BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most common cancer diagnosed in women worldwide. BC stem cells (BCSCs) have been known to be involved in the carcinogenesis of the breast and contribute to therapeutic resistance. The programmed death-ligand 1 (PD-L1) expression of BC correlated with a poor prognosis. Immunotherapies that target PD-L1 have great potential and have been successful when applied to cancer treatment. However, whether PD-L1 regulates BCSC formation is unknown. METHODS: BCSCs were enriched by serum-free suspension culture. The properties of BCSCs were examined by mammosphere formation assay, CD44+/Cd24-, aldehyde dehydrogenase (ALDH) assay, CSC marker analysis, and mammosphere growth assay. To elucidate the functions of bromodomain-containing protein 4 (BRD4), nuclear PD-L1, and RelB proteins in the stemness of BCSCs, mammosphere formation was examined using BRD4 inhibitor and degrader, PD-L1 degrader, and RelB inhibitor. The antitumor function of 3',4',7,8-tetrahydroxyflavone (THF), a specific BRD4 inhibitor, was studied through in vivo tumor model and mouse studies, and the protein levels of c-Myc, PD-L1, and RelB were examined in tumor model under THF treatment. RESULTS: BRD4 was upregulated in breast CSCs and regulates the stemness of BCs. The downregulation of BRD4 using BRD4 PROTAC, ARV-825, and BRD4 inhibitor, (+)-JQ1, inhibits mammosphere formation and reduces the levels of breast CSC markers (CD44+/CD24- and ALDH1), stem cell marker genes, and mammosphere growth. BRD4 inhibitor (JQ1) and degrader (ARV825) downregulate membrane and nuclear fractions of PD-L1 through the inhibition of PD-L1 transcript levels. The knockdown of PD-L1 inhibits mammosphere formation. Verteporfin, a PD-L1 degrader, inhibits the transcripts and protein levels of PD-L1 and downregulates the transcript and protein levels of RelB. Calcitriol, a RelB inhibitor, and the knockdown of RelB using si-RelB regulate mammosphere formation through interleukin-6 (IL-6) expression. THF is a natural product and a potent selective BRD4 inhibitor, inhibits mammosphere formation, and reduces the levels of CD44+/CD24- and mammosphere growth by downregulating c-Myc, PD-L1, and RelB. 3',4',7,8-THF shows tumoricidal activity and increased levels of CD3+CD4+ and CD3+CD8+ T-cells in the tumor and tumor-draining lymph nodes (TDLNs) in the murine tumor model using 4T1 and MC38 cells. CONCLUSIONS: The results show the first evidence of the essential role of the BRD4/nuclear PD-L1/RelB axis in breast CSC formation. The nuclear PD-L1 regulates RelB, and the RelB/p65 complex induces IL6 and breast CSC formation. Targeting nuclear PD-L1 represents a potential and novel tool for immunotherapies of intractable BC. Video Abstract.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic Acid Overcomes Chemoresistance of 5-Fluorouracil by Suppressing the Infiltration of Tumor-Associated Macrophages and Inhibiting the Activation of Cancer Stem Cells in a Colorectal Cancer Xenograft Model.

Although the tumor bulk is initially reduced by 5-fluorouracil (5-FU), chemoresistance developed due to prolonged chemotherapy in colorectal cancer (CRC). The enrichment of cancer stem cells (CSCs) and the infiltration of tumor-associated macrophages (TAMs) contribute to chemoresistance and poor outcomes. A docosahexaenoic acid derivative developed by our group, 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), exerts antitumor effects against TAMs infiltration and CSCs enrichment in our previous study. The current study aimed to investigate whether diHEP-DPA was able to overcome chemoresistance to 5-FU in CRCs, together with the potential synergistic mechanisms in a CT26-BALB/c mouse model. Our results suggested that although 5-FU inhibited tumor growth, 5-FU enriched CSCs via the WNT/ß-catenin signaling pathway, resulting in chemoresistance in CRCs. However, we revealed that 5-FU promoted the infiltration of TAMs via the NF-kB signaling pathway and improved epithelial-mesenchymal transition (EMT) via the signal transducer and activator of the transcription 3 (STAT3) signaling pathway; these traits were believed to contribute to CSC activation. Furthermore, supplementation with diHEP-DPA could overcome drug resistance by decreasing the CSCs, suppressing the infiltration of TAMs, and inhibiting EMT progression. Additionally, the combinatorial treatment of diHEP-DPA and 5-FU effectively enhanced phagocytosis by blocking the CD47/signal regulatory protein alpha (SIRPα) axis. These findings present that diHEP-DPA is a potential therapeutic supplement to improve drug outcomes and suppress chemoresistance associated with the current 5-FU-based therapies for colorectal cancer.

Primaquine Inhibits the Endosomal Trafficking and Nuclear Localization of EGFR and Induces the Apoptosis of Breast Cancer Cells by Nuclear EGFR/Stat3-Mediated c-Myc Downregulation.

Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.

Physalin A, 13,14-Seco-16, 24-Cyclo-Steroid, Inhibits Stemness of Breast Cancer Cells by Regulation of Hedgehog Signaling Pathway and Yes-Associated Protein 1 (YAP1).

The Hedgehog (HH) signaling pathway plays an important role in embryonic development and adult organ homeostasis. Aberrant activity of the Hedgehog signaling pathway induces many developmental disorders and cancers. Recent studies have investigated the relationship of this pathway with various cancers. GPCR-like protein Smoothened (SMO) and the glioma-associated oncogene (GLI1) are the main effectors of Hedgehog signaling. Physalin A, a bioactive substance derived from Physalis alkekengi, inhibits proliferation and migration of breast cancer cells and mammospheres formation. Physalin A-induced apoptosis and growth inhibition of mammospheres, and reduced transcripts of cancer stem cell (CSC) marker genes. Physalin A reduced protein expressions of SMO and GLI1/2. Down-regulation of SMO and GLI1 using siRNA inhibited mammosphere formation. Physalin A reduced mammosphere formation by reducing GLI1 gene expression. Down-regulation of GLI1 reduced CSC marker genes. Physalin A reduced protein level of YAP1. Down-regulation of YAP1 using siRNA inhibited mammosphere formation. Physalin A reduced mammosphere formation through reduction of YAP1 gene expression. Down-regulation of YAP1 reduced CSC marker genes. We showed that treatment of MDA-MB-231 breast cancer cells with GLI1 siRNA induced inhibition of mammosphere formation and down-regulation of YAP1, a Hippo pathway effector. These results show that Hippo signaling is regulated by the Hedgehog signaling pathway. Physalin A also inhibits the canonical Hedgehog and Hippo signaling pathways, CSC-specific genes, and the formation of mammospheres. These findings suggest that physalin A is a potential therapeutic agent for targeting CSCs.

The FDA-Approved Anti-Asthma Medicine Ciclesonide Inhibits Lung Cancer Stem Cells through Hedgehog Signaling-Mediated SOX2 Regulation.

Ciclesonide is an FDA-approved glucocorticoid (GC) used to treat asthma and allergic rhinitis. However, its effects on cancer and cancer stem cells (CSCs) are unknown. Our study focuses on investigating the inhibitory effect of ciclesonide on lung cancer and CSCs and its underlying mechanism. In this study, we showed that ciclesonide inhibits the proliferation of lung cancer cells and the growth of CSCs. Similar glucocorticoids, such as dexamethasone and prednisone, do not inhibit CSC formation. We show that ciclesonide is important for CSC formation through the Hedgehog signaling pathway. Ciclesonide reduces the protein levels of GL1, GL2, and Smoothened (SMO), and a small interfering RNA (siRNA) targeting SMO inhibits tumorsphere formation. Additionally, ciclesonide reduces the transcript and protein levels of SOX2, and an siRNA targeting SOX2 inhibits tumorsphere formation. To regulate breast CSC formation, ciclesonide regulates GL1, GL2, SMO, and SOX2. Our results unveil a novel mechanism involving Hedgehog signaling and SOX2 regulated by ciclesonide in lung CSCs, and also open up the possibility of targeting Hedgehog signaling and SOX2 to prevent lung CSC formation.

The Antiasthma Medication Ciclesonide Suppresses Breast Cancer Stem Cells through Inhibition of the Glucocorticoid Receptor Signaling-Dependent YAP Pathway.

Ciclesonide is an FDA-approved glucocorticoid used to treat asthma and allergic rhinitis. However, whether it has anticancer and anti-cancer stem cell (CSC) effects is unknown. This study focused on investigating the effect of ciclesonide on breast cancer and CSCs and determining its underlying mechanism. Here, we showed that ciclesonide inhibits breast cancer and CSC formation. Similar glucocorticoids-dexamethasone and prednisone-did not inhibit CSC formation. Ciclesonide-induced glucocorticoid receptor (GR) degradation was dependent on ubiquitination. We showed via GR small interfering RNA (siRNA) that GR plays an important role in CSC formation. We showed via western blot and immunofluorescence assays that ciclesonide reduces the nuclear level of GR. The GR antagonist RU-486 also inhibited CSC formation. Ciclesonide reduced the protein level of the Hippo transducer Yes-associated protein (YAP). GR siRNA induced a decrease in YAP protein expression and inhibited mammosphere formation. The YAP inhibitor verteporfin inhibited CSC formation and transcription of the connective tissue growth factor and cysteine-rich protein 61 genes. The GR/YAP1 pathway regulated breast CSC formation. We showed that the GR/YAP signaling pathway regulates breast CSC formation and revealed a new approach for targeting GR and YAP to inhibit CSC formation.

Coriolic Acid (13-(S)-Hydroxy-9Z, 11E-octadecadienoic Acid) from Glasswort (Salicornia herbacea L.) Suppresses Breast Cancer Stem Cell through the Regulation of c-Myc.

Cancer stem cells have certain characteristics, such as self-renewal, differentiation, and drug resistance, which are related to tumor progression, maintenance, recurrence, and metastasis. In our study, we targeted breast cancer stem cells (BCSCs) using a natural compound, coriolic acid, from Salicornia herbacea L. This compound was isolated by mammosphere formation inhibition bioassay-guided fractionation and identified by using NMR spectroscopy and electrospray ionization mass spectrometry. Coriolic acid inhibited the formation of mammospheres and induced BCSC apoptosis. It also decreased the subpopulation of CD44high/CD24low cells, a cancer stem cell (CSC) phenotype, and specific genes related to CSCs, such as Nanog,Oct4, and CD44. Coriolic acid decreased the transcriptional and translational levels of the c-Myc gene, which is a CSC survival factor. These results indicated that coriolic acid could be a novel compound to target BCSCs via regulation of c-Myc.

Inhibitory Effects of Tangeretin, A Citrus Peel-Derived Flavonoid, on Breast Cancer Stem Cell Formation through Suppression of Stat3 Signaling.

Breast cancer stem cells (BCSCs) are responsible for tumor chemoresistance and recurrence. Targeting CSCs using natural compounds is a novel approach for cancer therapy. A CSC-inhibiting compound was purified from citrus extracts using silica gel, gel filtration and high-pressure liquid chromatography. The purified compound was identified as tangeretin by using nuclear magnetic resonance (NMR). Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The frequency of a subpopulation with a CSC phenotype (CD44+/CD24-) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3). Our results in this study show that tangeretin inhibits the Stat3 signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound that targets breast cancer cells and CSCs.

6-Methoxymellein Isolated from Carrot (Daucus carota L.) Targets Breast Cancer Stem Cells by Regulating NF-κB Signaling.

The presence of breast cancer stem cells (BCSCs) induces the aggressive progression and recurrence of breast cancer. These cells are drug resistant, have the capacity to self-renew and differentiate and are involved in recurrence and metastasis, suggesting that targeting BCSCs may improve treatment efficacy. In this report, methanol extracts of carrot root were purified by means of silica gel, Sephadex LH-20, and preparative high-performance liquid chromatography to isolate a compound targeting mammosphere formation. We isolated the compound 6-methoxymellein, which inhibits the proliferation and migration of breast cancer cells, reduces mammosphere growth, decreases the proportion of CD44+/CD24- cells in breast cancer cells and decreases the expression of stemness-associated proteins c-Myc, Sox-2 and Oct4. 6-Methoxymellein reduces the nuclear localization of nuclear factor-κB (NF-κB) subunit p65 and p50. Subsequently, 6-methoxymellein decreases the mRNA transcription and secretion of IL-6 and IL-8. Our data suggest that 6-methoxymellein may be an anticancer agent that inhibits BCSCs via NF-κB/IL-6 and IL-8 regulation.

Betavulgarin Isolated from Sugar Beet (Beta vulgaris) Suppresses Breast Cancer Stem Cells through Stat3 Signaling.

Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells (BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal, recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected to silica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) for isolation of one compound, based on activity-guided purification using tumorsphere formation assays. The purified compound was identified as betavulgarin, using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) mass spectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, and mammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24- subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4. This compound decreased the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sex determining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest that betavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicating betavulgarin might be an anticancer agent against breast cancer cells and BCSCs.

Triterpene Acid (3-O-p-Coumaroyltormentic Acid) Isolated From Aronia Extracts Inhibits Breast Cancer Stem Cell Formation through Downregulation of c-Myc Protein.

Cancer stem cells (CSCs) are drug-resistant and radiation-resistant cancer cells that are responsible for tumor progression and maintenance, cancer recurrence, and metastasis. Targeting breast CSCs with phytochemicals is a new paradigm for cancer prevention and treatment. In this study, activity-guided fractionation from mammosphere formation inhibition assays, repeated chromatographic preparations over silica gel, preparatory thin layer chromatography, and HPLC using aronia extracts led to the isolation of one compound. Using ¹H and 13C 2-dimensional nuclear magnetic resonance (NMR) as well as electrospray ionization (ESI) mass spectrometry, the isolated compound was identified as 3-O-p-coumaroyltormentic acid. This compound inhibits breast cancer cell proliferation and mammosphere formation in a dose-dependent manner and reduces the CD44high/CD24low subpopulation and aldehyde dehydrogenase (ALDH)-expressing cell population as well as the expression of the self-renewal-related genes CD44, SOX2, and OCT4.3-O-p-Coumaroyltormentic acid preferentially reduced the protein levels of c-Myc, which is a CSC survival factor, by inducing c-Myc degradation. These findings indicate the novel utilization of 3-O-p-coumaroyltormentic acid for breast cancer therapy via disruption of c-Myc protein, which is a CSC survival factor.

Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1.

Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5'-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). J. Cell. Physiol. 232: 576-584, 2017. © 2016 Wiley Periodicals, Inc.

Screening of breast cancer stem cell inhibitors using a protein kinase inhibitor library.

BACKGROUND: Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)'s Integrated Colony Enumerator (NICE) with a screening of protein kinase library. METHODS: Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated. RESULTS: Automated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IκB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-ß1-induced migration, colony formation and EMT of MCF-7 cells. CONCLUSIONS: We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.

Baicalin and baicalein inhibit transforming growth factor-ß1-mediated epithelial-mesenchymal transition in human breast epithelial cells.

Since the epithelial-mesenchymal transition (EMT) is involved in many crucial functions of cancer cells, we set out to identify a natural compound capable of inhibiting EMT processes. TGF-ß1 treatment induces EMT among normal mammary epithelial cells (MCF10A cells), as reflected by characteristic morphological changes into the fibroblastic phenotype, reduced expression of E-cadherin. Interestingly, butanol extracts of Scutellaria baicalensis Georgi significantly reduced the TGF-ß1-mediated EMT of MCF10A cells. Further analysis revealed that baicalin and baicalein, the major flavones of these butanol extracts, inhibited TGF-ß1-mediated EMT by reducing the expression level of the EMT-related transcription factor, Slug via the NF-κB pathway, and subsequently increased migration in MCF10A cells. Finally, both compounds reduced the TGF-ß1-mediated EMT, anchorage-independent growth and cell migration of human breast cancer cells (MDA-MB-231 cells). Taken together, these results suggest that baicalin and baicalein of Scutellaria baicalensis Georgi may suppress the EMT of breast epithelial cells and the tumorigenic activity of breast cancer cells. Thus, these compounds could have potential as therapeutic or supplementary agents for the treatment of breast cancer.

Renoprotective effect of red ginseng in gentamicin-induced acute kidney injury.

Aminoglycoside-induced nephrotoxicity is one of the prevalent causes of acute kidney injury (AKI). Oxidative stress-mediated apoptosis of renal tubular cells is known to be a major mechanism of renal injury. Red ginseng extract (RGE) has been reported to possess antioxidant and immune-modulatory activities. We investigated the effect of RGE on gentamicin (GM)-induced apoptosis and oxidative stress in cultured renal tubular cells and animal model of GM-induced AKI. GM induced the generation of reactive oxygen species (ROS) with an increase in NADPH oxidase (NOX) activity and mitochondrial oxidation in NRK-52E cells that were ameliorated with RGE. GM-induced apoptosis of NRK-52E cells, which was associated with an increased expression of mitochondrial Bax, cytosolic cytochrome c, and cleaved caspase-9 and -3, along with a decrease in bcl-2 expression, was also blocked by RGE. In an animal model of GM-induced AKI, RGE treatment significantly attenuated renal dysfunction, cell apoptosis, and tubular damage. RGE ameliorated ROS production in rats with GM-induced AKI, as demonstrated by an increase in the reduced form of glutathione in renal cortex and a decrease in urinary excretion of 8-hydroxy-2'-deoxyguanosine. Our results suggest that RGE protects the kidney from GM-induced AKI via the mechanism of modulation of oxidative stress.

Uric acid induces fat accumulation via generation of endoplasmic reticulum stress and SREBP-1c activation in hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common types of chronic liver injury. Elevated serum uric acid is a strong predictor of the development of fatty liver as well as metabolic syndrome. Here we demonstrate that uric acid induces triglyceride accumulation by SREBP-1c activation via induction of endoplasmic reticulum (ER) stress in hepatocytes. Uric acid-induced ER stress resulted in an increase of glucose-regulated protein (GRP78/94), splicing of the X-box-binding protein-1 (XBP-1), the phosphorylation of protein kinase RNA-like ER kinase (PERK), and eukaryotic translation initiation factor-2α (eIF-2α) in cultured hepatocytes. Uric acid promoted hepatic lipogenesis through overexpression of the lipogenic enzyme, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) via activation of SREBP-1c, which was blocked by probenecid, an organic anion transport blocker in HepG2 cells and primary hepatocytes. A blocker of ER stress, tauroursodeoxycholic acid (TUDCA), and an inhibitor of SREBP-1c, metformin, blocked hepatic fat accumulation, suggesting that uric acid promoted fat synthesis in hepatocytes via ER stress-induced activation of SREBP-1c. Uric acid-induced activation of NADPH oxidase preceded ER stress, which further induced mitochondrial ROS production in hepatocytes. These studies provide new insights into the mechanisms by which uric acid stimulates fat accumulation in the liver.

Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by persistent inflammation and joint destruction. A lipid mediator (LM, namely, 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA, exhibits anti-inflammatory activity. In this study, we determined the effect of LM on collagen antibody-induced arthritis (CAIA) in mice and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in RAW264.7 cells. LM effectively downregulated the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K, inhibited osteoclast formation, and suppressed the NF-κB signaling pathway in vitro. In vivo, LM at 10 µg/kg/day significantly decreased paw swelling and inhibited progression of arthritis in CAIA mice. Moreover, proinflammatory cytokine (tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, IL-17, and interferon-γ) levels in serum were decreased, whereas IL-10 levels were increased following LM treatment. Furthermore, LM alleviated joint inflammation, cartilage erosion, and bone destruction in the ankles, which may be related to matrix metalloproteinase and Janus kinase (JAK)-signal transducer and activators of transcription (STAT) signaling pathway. Our findings suggest that LM attenuates arthritis severity, restores serum imbalances, and modifies joint damage. Thus, LM represents a promising therapy for relieving RA symptoms.

Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease.

Recent experimental and clinical studies suggest a causal role of uric acid in the development of chronic kidney disease. Most studies have focused on uric acid-induced endothelial dysfunction, oxidative stress, and inflammation in the kidney. The direct effects of uric acid on tubular cells have not been studied in detail, and whether uric acid can mediate phenotypic transition of renal tubular cells such as epithelial-to-mesenchymal transition (EMT) is not known. We therefore investigated whether uric acid could alter E-cadherin expression and EMT in the kidney of hyperuricemic rats and in cultured renal tubular cells (NRK cells). Experimental hyperuricemia was associated with evidence of EMT before the development of significant tubulointerstitial fibrosis at 4 wk, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited uric acid-induced changes in E-cadherin and α-SMA with an amelioration of renal fibrosis at 6 wk. In cultured NRK cells, uric acid induced EMT, which was blocked by the organic anion transport inhibitor probenecid. Uric acid increased expression of transcriptional factors associated with decreased synthesis of E-cadherin (Snail and Slug). Uric acid also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of E-cadherin is considered to be a triggering mechanism for EMT. In conclusion, uric acid induces EMT of renal tubular cells decreasing E-cadherin synthesis via an activation of Snail and Slug as well as increasing the degradation of E-cadherin.

Vimentin interacts with the 5'-untranslated region of mouse mu opioid receptor (MOR) and is required for post-transcriptional regulation.

The opioid receptors are among the most highly studied members of the superfamily of G-protein coupled receptors. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Expression of opioid receptor proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. Here, we demonstrate for the first time the role of vimentin as a post-transcriptional repressor in MOR gene regulation. To identify potential regulators of the mouse MOR gene, we performed affinity column chromatography using 5'-UTR-specific RNA oligonucleotides using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified an intermediate filament protein, vimentin, which bound specifically to the region between -175 and -150 (175-150) of the MOR 5'-UTR. Binding was confirmed by western blot analysis and RNA supershift assay. Furthermore, a cotransfection study demonstrated that the presence of vimentin resulted in reduced expression of the mouse MOR. Our data suggest that vimentin functions as a repressor of MOR translation, dependent on 175-150 of the MOR 5'-UTR.

Post-transcriptional regulation of mu-opioid receptor: role of the RNA-binding proteins heterogeneous nuclear ribonucleoprotein H1 and F.

Classical opioids have been historically used for the treatment of pain and are among the most widely used drugs for both acute severe pain and long-term pain. Morphine and endogenous mu-opioid peptides exert their pharmacological actions mainly through the mu-opioid receptor (MOR). However, the expression of opioid receptor (OR) proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. To identify proteins binding to the 5'-UTR as potential regulators of the mouse MOR gene, affinity column chromatography using 5'-UTR-specific RNA oligonucleotides was performed using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified two heterogeneous ribonucleoproteins (hnRNPs) that bound to RNA sequences of interest: hnRNP H1 and hnRNP F. Binding of these proteins to the RNA region was M4-region sequence-specific as confirmed by Western-blot analysis and RNA supershift assay. Furthermore, a cotransfection study showed that the presence of hnRNP H1 and F resulted in repressed expression of the mouse MOR. Our data suggest that hnRNP H1 and F can function as repressors of MOR translation dependent on the M4 (-75 to -71 bp upstream of ATG) sequences. We demonstrate for the first time a role of hnRNPs as post-transcriptional repressors in MOR gene regulation.