Identification and characterization of bioactive metabolites of 12-hydroxyheptadecatrienoic acid, a ligand for leukotriene B4 receptor 2.

12(S)-hydroxyheptadecatrienoic acid (12-HHT) is a bioactive fatty acid synthesized from arachidonic acid via the cyclooxygenase pathway and serves as an endogenous ligand for the low-affinity leukotriene B4 receptor 2 (BLT2). Although the 12-HHT/BLT2 axis contributes to the maintenance of epithelial homeostasis, 12-HHT metabolism under physiological conditions is unclear. In this study, 12-keto-heptadecatrienoic acid (12-KHT) and 10,11-dihydro-12-KHT (10,11dh-12-KHT) were detected as 12-HHT metabolites in the human megakaryocytic cell line MEG01s. We found that 12-KHT and 10,11dh-12-KHT are produced from 12-HHT by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and prostaglandin reductase 1 (PTGR1), key enzymes in the degradation of prostaglandins, respectively. The 15-PGDH inhibitor SW033291 completely suppressed the production of 12-KHT and 10,11dh-12-KHT in MEG01s cells, resulting in a 9-fold accumulation of 12-HHT. 12-KHT and 10,11dh-12-KHT were produced in mouse skin wounds, and the levels were significantly suppressed by SW033291. Surprisingly, the agonistic activities of 12-KHT and 10,11dh-12-KHT on BLT2 were comparable to that of 12-HHT. Taken together, 12-HHT is metabolized into 12-KHT by 15-PGDH, and then 10,11dh-12-KHT by PTGR1 without losing the agonistic activity.

Dietary intake of n-3 polyunsaturated fatty acids alters the lipid mediator profile of the kidney but does not attenuate renal insufficiency.

The efficacy of n-3 polyunsaturated fatty acids (PUFAs) in improving outcomes in a renal ischemia-reperfusion injury (IRI) model has previously been reported. However, the underlying mechanisms remain poorly understood and few reports demonstrate how dietary n-3 PUFAs influence the composition of membrane phospholipids in the kidney. Additionally, it has not been elucidated whether perilla oil (PO), which is mainly composed of the n-3 alpha-linolenic acid, mitigates renal IRI. In this study, we investigated the effect of dietary n-3 PUFAs (PO), compared with an n-6 PUFA-rich soybean oil (SO) diet, on IRI-induced renal insufficiency in a rat model. Levels of membrane phospholipids containing n-3 PUFAs were higher in the kidney of PO-rich diet-fed rats than the SO-rich diet-fed rats. Levels of blood urea nitrogen and serum creatinine were significantly higher in the ischemia-reperfusion group than the sham group under both dietary conditions. However, no significant differences were observed in blood urea nitrogen, serum creatinine, or histological damage between PO-rich diet-fed rats and SO-rich diet-fed rats. In the kidney of PO-rich diet-fed rats, levels of arachidonic acid and arachidonic acid-derived pro-inflammatory lipid mediators were lower than SO-rich diet-fed rats. Eicosapentaenoic acid and eicosapentaenoic acid-derived lipid mediators were significantly higher in the kidney of PO-rich than SO-rich diet-fed rats. These results suggest that dietary n-3 PUFAs alter the fatty acid composition of membrane phospholipids and lipid mediators in the kidney; however, this does not attenuate renal insufficiency or histological damage in a renal IRI model.

Eicosanoids in Skin Wound Healing.

Wound healing is an important process in the human body to protect against external threats. A dysregulation at any stage of the wound healing process may result in the development of various intractable ulcers or excessive scar formation. Numerous factors such as growth factors, cytokines, and chemokines are involved in this process and play vital roles in tissue repair. Moreover, recent studies have demonstrated that lipid mediators derived from membrane fatty acids are also involved in the process of wound healing. Among these lipid mediators, we focus on eicosanoids such as prostaglandins, thromboxane, leukotrienes, and specialized pro-resolving mediators, which are produced during wound healing processes and play versatile roles in the process. This review article highlights the roles of eicosanoids on skin wound healing, especially focusing on the biosynthetic pathways and biological functions, i.e., inflammation, proliferation, migration, angiogenesis, remodeling, and scarring.

Shared transcriptional responses to con- and heterospecific behavioral antagonists in a wild songbird.

The recognition of and differential responses to salient stimuli are among the main drivers of behavioral plasticity, yet, how animals evolve and modulate functional responses to novel classes of antagonistic stimuli remain poorly understood. We studied free-living male red-winged blackbirds (Agelaius phoeniceus) to test whether gene expression responses in blood are distinct or shared between patterns of aggressive behavioral responses directed at simulated conspecific versus heterospecific intruders. In this species, males defend territories against conspecific males and respond aggressively to female brown-headed cowbirds (Molothrus ater), a brood parasite that commonly lays eggs in blackbird nests. Both conspecific songs and parasitic calls elicited aggressive responses from focal subjects and caused a downregulation in genes associated with immune system response, relative to control calls of a second, harmless heterospecific species. In turn, only the conspecific song treatment elicited an increase in singing behavior and an upregulation of genes associated with metabolic processes relative to the two heterospecific calls. Our results suggest that aspects of antagonistic behaviors to both conspecifics and brood parasites can be mediated by similar physiological responses, suggestive of shared molecular and behavioral pathways involved in the recognition and reaction to both evolutionarily old and new enemies.

MicroRNA-493-5p-mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion.

Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer-related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR-493-5p, a major tumor suppressor miRNA that inactivates miR-483-3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR-493-5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR-493-5p given its significant inhibition through 3'-UTR targeting in miR-493-5p-rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR-493-5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR-493-5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR-493-5p activity. In summary, miR-493-5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies.

MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells.

Numerous studies have described the critical role played by microRNAs (miRNAs) in cancer progression and the potential of these small non-coding RNAs for diagnostic or therapeutic applications. However, the mechanisms responsible for the altered expression of miRNAs in malignant cells remain poorly understood. Herein, via epigenetic unmasking, we identified a group of miRNAs located in the imprinted delta like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3 (MEG3) locus that were repressed in hepatic tumor cells. Notably, miR-493-5p epigenetic silencing was correlated with hypermethylation of the MEG3 differentially regulated region (DMR) in liver cancer cell lines and tumor tissues from patients. Experimental rescue of miR-493-5p promoted an anti-cancer response by hindering hepatocellular carcinoma (HCC) cell growth in vitro and tumor progression in vivo. We found that miR-493-5p mediated part of its tumor-suppressor activity by abrogating overexpression of insulin-like growth factor 2 (IGF2) and the IGF2-derived intronic oncomir miR-483-3p in HCC cells characterized by IGF2 loss of imprinting (LOI). In summary, this study describes an unknown miRNA-dependent regulatory mechanism between two distinct imprinted loci and a possible therapeutic window for liver cancer patients exhibiting IGF2-miR-483 LOI and amplification.

Comparison of the effects of pachymic acid, moronic acid and hydrocortisone on the polysome loading of RNAs in lipopolysaccharide-treated THP-1 macrophages.

We have proposed that analysis of ribosome-loaded mRNAs (i.e., the translatome) is useful for elucidation of pharmacological effects of phytocompounds in immune cells, regarding the involvement of post-transcriptional regulation mechanisms. In the present study, we compared the effects of pachymic acid from Poria cocos fungus and moronic acid from propolis with those of hydrocortisone on the translatomes of THP-1 macrophages exposed to bacterial lipopolysaccharide (LPS) to find clues to their biological effects. Polysome-associated RNAs collected from cells treated for 3 h with LPS plus each of the compounds were analyzed by DNA microarray followed by analyses of pathways/gene ontologies (GO). Upregulated mRNAs in enriched pathways that were found to contain AUUUA (AU)-rich motifs were checked by real-time PCR, and expression of candidate RNA-binding proteins stabilizing/destabilizing such AU-rich mRNAs was checked by Western blotting. The numbers of upregulated and downregulated genes (fold-changes ± 2.0 versus vehicle-control) were, respectively, 209 and 125 for moronic acid, 23 and 2 for pachymic acid, and 214 and 59 for hydrocortisone treatment. Overlapping with hydrocortisone treatment for upregulation were 158 genes in moronic acid and 17 in pachymic acid treatment; of these, 16 overlapped within all treatments (C-X-C motif chemokine ligands, interferon-induced protein with tetratricopeptide repeats, etc.). Pathway analyses showed GO enrichments such as 'immune response', 'receptor binding', 'extracellular space' etc. The pachymic acid-upregulated mRNAs (highly overlapped with the other 2 treatments) showed the presence of signal peptides and AU-rich motifs, suggesting regulation by AU-rich element (ARE)-binding proteins. The expression of ARE-binding protein HuR/ELAV-1 was increased by the 3 compounds, and AUF1/hnRNP D was decreased by pachymic acid. These results suggested that pachymic acid and moronic acid effects may involve as yet unknown post-transcriptional modulation via ARE-binding proteins resembling that of glucocorticoids.

Solvent-Free Synthesis of 2-Alkylbenzothiazoles and Bile Acid Derivatives Containing Benzothiazole Ring by Using Active Carbon/Silica Gel and Microwave.

A highly efficient and simple method for the synthesis of 2-alkylbenzothiazoles through the condensation of 2-aminothiophenol and aliphatic aldehydes in the presence of active carbon/silica gel is described. The reaction proceeded under solvent-free and microwave irradiation to afford 2-alkylbenzothiazoles in high yields. This method was extended to the synthesis of bile acid derivatives containing a benzothiazole ring and obtained the desired products in high yields. The anti-inflammatory activity and cytotoxicity of the newly synthesized benzothiazole derivatives of bile acid were tested; the results showed that anti-inflammatory activities of all tested compounds tested were higher than that of standard drugs, such as indomethacin.

Triterpenoids from Euphorbia maculata and Their Anti-Inflammatory Effects.

Euphorbia maculata is a medicinal plant of the Euphorbiaceae family, which can produce anti-inflammatory and cancer chemopreventive agents of triterpenoids. The present study reports on the bioactive triterpenoids of this plant. Two new lanostane-type triterpenoids, named (3S,4S,7S,9R)-4-methyl-3,7-dihydroxy-7(8→9) abeo-lanost-24(28)-en-8-one (1) and 24-hydroperoxylanost-7,25-dien-3ß-ol (2), together with 15 known triterpene derivatives, were isolated from Euphorbia maculata. The structures of the new compounds were determined on the basis of extensive spectroscopic data (UV, MS, ¹H and 13C-NMR, and 2D NMR) analysis. All tetracyclic triterpenoids (1⁻11) were evaluated for their anti-inflammatory effects in the test of TPA-induced inflammation (1 µg/ear) in mice. The triterpenes exhibited significant anti-inflammatory activities.

Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells.

Primary liver tumors are mainly represented by hepatocellular carcinoma (HCC), one of the most aggressive and resistant forms of cancer. Liver tumorigenesis is characterized by an accumulation of epigenetic abnormalities, leading to gene extinction and loss of hepatocyte differentiation. The aim of this work was to investigate the feasibility of converting liver cancer cells toward a less aggressive and differentiated phenotype using a process called epigenetic reconditioning. Here, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine (5-AZA) promoted an anti-cancer response by inhibiting HCC cell tumorigenicity. Furthermore, epigenetic reconditioning improved sorafenib response. Remarkably, epigenetic treatment was associated with a significant restoration of differentiation, as attested by the increased expression of characteristic hepatocyte markers in reconditioned cells. In particular, we showed that reexpression of these epigenetically silenced liver genes following 5-AZA treatment or after knockdown of DNA methyltransferase 1 (DNMT1) was the result of regional CpG demethylation. Lastly, we confirmed the efficacy of HCC differentiation therapy by epigenetic reconditioning using an in vivo tumor growth model. In summary, this work demonstrates that epigenetic reconditioning using the demethylating compound 5-AZA shows therapeutic significance for liver cancer and is potentially attractive for the treatment of solid tumors.

Anti-inflammatory effects of alpinone 3-acetate from Alpinia japonica seeds.

We aimed to investigate the bioactive components of Alpinia japonica as anti-inflammatory compounds using searches of the Alpinia genus, and subsequently demonstrated that alpinone 3-acetate markedly inhibits 12-O-tetradecanoyiphorbol 13-acetate-induced inflammation in a mouse model of ear edema. To assess other bioactivities of alpinone 3-acetate, we performed translatome analyses and compared them with those of hydrocortisone. Polysome-associated mRNAs were prepared from alpinone 3-acetate- or hydrocortisone-treated and control cells from 12-O-tetradecanoyiphorbol 13-acetate-induced THP-1-derived macrophages cultured in the presence of Escherichia coli O-111 lipopolysaccharide. Subsequent microarray analysis revealed that alpinone 3-acetate and hydrocortisone upregulated and downregulated the same 155 and 41 genes, respectively. Moreover, direct comparisons of translationally regulated genes indicated 5 and 10 gene probes that were upregulated and downregulated by alpinone 3-acetate and hydrocortisone, respectively. In conclusion, assays of 12-O-tetradecanoyiphorbol 13-acetate-induced inflammation ear edema in mice and polysome profiling of alpinone 3-acetate bioactivities indicated similar medicinal possibilities to those of hydrocortisone.

Inhibitory effects of compounds isolated from the dried branches and leaves of murta (Myrceugenia euosma) on lipid accumulation in 3T3-L1 cells.

As obesity is a global health concern the demand for anti-obesity drugs is high. In this study, we investigated the anti-obesity effect of the dried branches and leaves of murta (Myrceugenia euosma Legrand, Myrtaceae). A methanol extract of the dried branches and leaves of murta inhibited adipogenesis in 3T3-L1 cells. Three known flavanones-cryptostrobin (1), pinocembrin (4), and 5,7-dihydroxy-6,8-dimethylflavanone (6), and three chalcones-2',6'-dihydroxy-3'-methyl-4'-methoxychalcone (2), pinostrobin chalcone (3), and 2',6'-dihydroxy-4'-methoxy-3',5'-dimethylchalcone (5) were isolated from the active fraction. Structures of these compounds were identified using various spectral data. Each of these compounds also inhibited adipogenesis in 3T3-L1 cells. In particular, compound 3 was a more potent inhibitor of triglyceride accumulation than the positive control berberine. Gene expression studies revealed that treatment of 3T3-L1 cells with 3 lowers the expression levels of CCAAT/enhancer-binding protein α and peroxisome proliferator activator γ2 during adipogenesis without affecting cell viability. Treatment of 3T3-L1 cells with 3 reduced the expression levels of mRNAs encoding sterol regulatory element-binding protein 1c and several lipogenic enzymes, including fatty acid synthase and stearoyl CoA desaturase-1. These results indicate that the methanol extract and compounds isolated from the dried branches and leaves of murta exert their anti-obesity effects through the inhibition of adipogenesis.

A robust screening method for dietary agents that activate tumour-suppressor microRNAs.

Certain dietary agents, such as natural products, have been reported to show anti-cancer effects. However, the underlying mechanisms of these substances in human cancer remain unclear. We recently found that resveratrol exerts an anti-cancer effect by upregulating tumour-suppressor microRNAs (miRNAs). In the current study, we aimed to identify new dietary products that have the ability to activate tumour-suppressor miRNAs and that therefore may serve as novel tools for the prevention and treatment of human cancers. We describe the generation and use of an original screening system based on a luciferase-based reporter vector for monitoring miR-200c tumour-suppressor activity. By screening a library containing 139 natural substances, three natural compounds - enoxolone, magnolol and palmatine chloride - were identified as being capable of inducing miR-200c expression in breast cancer cells at 10 µM. Moreover, these molecules suppressed the invasiveness of breast cancer cells in vitro. Next, we identified a molecular pathway by which the increased expression of miR-200c induced by natural substances led to ZEB1 inhibition and E-cadherin induction. These results indicate that our method is a valuable tool for a fast identification of natural molecules that exhibit tumour-suppressor activity in human cancer through miRNA activation.

Melanogenesis-inhibitory activity and cancer chemopreventive effect of glucosylcucurbic acid from shea (Vitellaria paradoxa) kernels.

Two jasmonate derivatives, glucosylcucurbic acid (1) and methyl glucosylcucurbate (2), were isolated from the MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels. These and their deglucosylated derivatives, cucurbic acid (3) and methyl cucurbate (4), were evaluated for their melanogenesis-inhibitory and cancer chemopreventive potencies. Compounds 1, 3, and 4 exhibited potent melanogenesis-inhibitory activities in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells. Western-blot analysis revealed that compounds 1 and 3 reduced the protein levels of MITF (=microphthalmia-associated transcription factor), tyrosinase, TRP-1 (=tyrosine-related protein 1), and TRP-2 mostly in a concentration-dependent manner. In addition, compound 1 exhibited inhibitory effects against Epstein-Barr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, against TPA-induced inflammation in mice, and against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.

Triterpene glycosides and other polar constituents of shea (Vitellaria paradoxa) kernels and their bioactivities.

The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two pentane-2,4-diol glucosides, seven phenolic compounds, and three sugars, were isolated. The structures of five triterpene glycosides were elucidated on the basis of spectroscopic and chemical methods. Upon evaluation of the bioactivity of the isolated compounds, it was found that some or most of the compounds have potent or moderate inhibitory activities against the following: melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH); generation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-teradecanoylphorbol 13-acetate (TPA) in Raji cells; t TPA-induced inflammation in mice, and proliferation of one or more of HL-60, A549, AZ521, and SK-BR-3 human cancer cell lines, respectively. Western blot analysis established that paradoxoside E inhibits melanogenesis by regulation of expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2. In addition, tieghemelin A was demonstrated to exhibit cytotoxic activity against A549 cells (IC50 13.5 µM) mainly due to induction of apoptosis by flow cytometry. The extract of defatted shea kernels and its constituents may be, therefore, valuable as potential antioxidant, anti-inflammatory, skin-whitening, chemopreventive, and anticancer agents.

Plant science. Biosynthesis, regulation, and domestication of bitterness in cucumber.

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.

Elevated CXCL1 expression in breast cancer stroma predicts poor prognosis and is inversely associated with expression of TGF-ß signaling proteins.

BACKGROUND: CXCL1 is a chemotactic cytokine shown to regulate breast cancer progression and chemo-resistance. However, the prognostic significance of CXCL1 expression in breast cancer has not been fully characterized. Fibroblasts are important cellular components of the breast tumor microenvironment, and recent studies indicate that this cell type is a potential source of CXCL1 expression in breast tumors. The goal of this study was to further characterize the expression patterns of CXCL1 in breast cancer stroma, determine the prognostic significance of stromal CXCL1 expression, and identify factors affecting stromal CXCL1 expression. METHODS: Stromal CXCL1 protein expression was analyzed in 54 normal and 83 breast carcinomas by immunohistochemistry staining. RNA expression of CXCL1 in breast cancer stroma was analyzed through data mining in http://www.Oncomine.org. The relationships between CXCL1 expression and prognostic factors were analyzed by univariate analysis. Co-immunofluorescence staining for CXCL1, α-Smooth Muscle Actin (α-SMA) and Fibroblast Specific Protein 1 (FSP1) expression was performed to analyze expression of CXCL1 in fibroblasts. By candidate profiling, the TGF-ß signaling pathway was identified as a regulator of CXCL1 expression in fibroblasts. Expression of TGF-ß and SMAD gene products were analyzed by immunohistochemistry and data mining analysis. The relationships between stromal CXCL1 and TGF-ß signaling components were analyzed by univariate analysis. Carcinoma associated fibroblasts isolated from MMTV-PyVmT mammary tumors were treated with recombinant TGF-ß and analyzed for CXCL1 promoter activity by luciferase assay, and protein secretion by ELISA. RESULTS: Elevated CXCL1 expression in breast cancer stroma correlated with tumor grade, disease recurrence and decreased patient survival. By co-immunofluorescence staining, CXCL1 expression overlapped with expression of α-SMA and FSP1 proteins. Expression of stromal CXCL1 protein expression inversely correlated with expression of TGF-ß signaling components. Treatment of fibroblasts with TGF-ß suppressed CXCL1 secretion and promoter activity. CONCLUSIONS: Increased CXCL1 expression in breast cancer stroma correlates with poor patient prognosis. Furthermore, CXCL1 expression is localized to α-SMA and FSP1 positive fibroblasts, and is negatively regulated by TGF-ß signaling. These studies indicate that decreased TGF-ß signaling in carcinoma associated fibroblasts enhances CXCL1 expression in fibroblasts, which could contribute to breast cancer progression.

Diarylheptanoids from Alpinia officinarum Cause Distinct but Overlapping Effects on the Translatome of B Lymphoblastoid Cells.

Diarylheptanoids (AO-0001, AO-0002, and AO-0003) isolated from Alpinia officinarum inhibit proinflammatory mediators and exhibit cytotoxic and antiviral activity. However, the precise mechanisms of action of these diarylheptanoids are unknown as are their effects on expression of specific genes. Here, we used a translatome analysis to investigate the mechanisms and modes of action of these three diarylheptanoids. Polysome-associated messenger RNAs (mRNAs) were prepared from diarylheptanoids-treated and control cells from a human B lymphoblastoid cell line; these mRNA samples were then used for microarray analysis. Microarray Data Analysis Tool version 3.2 was used to analyze the microarray data analysis; this software uses pathway information of the WikiPathways for gene ontology analysis. Each of the diarylheptanoids caused upregulation or downregulation of the same 37 and 286 genes, respectively. Among the 37 upregulated genes, 16 were related to mRNA processing based on the WikiPathways analysis. Our findings provided new insights into the mode of action of diarylheptanoids from A. officinarum.