PERK signaling through C/EBPδ contributes to ER stress-induced expression of immunomodulatory and tumor promoting chemokines by cancer cells.

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.

EGCG Inhibits Adipose-Derived Mesenchymal Stem Cells Differentiation into Adipocytes and Prevents a STAT3-Mediated Paracrine Oncogenic Control of Triple-Negative Breast Cancer Cell Invasive Phenotype.

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells.

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

Sulforaphane controls the release of paracrine factors by keratinocytes and thus mitigates particulate matter-induced premature skin aging by suppressing melanogenesis and maintaining collagen homeostasis.

BACKGROUND: Skin aging, potentially caused by exposure to particulate matter (PM)2.5, is characterized by wrinkling, abnormal pigmentation, and skin dryness triggered by several keratinocyte-derived paracrine factors. Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN), commonly found in cruciferous vegetables, has diverse biological effects on skin tissue. PURPOSE: In the present study, we have investigated whether SFN may alleviate PM2.5-induced premature skin aging. METHODS: We used keratinocyte/melanocyte or keratinocyte/fibroblast coculture models of skin cells and measured the parameters of melanogenesis, collagen homeostasis and inflammation. RESULTS: SFN inhibited the development of reactive oxygen species in keratinocytes exposed to PM2.5. In keratinocyte/melanocyte cocultures, it significantly inhibited the upregulation of melanogenic paracrine mediators (including endothelin-1 and prostaglandin E2) in keratinocytes exposed to PM2.5; the synthesis of melanogenic proteins including microphthalmia-associated transcription factor, tyrosinase-related protein 1, and tyrosinase; and the levels of melanin in melanocytes. SFN treatment of keratinocyte/fibroblast cocultures significantly reduced the PM2.5-induced expression of NF-κB-mediated cytokines including interleukin-1ß, interleukin-6, tumor necrosis factor α, and cyclooxygenase-2. In fibroblasts of the keratinocyte/fibroblast coculture system, the expression levels of phospho-NF-κB, cysteine-rich protein 61, and matrix metalloproteinase-1 were significantly decreased whereas procollagen type I synthesis was significantly increased. CONCLUSION: Collectively, our results suggest that SFN mitigates PM2.5-induced premature skin aging by suppressing melanogenesis and maintaining collagen homeostasis. It acts by regulating the release of paracrine factors from keratinocytes.

Nitric-Oxide-Releasing Biomaterial Regulation of the Stem Cell Microenvironment in Regenerative Medicine.

Stem cell therapy has proven to be an attractive solution for the treatment of degenerative diseases or injury. However, poor cell engraftment and survival within injured tissues limits the successful use of stem cell therapy within the clinical setting. Nitric oxide (NO) is an important signaling molecule involved in various physiological processes. Emerging evidence supports NO's diverse roles in modulating stem cell behavior, including survival, migration, differentiation, and paracrine secretion of proregenerative factors. Thus, there has been a shift in research focus to concentrate efforts on the delivery of therapeutic concentration ranges of NO to the target tissue sites. Combinatory therapies utilizing biomaterials that control NO generation and support stem cell delivery can be holistic and synergistic approaches to significantly improve tissue regeneration. Here, the focus is on recent developments of various therapeutic platforms, engineered to both transport NO and to enhance stem-cell-mediated regeneration of damaged tissues. New and emerging revelations of how the stem cell microenvironment can be regulated by NO-releasing biomaterials are also highlighted.

Ganoderma lucidum polysaccharide reduces melanogenesis by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway.

Our previous study found that Ganoderma lucidum polysaccharide (GLP), bioactive ingredients from Ganoderma lucidum, protected fibroblasts from photoaging. However, whether GLP can affect melanogenesis in melanocytes through regulating paracrine mediators that secreted by keratinocytes and fibroblasts is unclear. We aimed to investigate the efficacy and mechanisms of action of GLP in melanogenesis by regulating paracrine effects of keratinocytes and fibroblasts. The effect of GLP on cell viability affected by GLP was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After an immortal keratinocyte line (HaCaT) and primary fibroblasts (FB) were treated with GLP, the supernatants of HaCaT and FB cells were collected and cocultured with an immortalized melanocyte line (PIG1). The expression levels of melanogenesis-associated genes in PIG1 cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Furthermore, FRS-2, ERK, JNK, and p38 phosphorylation levels were measured. Then, major melanogenic paracrine mediators in HaCaT and FB cells treated with GLP were evaluated by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). In addition, the expression of IL-6 and STAT3 was examined in HaCaT and FB cells. GLP was not cytotoxic to HaCaT and FB cells. The supernatants of GLP-treated HaCaT and FB cells downregulated the expression levels of MITF, TYR, TYRP1, TYRP2, RAB27A, and FSCN1 genes and inhibited the phosphorylation of FRS-2, ERK, JNK, and p38 in PIG1 cells. GLP also decreased FGF2 secretion in HaCaT and FB cells. Moreover, GLP reduced IL-6 expression and STAT3 phosphorylation in HaCaT and FB cells. GLP reduced melanogenesis in melanocytes by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway.

Serenoa repens and N-acetyl glucosamine/milk proteins complex differentially affect the paracrine communication between endothelial and follicle dermal papilla cells.

Current treatments for hair follicle (HF) disruption are based on 5-α reductase inhibitors and prostaglandin modulators. Botanicals and nutraceutical compounds interfere with hair loss or stimulate its partial regrowth. Here, we used in vitro cocultures to investigate the activity of Serenoa repens ( SR) and N-acetyl glucosamine + milk proteins (NAG/Lac) on the paracrine interactions between human microvascular endothelial cells (HMVEC) and HF dermal papilla cells (FDPC). Both SR and NAG/Lac-induced endothelial tubulogenesis were enhanced by FDPC. SR promoted proliferation of both the cell types, while NAG/Lac was effective on endothelium. Vascular endothelial growth factor production, enhanced by SR, was further augmented by FDPC. In FDPC 5-α reductase-II and ß-catenin expressions were modified by SR and less by NAG/Lac, with no additional effect by HMVEC. SR and NAG/Lac prevented lipid peroxidation, whereas NAG/Lac was effective on interleukin 1ß production. Finally, SR and NAG/Lac differentially affected HMVEC permeability and tight junction proteins content. These data provide a mechanistic background for the potential use of these compounds as promoters of HF vascularization.

Additive effect of bFGF and selenium on expansion and paracrine action of human amniotic fluid-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell-derived conditioned medium (MSC-CM) has emerged as a promising cell-free tool for restoring degenerative diseases and treating traumatic injuries. The present study describes the effect of selenium as a reactive oxygen species (ROS) scavenger and its additive effect with basic fibroblast growth factor (bFGF) on in vitro expansion of amniotic fluid (AF)-MSCs and the paracrine actions of AF-MSC-CM as well as the associated cellular and molecular mechanisms. METHODS: In this study, we obtained CM from human AF-MSCs cultured with selenium. The stemness of selenium-treated AF-MSCs was evaluated by cell growth and differentiation potential. Human fibroblasts were treated with AF-MSC-CM and analyzed for cell signaling changes. For in vivo wound healing assay, ICR mice with a full-thickness skin wound were used. RESULTS: Selenium played a critical role in in vitro expansion of AF-MSCs through activation of the AKT-ERK1/2, Smad2, and Stat3 signaling pathways along with inactivation of GSK3ß. When administered together with bFGF, it showed remarkable effect in inhibiting ROS accumulation and preserving their multipotency. Proliferation and migration of human dermal fibroblasts and in vivo wound healing were improved in the CMs derived from AF-MSCs exposed to selenium and bFGF, which was caused by the Smad2, AKT-MEK1/2-ERK, and NFκB signaling triggered by the paracrine factors of AF-MSCs, such as TGF-ß, VEGF, and IL-6. Our results suggest the following: (a) supplementation of selenium in AF-MSC culture contributes to in vitro expansion and preservation of multipotency, (b) ROS accumulation causes progressive losses in proliferative and differentiation potential, (c) the separate activities of bFGF and selenium in MSCs exert an additive effect when used together, and (d) the additive combination improves the therapeutic effects of AF-MSC-derived CMs on tissue repair and regeneration. CONCLUSION: Antioxidants, such as selenium, should be considered as an essential supplement for eliciting the paracrine effects of MSC-CMs.

Marina crystal minerals (MCM) activate human dendritic cells to induce CD4+ and CD8+ T cell responses in vitro.

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs' maturation and function in vitro. Human monocyte-derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1ß, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

1,3,6,7-Tetrahydroxy-8-prenylxanthone ameliorates inflammatory responses resulting from the paracrine interaction of adipocytes and macrophages.

BACKGROUND AND PURPOSE: Chronic inflammation in adipose tissue is critical in the onset and development of insulin resistance and type 2 diabetes. Macrophage infiltration into adipose tissue and pro-inflammatory polarization play key roles in adipose tissue inflammation. The fruit hull of mangosteen (Garcinia mangostana) is used in traditional medicine to treat various inflammatory diseases. However, its role in regulating adipose tissue inflammation is unexplored. This study was designed to identify xanthones from G. mangostana, which could ameliorate adipose tissue inflammation. EXPERIMENTAL APPROACH: Expressions of inducible NOS, cytokines, chemokines and components of the NF-κB and MAPKs pathways were evaluated using Western blotting, immunofluorescence, quantitative real-time PCR or ELISA. The migration of macrophages towards adipocytes was tested using Transwell experiments in vitro. A murine model of LPS-induced acute inflammation was used to examine effects of 1,3,6,7-tetrahydroxy-8-prenylxanthone (TPX) on inflammatory responses in adipose tissue in vivo. KEY RESULTS: From a series of xanthones isolated from G. mangostana, TPX was identified as a potent inhibitor of LPS-induced NO production and IL-6 secretion in RAW264.7 macrophages. TPX ameliorated LPS-induced inflammatory responses in RAW264.7 macrophages, and TNF-α-mediated inflammation in 3T3-L1 adipocytes, through inhibiting MAPKs and NF-κB activation and promoting sirtuin 3 expression. TPX also blocked RAW264.7 macrophages migration towards 3T3-L1 adipocytes in co-cultures. Furthermore, TPX alleviated LPS-induced adipose tissue inflammation in vivo by reducing pro-inflammatory cytokines and preventing the pro-inflammatory polarization of macrophages. CONCLUSIONS AND IMPLICATIONS: Taken together, our results indicate that TPX disrupts the inflammatory responses between macrophages and adipocytes, and attenuates adipose tissue inflammation.

Luhong formula inhibits myocardial fibrosis in a paracrine manner by activating the gp130/JAK2/STAT3 pathway in cardiomyocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Luhong formula (LHF)-a traditional Chinese medicine containing Cervus nippon Temminck, Carthamus tinctorius L., Cinnamomum cassia Presl, Codonopisis pilosula( Franch.) Nannf., Astragalus membranaceus ( Fisch.) Bge. var. mongholicus ( Bge.) Hsiao, Lepidium apetalum Willd-is used in the treatment of heart failure. AIM OF THE STUDY: To investigate the antifibrotic efficacy of LHF in a myocardial infarction-induced rat model of heart failure and to determine its mechanism of action. MATERIAL AND METHODS: Myocardial infarction was induced in rats by coronary artery ligation, and cardiac fibroblasts were isolated. Neonatal rat cardiomyocytes (NRCMs) were isolated from 2 to 3-day-old Sprague-Dawley male rats, and cardiomyocyte hypertrophy was induced by isoprenaline. Histological examination was carried out to estimate the degree of myocardial fibrosis. Expression of gp130/JAK2/STAT3 pathway proteins was measured by western blot. The mRNA levels of downstream genes of gp130/JAK2/STAT3 pathway (i.e., CTGF, TSP-1, and TIMP1) were determined by RT-PCR; while CTGF, TSP-1, and TIMP1 protein levels were measured by ELISA. To investigate paracrine effects, cell proliferation and collagen synthesis was measured after treating cardiac fibroblasts with the conditioned media from isoprenaline-treated NRCMs. RESULTS: Histopathological changes showed that LHF inhibited myocardial fibrosis in heart failure rats. Treatment with LHF up-regulated gp130, JAK2, and STAT3 protein expression in heart tissue, and down-regulated CTGF, TSP-1, and TIMP1 gene expression. Isoprenaline-treated NRCMs displayed lower expression of the gp130, JAK2, and STAT3 pathway proteins and higher secretion of its downstream signaling molecules (CTGF, TSP-1, TIMP1). LHF inhibited cardiac fibroblast proliferation and collagen synthesis after treatment with the conditioned media from isoprenaline-treated NRCMs. CONCLUSION: LHF treatment attenuates myocardial fibrosis in vivo. LHF inhibits cardiac fibroblasts proliferation and collagen synthesis in a paracrine manner by activating the gp130/JAK2/STAT3 pathway in cardiomyocytes, thereby inhibiting the secretion of downstream profibrogenic cytokines.

Conditioned medium from LS 174T goblet cells treated with oxyresveratrol strengthens tight junctions in Caco-2 cells.

BACKGROUND: Strengthening of intestinal tight junctions provides an effective barrier from the external environment. Goblet cell-derived trefoil factor 3 (TFF3) increases transepithelial resistance by upregulating the expression of tight junction proteins. Oxyresveratrol (OXY) is a hydroxyl-substituted stilbene found in the roots, leaves, stems, and fruit of many plants and known to have various biological activities. In this study, we investigated the strengthening effect of OXY on intestinal tight junctions through stimulation of TFF production in goblet cells. METHODS: We prepared conditioned medium from LS 174T goblet cells treated with OXY (GCO-CM) and investigated the effect of GCO-CM on strengthening tight junctions of Caco-2 cells. The mRNA and protein expression levels of major tight junction components (claudin-1, occludin, and ZO-1) were measured by quantitative real-time PCR and western blotting, respectively. Transepithelial electric resistance (TEER) was measured using an ohm/V meter. Monolayer permeability was evaluated by paracellular transport of fluorescein isothiocyanate-dextran. RESULTS: OXY showed a strong antioxidant activity. It significantly increased the expression level of TFF3 in LS 174T goblet cells. GCO-CM prepared by treatment with 2.5, 5, and 10µg/ml OXY did not show cytotoxicity in Caco-2 cells. GCO-CM increased the mRNA and protein expression levels of claudin-1, occludin, and ZO-1. It also significantly increased tight junction integrity and reduced permeability in a dose-dependent manner. CONCLUSION: OXY stimulates the expression of TFF3 in goblet cells, which might increase the integrity of the intestinal tight junction barrier.

Frontline Science: D1 dopaminergic receptor signaling activates the AMPK-bioenergetic pathway in macrophages and alveolar epithelial cells and reduces endotoxin-induced ALI.

Catecholamines, including ß-adrenergic and dopaminergic neurotransmitters, have an essential role in regulating the "fight or flight" reflex and also affects immune cell proinflammatory action. However, little is known about whether catecholamines prevent dysfunction of metabolic pathways associated with inflammatory organ injury, including development of acute lung injury (ALI). We hypothesize that selected catecholamines may reduce metabolic alterations in LPS-stimulated macrophages and in the lungs of mice subjected to endotoxin-induced ALI, a situation characterized by diminished activity of AMP-activated protein kinase (AMPK). We found that activation of the dopamine 1 receptor (D1R) with fenoldopam, but not stimulation of adrenergic receptors with norepinephrine, resulted in a robust activation of AMPK in peritoneal macrophages, human monocytes, or alveolar epithelial cells (AECs). Such AMPK activation was mediated by a phospholipase C (PLC)-dependent mechanism. Unlike norepinephrine, D1R activation also prevented Thr172-AMPK dephosphorylation and kinase inactivation in LPS-treated macrophages. Furthermore, we show that a culture of AECs with either fenoldopam or the AMPK activator metformin effectively diminished IL-1ß-induced release of adverse paracrine signaling, which promotes the macrophage proinflammatory response. In vivo, fenoldopam reduced the severity of LPS-induced ALI, including development of pulmonary edema, lung permeability, and production of inflammatory cytokines TNF-α, MIP-2, or KC and HMGB1. Fenoldopam also prevented AMPK dephosphorylation in the lungs of LPS-treated mice and prevented loss of mitochondrial complexes NDUFB8 (complex I) and ATP synthase (complex V). Collectively, these results suggest that dopamine is coupled to AMPK activation, which provides a substantial anti-inflammatory and bioenergetic advantage and reduces the severity of endotoxin-induced ALI.

CP-25 attenuates the inflammatory response of fibroblast-like synoviocytes co-cultured with BAFF-activated CD4(+) T cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells. METHODS: The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1ß, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA). RESULTS: The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1ß, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells. CONCLUSION: The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability to inhibit the expression of BAFF-R in CD4(+) T cells in a co-culture. These observations might provide a scientific basis for the development of new drugs for the treatment of autoimmune diseases by CP-25.

Effect of YangZheng XiaoJi Extract, DME-25, on Endothelial Cells and their Response to Avastin.

BACKGROUND: Angiogenesis is a cellular process that has been identified as a key target for therapy in solid cancer. However, over the course of anti-angiogenic therapies, cancer cells acquire resistance to these therapies after an initial period of success. DME-25 is an extract from Yang Zheng Xiao Ji, a traditional Chinese medicine that has been reported to benefit patients with cancer by alleviating chemotherapy-associated symptoms and possibly inhibiting key cancer cell traits. This study aimed to explore if DME-25 on its own and in combination with avastin affected endothelial cell behaviour in vitro in the presence of hypoxic lung cancer-conditioned medium (CM). MATERIALS AND METHODS: Two lung cancer cell lines, A549 and SK-MES-1, were exposed to hypoxic conditions (O2 ≤1%) for 4 h, after which CM, and RNA were collected. Transcript expression of several influential angiogenic markers in lung cancer cells were assessed following hypoxic/normoxic conditions. Lung cancer CM was added in combination with avastin and DME-25, before or after vascular endothelial growth factor (VEGF) depletion, to endothelial cells (HECV) and cell migration and microtubule formation were assessed in vitro. RESULTS: HECV cell migration was reduced in the presence of avastin, although less efficiently in the presence of lung cancer CM. A combination of DME-25 and avastin with lung cancer CM significantly reduced HECV cell migration irrespective of culture under hypoxia or normoxia. Depletion of VEGF from the CM reduced the inhibitory capacity of avastin, however, it appeared to have little impact on the anti-angiogenic effects of DME-25. CONCLUSION: DME-25 inhibits tubule formation irrespectively of the factors secreted by normoxic or hypoxic lung cancer cell CM depleted of VEGF.

Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

The herbal compound Songyou Yin (SYY) inhibits hepatocellular carcinoma growth and improves survival in models of chronic fibrosis via paracrine inhibition of activated hepatic stellate cells.

Chronic fibrosis is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathological progression of hepatic fibrosis has been linked to cellular processes that promote tumor growth and metastasis. Several recent studies have highlighted the cross-talk between tumor cells and activated hepatic stellate cells (aHSCs) in HCC. The herbal compound Songyou Yin (SYY) is known to attenuate hepatoma cell invasion and metastasis via down-regulation of cytokine secretion by aHSCs. However the underlying mechanism of SYY treatment in reversal of hepatic fibrosis and metastasis of liver cancers is not known. In the current study, a nude mouse model with liver fibrosis bearing orthotopic xenograft was established and we found that SYY could reduce associated fibrosis, inhibit tumor growth and improve survival. In the subcutaneous tumor model with fibrosis, we found that SYY could inhibit liver cancer. In vitro, hepatoma cells incubated with conditioned media (CM) from SYY treated aHSCs showed reduced proliferation, decrease in colony formation and invasive potential. SYY treated group showed altered gene expression, with 1205 genes up-regulated and 1323 genes down-regulated. Gene cluster analysis indicated that phosphatidylinositol-3-kinase (PI3K) was one of the key genes altered in the expression profiles. PI3K related markers were all significantly down-regulated. ELISA also indicated decreased secretion of cytokines which were regulated by PI3K/AKT signaling after SYY treatment in the hepatic stellate cell line, LX2. These data clearly demonstrate that SYY therapy inhibits HCC invasive and metastatic potential and improves survival in nude mice models with chronic fibrosis background via inhibition of cytokine secretion by activated hepatic stellate cells.

Gonadotropin-releasing hormone stimulates biliary proliferation by paracrine/autocrine mechanisms.

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.

n-3 polyunsaturated fatty acids and mechanisms to mitigate inflammatory paracrine signaling in obesity-associated breast cancer.

Globally, the prevalence of obesity is increasing which subsequently increases the risk of the development of obesity-related chronic diseases. Low-grade chronic inflammation and dysregulated adipose tissue inflammatory mediator/adipokine secretion are well-established in obesity, and these factors increase the risk of developing inflammation-associated cancer. Breast cancer is of particular interest given that increased inflammation within the subcutaneous mammary adipose tissue depot can alter the local tissue inflammatory microenvironment such that it resembles that of obese visceral adipose tissue. Therefore, in obese women with breast cancer, increased inflammatory mediators both locally and systemically can perpetuate inflammation-associated pro-carcinogenic signaling pathways, thereby increasing disease severity. Herein, we discuss some of these inflammation-associated pro-carcinogenic mechanisms of the combined obese breast cancer phenotype and offer evidence that dietary long chain n-3 polyunsaturated fatty acids (PUFA) may have utility in mitigating the severity of obesity-associated inflammation and breast cancer.