisotanshinone I on prostate cancer: targeting the crosstalk between macrophages and cancer cells via inhibition of the STAT3/CCL2 signaling pathway.

Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. In our study, the in vivo protective effect of danshen in prostate cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro, we discovered that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the migration of both androgen-dependent and androgen-independent prostate cancer cells. In addition, we noted that DT substantially inhibited the migratory ability of prostate cancer cells in both a macrophage-conditioned medium and macrophage/prostate cancer coculture medium. Mechanistically, DT both diminished the ability of prostate cancer cells to recruit macrophages and reduced the secretion of chemokine (C-C motif) ligand 2 (CCL2) from both macrophages and prostate cancer cells in a dose-dependent manner. Moreover, DT inhibited the protein expression of p-STAT3 and decreased the translocation of STAT3 into nuclear chromatin. DT also suppressed the expression of tumor epithelial–mesenchymal transition genes, including RhoA and SNAI1. In conclusion, danshen can prolong the survival rate of prostate cancer patients in Taiwan. Furthermore, DT can inhibit the migration of prostate cancer cells by interrupting the crosstalk between prostate cancer cells and macrophages via the inhibition of the CCL2/STAT3 axis. These results may provide the basis for a new therapeutic approach toward the treatment of prostate cancer progression.

Purification and identification of 25-hydroxycholesterol in a reptile: Seasonal variation and hormonal regulation

The present in vitro study, for the first time, demonstrates the production of 25-hydroxycholestrol (25-HC) by testicular macrophages of a non-mammalian vertebrate. The ether extracts of testicular macrophage-conditioned medium (TMCM) were fractionated on a C18 reversed phase high-performance liquid chromatography (HPLC) column using methanol as the mobile phase. The mass spectrometry (MS) fragmentation pattern of HPLC-purified 25-HC was found to be identical to that of authentic 25-HC. Further, a significant seasonal variation in 25-HC concentration was observed with maximal level in regressed and minimal during breeding phase. To understand the hormonal control of 25-HC production, testicular macrophages from regressed phase testes were incubated with 0.5μg/ml of ovine follicle stimulating hormone (FSH) and 0.1, 1 and 10μg/ml of testosterone (T). FSH considerably enhanced 25-HC production by testicular macrophages. In contrast, T markedly inhibited 25-HC production in a dose-dependent manner. In addition, T significantly inhibited FSH-induced 25-HC production, though pre-treatment with T was more effective as compared to post-treatment with T to FSH. Our findings on production, seasonal variation and hormonal control of 25-HC suggest the functional significance of 25-HC in the testis of reptiles.

Rosmarinic acid suppresses adipogenesis, lipolysis in 3T3-L1 adipocytes, lipopolysaccharide-stimulated tumor necrosis factor-α secretion in macrophages, and inflammatory mediators in 3T3-L1 adipocytes

ABSTRACTBackground: Rosmarinic acid (RA) is a natural phenol carboxylic acid with many promising biological effects. It may be a suitable candidate for improving obesity-related adipose tissue dysfunction.Objective: We aimed to investigate the therapeutic use of RA as an anti-obesity agent by measuring its effects on adipogenesis, lipolysis, and messenger RNA (mRNA) expression of major adipokines in 3T3-L1 adipocytes; and its effects on lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) secretion in macrophages and inflammatory mediators in 3T3-L1 adipocytes incubated with macrophage-conditioned medium (MCM).Methods: 3T3-L1 preadipocytes were used to explore how RA affects adipogenesis, as well as the involvement of phosphorylated extracellular signal-regulated kinase-1/2 (p-ERK1/2) and mothers against decapentaplegic homolog 3 (p-Smad3). 3T3-L1 preadipocytes were also differentiated into mature adipocytes to explore how RA affects basal and isoproterenol- and forskolin-stimulated lipolysis; and how RA affects key adipokines’ mRNA expression. RAW 264.7 macrophages were stimulated with LPS in the absence or presence of RA to explore RA’s effects on TNF-α secretion. MCM was collected and 3T3-L1 adipocytes were incubated with MCM to explore RA’s effects on interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 (MCP-1), and RANTES mRNA expression.Results: During the preadipocyte differentiation process, RA suppressed peroxisome proliferator-activated receptor-γ and CCAAT/enhancer binding protein-α, and activated p-ERK1/2 and p-Smad3; inhibition of adipogenesis by RA was partially restored following treatment with p-ERK1/2 and p-Smad3 inhibitors. In mature adipocytes, RA inhibited basal lipolysis; phosphodiesterase-3 inhibitor reversed this. RA also inhibited isoproterenol- and forskolin-stimulated glycerol and free fatty acid release, and the phosphorylation of hormone-sensitive lipase and perilipin. RA had no effects on leptin, adiponectin, resistin, or visfatin mRNA expression. RA suppressed TNF-α mRNA expression and secretion in LPS-stimulated RAW 264.7 macrophages; and reduced LPS-MCM-induced IL-6, IL-1β, MCP-1, and RANTES mRNA expression in 3T3-L1 adipocytes.Conclusions: RA exerts inhibitory effects on adipogenesis, lipolysis, and inflammation. RA could be a promising natural product for improving adipose mobilization in obesity.

Heparanase-driven inflammation from the AGEs-stimulated macrophages changes the functions of glomerular endothelial cells.

Amounts of macrophages were infiltrated in glomeruli in diabetic nephropathy. Heparanase has been thought to be closely related to proteinuria. Our aims were to determine the effect of heparanase on the inflammation in AGEs-stimulated macrophages and its role on the functions of glomerular endothelial cells (GEnCs). The expression of inflammation cytokines in macrophages were assayed by q-RT PCR, western, and ELISA. Then western was used to measure the expression of RAGE and key proteins in NF-κB pathway in macrophages. The expression of the adherence molecules and tight junction proteins in GEnCs were assessed by western. The adherence of mononuclear cells to GEnCs were observed by HE staining and transendothelial FITC-BSA were tested for the permeability of GEnCs. HPA siRNA and heparanase inhibitor sulodexide could attenuate the increasing inflammatory factors (TNF-α and IL-1β) in AGEs-stimulated macrophages. NF-κB inhibitor PDTC could also decrease the augmented inflammation cytokines through inhibiting the activation of the NF-κB pathway induced by AGEs. The phosphorylation of NF-κB signaling pathway could be also attenuated by HPA siRNA and sulodexide, the same to the receptor of AGEs RAGE. When the macrophage-conditioned culture medium were added to the glomerular endothelial cells, we found HPA siRNA and sulodexide groups could decrease the increasing adherence and permeability of GEnCs induced by AGEs. Heparanase increases the inflammation in AGEs-stimulated macrophages through activating the RAGE-NF-κB pathway. Heparanase driven inflammation from AGEs-stimulated macrophages increases the adherence of GEnCs and augments the permeability of GEnCs.

Macrophages Enhance Migration in Inflammatory Breast Cancer Cells via RhoC GTPase Signaling.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC’s extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC’s hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM “primes” the IBC cells’ cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins −6, −8, and −10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

Inhibitory Effect of Methyl 2-(4'-Methoxy-4'-oxobutanamide) Benzoate from Jerusalem Artichoke (Helianthus tuberosus) on the Inflammatory Paracrine Loop between Macrophages and Adipocytes.

The interaction between macrophages and adipocytes is known to aggravate inflammation of the adipose tissue, leading to decreased insulin sensitivity. Hence, attenuation of the inflammatory paracrine loop between macrophages and adipocytes is deemed essential to ameliorate insulin resistance and diabetes mellitus type 2. Methyl 2-(4'-methoxy-4'-oxobutanamide) benzoate (compound 1), a newly isolated compound from Jerusalem srtichoke (JA), has not been biologically characterized yet. Here, we investigated whether JA-derived compound 1 attenuates the inflammatory cycle between RAW 264.7 macrophages and 3T3-L1 adipocytes. Compound 1 suppressed the inflammatory response of RAW 264.7 cells to lipopolysaccharide through decreased secretion of IL-1β, IL-6, and TNF-α. Moreover, the mRNA expression of TNF-α, IL-6, IL-1β, MCP-1, and Rantes and MAPK pathway activation in 3T3-L1 adipocytes, incubated in macrophage-conditioned media, were inhibited. These findings suggest an anti-inflammatory effect of a newly extracted compound against adipose tissue inflammation and insulin resistance.

AMPKα Is Suppressed in Bladder Cancer through Macrophage-Mediated Mechanisms

Bladder cancer presents as either low- or high-grade disease, each with distinct mutational profiles; however, both display prominent mTORC1 activation. One major negative regulator of mTORC1 is AMPK, which is a critical metabolic regulator that suppresses cellular growth in response to metabolic stress by negatively regulating mTORC1. Alterations in the activation and protein levels of AMPK have been reported in breast, gastric, and hepatocellular carcinoma. To investigate whether AMPK suppression is responsible for mTOR activation in bladder cancer, the levels of AMPKα were quantified in a cohort of primary human bladder cancers and adjacent nontumor tissues. The levels of p-AMPKα, AMPKα1, AMPKα2, and total AMPKα were significantly suppressed in both low- and high-grade disease when compared with nontumor tissue. To elucidate the AMPKα suppression mechanism, we focused on inflammation, particularly tumor-infiltrating macrophages, due to their reported role in regulating AMPK expression. Treatment of HTB2 cancer cells with varying doses of differentiated U937 macrophage conditioned medium (CM) demonstrated a dose-dependent reduction of AMPKα protein. Additionally, macrophage CM treatment of HTB2 and HT1376 bladder cells for various times also reduced AMPKα protein but not mRNA levels. Direct TNFα treatment also suppressed AMPKα at the protein but not RNA level. Finally, staining of the human cohort for CD68, a macrophage marker, revealed that CD68+ cell counts correlated with reduced AMPKα levels. In summary, these data demonstrate the potential role for inflammation and inflammatory cytokines in regulating the levels of AMPKα and promoting mTORC1 activation in bladder cancer.

MiR-146a-mediated suppression of the inflammatory response in human adipocytes.

The obesity-associated inflammation of white adipose tissue (WAT) is one of the factors leading to the development of related diseases such as insulin resistance and liver steatosis. Recently, microRNAs (miRNAs) were identified as important regulators of WAT functions. Herein, we cultured human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with macrophage-conditioned medium (MacCM) and performed an Affimetrix miRNA array to identify miRNAs differentially expressed under inflammatory conditions. We identified 24 miRNAs differentially expressed upon inflammation in human adipocytes and miR-146a was the most up-regulated miRNA species. In subcutaneous WAT, miR-146a was elevated in both human and murine obesity. Transfection of miR-146a mimics prevented the MacCM-induced inflammatory response in SGBS adipocytes as seen by reduced levels of IL-8 and MCP-1 mRNA and protein. We identified IRAK1 and TRAF6 as targets of miR-146a in human adipocytes and detected a reduced inflammation-induced activation of JNK and p38 upon miR-146a transfection. Taken together, we could show that miR-146a reduces the inflammatory response in human adipocytes. In a negative feedback loop miR-146a might contribute to the regulation of inflammatory processes in WAT and possibly prevent an overwhelming inflammatory response.

M2 macrophages induce ovarian cancer cell proliferation via a heparin binding epidermal growth factor/matrix metalloproteinase 9 intercellular feedback loop.

In ovarian cancer, a high ratio of anti-inflammatory M2 to pro-inflammatory M1 macrophages correlates with poor patient prognosis. The mechanisms driving poor tumor outcome as a result of the presence of M2 macrophages in the tumor microenvironment remain unclear and are challenging to study with current techniques. Therefore, in this study we utilized a micro-culture device previously developed by our lab to model concentrated paracrine signaling in order to address our hypothesis that interactions between M2 macrophages and ovarian cancer cells induce tumor cell proliferation. Using the micro-culture device, we determined that co-culture with M2-differentiated primary macrophages or THP-1 increased OVCA433 proliferation by 10–12%. This effect was eliminated with epidermal growth factor receptor (EGFR) or heparin-bound epidermal growth factor (HB-EGF) neutralizing antibodies and HBEGF expression in peripheral blood mononuclear cells from ovarian cancer patients was 9-fold higher than healthy individuals, suggesting a role for HB-EGF in tumor progression. However, addition of HB-EGF at levels secreted by macrophages or macrophage-conditioned media did not induce proliferation to the same extent, indicating a role for other factors in this process. Matrix metalloproteinase-9, MMP-9, which cleaves membrane-bound HB-EGF, was elevated in co-culture and its inhibition decreased proliferation. Utilizing inhibitors and siRNA against MMP9 in each population, we determined that macrophage-secreted MMP-9 released HB-EGF from macrophages, which increased MMP9 in OVCA433, resulting in a positive feedback loop to drive HB-EGF release and increase proliferation in co-culture. Identification of multi-cellular interactions such as this may provide insight into how to most effectively control ovarian cancer progression.

Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation.

Arthritic gout is caused by joint inflammation triggered by the damaging effects of monosodium uric acid (MSU) crystal accumulation in the synovial space. Neutrophils play a major role in mediating joint inflammation in gout. Along with neutrophils, other immune cells, such as macrophages, are present in inflamed joints and contribute to gout pathogenesis. Neutrophils form neutrophil extracellular traps (NETs) in response to MSU crystals. In the presence of MSU crystals, macrophages release IL-1β, a cytokine crucial to initiate gout pathogenesis and neutrophil recruitment. Our research investigated interactions between human macrophages and neutrophils in an in vitro model system and asked how macrophages affect NET formation stimulated by MSU crystals. Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF. Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1β or anakinraprior to stimulation by MSU crystals. Interaction of neutrophils with MSU crystals was evaluated by live imaging using confocal microscopy. The presence of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured by ELISA. NET formation was quantitated by Sytox Orange-based extracellular DNA release assay and NE-DNA ELISA. AggNET formation was assessed by macroscopic evaluation. We found that crystal- and cell-free supernatants of macrophages stimulated with MSU crystals promote MSU crystal-stimulated NET formation in human neutrophils. This observation was confirmed by additional assays measuring the release of MPO, NE, and the enzymatic activity of NE. MSU crystal-induced NET formation remained unchanged when neutrophil supernatants were tested. IL-1β is a crucial cytokine orchestrating the onset of inflammation in gout and is known to be released in large amounts from macrophages following MSU crystal stimulation. We found that recombinant IL-1β strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1β alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1β is mainly responsible for this effect. Macrophage-derived IL-1β enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1β affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra.

Macrophage-Mediated Effects of Airborne Fine Particulate Matter (PM2.5) on Hepatocyte Insulin Resistance in Vitro.

Fine particulate matter (PM2.5) pollution poses significant health risks worldwide, including metabolic syndrome-related diseases with the characteristic feature of insulin resistance. However, the mechanism and influencing factors of this effect are poorly understood. In this serial in vitro study, we aimed at testing the hypothesis that macrophage-mediated effects of PM2.5 on hepatic insulin resistance depend on its chemical composition. Mouse macrophages were exposed to PM2.5 that had been collected during summer or winter in Beijing, which represented different compositions of PM2.5. Thereafter, hepatocytes were treated with macrophage-conditioned medium (CM). PM2.5 induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 expression and secretion in macrophages, particularly after winter PM2.5 exposure. Correspondingly, winter CM weakened hepatocellular insulin-stimulated glucose consumption. Further investigation revealed that the normal insulin pathway was suppressed in winter CM-treated hepatocytes, with increased phosphorylation of insulin receptor substrate 1 at serine residue 307 (Ser307) and decreased phosphorylation of protein kinase B (PKB/AKT) and forkhead box transcription factor O1 (FoxO1). Moreover, c-Jun N-terminal kinase, a key moderator of the sensitivity response to insulin stimulation, was activated in hepatocytes treated with winter CM. Although further studies are warranted, this preliminary study suggested an association between PM composition and insulin resistance, thus contributing to our understanding of the systemic toxicity of PM2.5.

Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion.

BackgroundN-cadherin is a trans-membrane adhesion molecule associated with advanced carcinoma progression and poor prognosis. The effect of N-cadherin on matrix metalloproteinase 9 (MMP-9) regulation is implicated in human nasopharyngeal carcinoma (NPC) cell invasion.Methods and resultsExposure of NPC cells to phorbol-12-myristate-13-acetate (PMA) or macrophage conditioned media (CM) upregulated MMP-9 and N-cadherin cleavage, which resulted in NPC cell invasion. MMP-9 cleaved the extracellular domain of N-cadherin, which was further cleaved by γ-secretase with PMA or macrophage-CM treatment. The extracellular cleavage of N-cadherin was inhibited with treatment with an MMP inhibitor and MMP-9 siRNA, whereas the intracellular cleavage of N-cadherin was inhibited by treatment with a γ-secretase inhibitor (γI), which resulted in enhanced accumulation of N-cadherin C-terminal fragment (CTF1, ~40 kDa). CTF2/N-cad (CTF2), a product of the γ-secretase cleavage of N-cadherin, was released and translocated into the nuclear compartment in PMA-treated cells. Moreover, CTF2 enhanced the effect of PMA-mediated MMP-9 gene expression as assessed by treatment with γI or overexpression with exogenous CTF2. Additionally, siRNA silencing of N-cadherin decreased PMA-mediated MMP-9 expression and cell invasion. The outside-in signaling effect of MMP-9 in macrophage CM- or PMA-treated cell cultures significantly enhanced NPC cell invasion via N-cadherin cleavage.ConclusionExtracellular and intracellular cleavage of N-cadherin might be involved in elevated MMP-9 expression enhancing tumor cell invasion. Furthermore, N-cadherin–affected tumor progression might be via enhanced MMP-9 signaling in a cross-talk regulatory mechanism. N-cadherin might contribute to the invasive characteristics of carcinoma cells by upregulating MMP-9, thereby leading to increased aggressive metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2846-4) contains supplementary material, which is available to authorized users.

Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury.

WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation.

N-acetylcysteine attenuates lipopolysaccharide-induced impairment in lamination of Ctip2-and Tbr1- expressing cortical neurons in the developing rat fetal brain.

Oxidative stress and inflammatory insults are the major instigating events of bacterial intrauterine infection that lead to fetal brain injury. The purpose of this study is to investigate the remedial effects of N-acetyl-cysteine (NAC) for inflammation-caused deficits in brain development. We found that lipopolysaccharide (LPS) induced reactive oxygen species (ROS) production by RAW264.7 cells. Macrophage-conditioned medium caused noticeable cortical cell damage, specifically in cortical neurons. LPS at 25 μg/kg caused more than 75% fetal loss in rats. An increase in fetal cortical thickness was noted in the LPS-treated group. In the enlarged fetal cortex, laminar positioning of the early born cortical cells expressing Tbr1 and Ctip2 was disrupted, with a scattered distribution. The effect was similar, but minor, in later born Satb2-expressing cortical cells. NAC protected against LPS-induced neuron toxicity in vitro and counteracted pregnancy loss and alterations in thickness and lamination of the neocortex in vivo. Fetal loss and abnormal fetal brain development were due to LPS-induced ROS production. NAC is an effective protective agent against LPS-induced damage. This finding highlights the key therapeutic impact of NAC in LPS-caused abnormal neuronal laminar distribution during brain development.

Cartilage inflammation and degeneration is enhanced by pro-inflammatory (M1) macrophages in vitro, but not inhibited directly by anti-inflammatory (M2) macrophages

Macrophages play a crucial role in the progression of osteoarthritis (OA). Their phenotype may range from pro-inflammatory to anti-inflammatory. The aim of this study was to evaluate the direct effects of macrophage subtypes on cartilage by culturing macrophage conditioned medium (MCM) on human articular cartilage. Human OA cartilage explants were cultured with MCM of pro-inflammatory M(IFNγ+TNFα), or anti-inflammatory M(IL-4) or M(IL-10) human monocyte-derived macrophages. To assess effects of anti-inflammatory macrophages, the cartilage was cultured with a combination of MCM phenotypes as well as pre-stimulated with IFNγ+TNFα cartilage before culture with MCM. The reactions of the explants were assessed by gene expression, nitric oxide (NO) production and release of glycosaminoglycans (GAGs). M(IFNγ+TNFα) MCM affected OA cartilage by upregulation of IL1B (Interleukin 1β), IL6, MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-5), while inhibiting ACAN (aggrecan) and COL2A1 (collagen type II). M(IL-10) upregulated IL1B and Suppressor of cytokine signaling 1 (SOCS1). NO production and GAG release by the cartilage was increased when cultured with M(IFNγ+TNFα) MCM. M(IL-4) and M(IL-10) did not inhibit the effects of M(IFNγ+TNFα) MCM of neither phenotype affected IFNγ+TNFα pre-stimulated cartilage, in which an inflammatory gene response was deliberately induced. M(IFNγ+TNFα) macrophages have a prominent direct effect on OA cartilage, while M(IL-4) and M(IL-10) do not inhibit the effects of M(IFNγ+TNFα), or IFNγ+TNFα induced inflammation of the cartilage. Therapies aiming at inhibiting cartilage degeneration may take this into account by directing suppression of pro-inflammatory macrophages or stimulation of anti-inflammatory macrophages.

Immune Cell-Conditioned Media Suppress Prostate Cancer PC-3 Cell Growth Correlating With Decreased Proinflammatory/Anti-inflammatory Cytokine Ratios in the Media Using 5 Selected Crude Polysaccharides.

Five different crude polysaccharides from guava seed (GSPS), bitter buckwheat (BBPS), common buckwheat (CBPS), red Formosa lambsquarters (RFLPS), and yellow Formosa lambsquarters (YFLPS) were isolated to treat human prostate cancer PC-3 cells via direct action or tumor immunotherapy. The splenocyte- and macrophage-conditioned media (SCM and MCM) were prepared using individual selected polysaccharides, and then SCM or MCM was further collected to treat PC-3 cells. The relationship between PC-3 cell growth and Th1/Th2 cytokines in SCM as well as proinflammatory/anti-inflammatory cytokine secretion profiles in MCM were delineated. The results showed that all 5 selected polysaccharides did not significantly inhibit PC-3 cell growth via direct action. However, SCM or MCM cultured in the absence or presence of 5 selected polysaccharides significantly (P < .05) inhibited PC-3 cell growth. MCM cultured with 5 polysaccharides dose dependently enhanced their inhibitory effects on the viabilities of PC-3 cells than those cultured without polysaccharides. There was a significant (P < .05) negative correlation between PC-3 cell viabilities and (interleukin [IL]-6 + tumor necrosis factor [TNF]-α)/IL-10 level ratios in the corresponding MCM, implying that macrophages suppress PC-3 cell growth through decreasing secretion ratios of proinflammatory/anti-inflammatory cytokines in a tumor microenvironment.

Abstract 3236: Macrophages increase the expression of RhoC in inflammatory breast cancer leading to increased migration

Abstract Inflammatory breast cancer (IBC) is considered the most lethal form of breast cancer due to its ability to progress quickly and the frequent presence of metastasis at diagnosis. African Americas are disproportionally diagnosed with IBC and often have worse outcomes than Caucasians. By investigating IBC in both African American and Caucasian cell lines we seek to understand the differences in IBC progression and help address disparities by providing new anti-IBC strategies. RhoC GTPase is overexpressed in 90% of IBC tumors and is known to increase cell motility. Sites of inflammation, as seen in IBC, attract tumor associated macrophages (TAMs), which have been found to facilitate the movement and invasion of many breast cancers. We hypothesize that TAMs play a role in increasing RhoC expression in IBC cell lines, consequently leading to IBC's severe migratory and metastatic potential. A novel microfluidic device created by our team was used to measure the migratory phenotype of IBC cell lines in response to macrophage conditioned media (CM) and cytokine stimulation. IBC cell lines were treated with CM, cytokines, or pathway inhibitors then Western blotting was used to determine protein expression and phosphorylation to identify important signaling pathways. We found the expression of RhoC significantly increased in two different IBC cell lines, SUM149 (African American) and SUM190 (Caucasian), after culturing with conditioned media from the macrophage-differentiated U937 monocytic cell line. This increase was not detected in either the normal-like MCF-10A breast epithelial cell line or the non-IBC MDA-MB-231 triple negative breast cancer cell line. CM caused a significant increase in the migration distance and frequency of both SUM149 and SUM190 cell lines. Analysis of the CM determined CCL2, CCL5, and IL-8 to be the key mediators in the macrophage CM. Western blotting proposes that CCL2, CCL5, and IL-8 stimulation causes twice as much RhoC expression compared to the control. Further analysis suggests a role for the MAPK pathway in controlling RhoC expression and migration. Macrophage conditioned media causes an increase in RhoC expression in IBC cell lines and stimulates migration. Individual cytokines can lead to an increase in RhoC possibly through the MAPK pathway. Studies involving RhoC inhibitors are ongoing and could yield promising therapies for the prevention of metastasis in IBC. By understanding the specific mechanism of TAMs’ effects on IBC, we hope to learn how to control the lethal metastatic nature of IBC and improve outcomes for patients of all ethnicities. Citation Format: Julie Madden, Steve Allen, Yu-Chi Shen, Chelsea Fournier, ZhiFen Wu, Liwei Bao, Sofia Merajver. Macrophages increase the expression of RhoC in inflammatory breast cancer leading to increased migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3236.

Effect of High Glucose Concentration on Human Preadipocytes and Their Response to Macrophage-Conditioned Medium.

Adipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown whether these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. Our aim was to compare the effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation. Human monocyte-derived macrophages (MDMs) were placed in 5 mmol/L (NG) or 25 mmol/L (HG) glucose for 24 hours. MacCM was collected and its effect on differentiation of human subcutaneous abdominal preadipocytes and adipocyte inflammation was evaluated. HG-MacCM, but not NG-MacCM, inhibited triacylglycerol (TG) accumulation and protein expression of peroxisome proliferator-activated receptor γ (PPARgamma) during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased interleukin-6 and monocyte chemotactic protein-1 (MCP-1), as well as reduced adiponectin mRNA expression. In MDMs, HG increased phosphorylation of inhibitor of kappaB kinase (IKK)-beta and decreased protein expression of inhibitor of kappaB alpha. HG also reduced protein expression of PPARgamma in MDMs. However, no MDM changes in mRNA expression of MCP-1, interleukin-1beta or tumor necrosis factor-alpha were detected. The stimulatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKbeta inhibitor). High glucose concentration accentuates the anti-adipogenic and pro-inflammatory effects of MacCM on human preadipocytes.

MP44-11 GROWTH HORMONE-RELEASING HORMONE (GHRH) ANTAGONISTS REDUCE INFLAMMATION- AND TRANSFORMING GROWTH FACTOR (TGF)-?2-INDUCED PROLIFERATION OF HUMAN BPH-1 PROSTATE EPITHELIAL CELLS GROWN IN 3D CULTURE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2016MP44-11 GROWTH HORMONE-RELEASING HORMONE (GHRH) ANTAGONISTS REDUCE INFLAMMATION- AND TRANSFORMING GROWTH FACTOR (TGF)-?2-INDUCED PROLIFERATION OF HUMAN BPH-1 PROSTATE EPITHELIAL CELLS GROWN IN 3D CULTURE Petra Popovics, Andrew V. Schally, Roberto Perez, Rhenzi Cai, and Ferenc G. Rick Petra PopovicsPetra Popovics More articles by this author , Andrew V. SchallyAndrew V. Schally More articles by this author , Roberto PerezRoberto Perez More articles by this author , Rhenzi CaiRhenzi Cai More articles by this author , and Ferenc G. RickFerenc G. Rick More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.267AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Benign prostatic hyperplasia (BPH) is frequently associated with chronic inflammation. Epithelial-to-mesenchymal transition (EMT) has been recently indicated as a major factor in the pathogenesis of BPH. Evidence also suggests that inflammatory cytokines may induce BPH. Hypothalamic neuropeptides, GHRH, luteinizing hormone-releasing hormone (LHRH) and their receptors are expressed in experimental models of BPH in which antagonists of GHRH and LHRH have been shown to suppress the levels of proinflammatory cytokines and alter the expression of genes related to EMT. These findings imply a role of GHRH/LHRH in the development of prostatitis, however, this has not been investigated to date. METHODS Human BPH-1 prostatic cell line was used to generate matrigel-embedded 3D cultures in which average sphere diameters were evaluated. Chronic inflammation was induced by treating cells with THP-1 macrophage-conditioned medium whereas EMT was triggered with TGF-ß1 or TGF-ß2 peptides. The role of secreted LHRH and GHRH in inflammation-induced proliferation was determined by using the LHRH antagonist, degarelix, and a specific GHRH antagonist developed in our lab. The presence of receptors for GHRH and LHRH was confirmed in 3D cultures by immunocytochemistry and quantitative RT-PCR. RESULTS BPH-1-derived spherical structures expressed cell membrane receptors for LHRH and GHRH. Macrophage-conditioned medium induced a 26% increase (P<0.001) in the average diameter of cells and a 2-fold elevation in the expression of the EMT markers N-cadherin (p<0.01) and Snail (p<0.001) as detected by qPCR. The expression of GHRH was also increased 2.7-fold (p<0.001), whereas mRNA levels of LHRH were slightly reduced (ns). GHRH antagonist reduced inflammation-induced increase in diameter by 64% (P<0.01), while no significant change was observed after degarelix treatment. TGF-ß2 increased average sphere diameter by 32%, but this effect was reduced by 67% by the GHRH antagonist (P<0.001). In contrast, TGF-ß1 did not significantly affect sphere diameter. CONCLUSIONS This study identifies GHRH as an important factor in inflammation-induced proliferation and TGF-ß2-triggered EMT of prostate epithelial cells and suggests the merit of further investigation to elucidate the effects of GHRH antagonists in the reduction of inflammation-induced BPH. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e602-e603 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Petra Popovics More articles by this author Andrew V. Schally More articles by this author Roberto Perez More articles by this author Rhenzi Cai More articles by this author Ferenc G. Rick More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.

Prostanoids derived from COX-2 and EP receptors are involved in vascular remodelling in different cardiovascular pathologies. This study evaluates the contribution of COX-2 and EP1 receptors to vascular remodelling and function in hypertension. Spontaneously hypertensive rats (SHR) and angiotensin II (AngII)-infused (1.44 mg · kg(-1) · day(-1), 2 weeks) mice were treated with the COX-2 inhibitor celecoxib (25 mg · kg(-1) · day(-1) i.p) or with the EP1 receptor antagonist SC19220 (10 mg · kg(-1) · day(-1) i.p.). COX-2(-/-) mice with or without AngII infusion were also used. Celecoxib and SC19220 treatment did not modify the altered lumen diameter and wall : lumen ratio in mesenteric resistance arteries from SHR-infused and/or AngII-infused animals. However, both treatments and COX-2 deficiency decreased the augmented vascular stiffness in vessels from hypertensive animals. This was accompanied by diminished vascular collagen deposition, normalization of altered elastin structure and decreased connective tissue growth factor and plasminogen activator inhibitor-1 gene expression. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses and endothelial dysfunction induced by AngII infusion. Hypertensive animals showed increased mPGES-1 expression and PGE2 production in vascular tissue, normalized by celecoxib. Celecoxib treatment also decreased AngII-induced macrophage infiltration and TNF-α expression. Macrophage conditioned media (MCM) increased COX-2 and collagen type I expression in vascular smooth muscle cells; the latter was reduced by celecoxib treatment. COX-2 and EP1 receptors participate in the increased extracellular matrix deposition and vascular stiffness, the impaired vascular function and inflammation in hypertension. Targeting PGE2 receptors might have benefits in hypertension-associated vascular damage.