Two new alkaloids from Crinum asiaticum var. japonicum.

A new crinine-type alkaloid crijaponine A (1), a new galanthamine-type alkaloid crijaponine B (2), and 11 known alkaloids-ungeremine (3), lycorine (4), 2-O-acetyllycorine (5), 1, 2-O-diacetyllycorine (6), (-)-crinine (7), 11-hydroxyvittatine (8), hamayne (9), (+)-epibuphanisine (10), crinamine (11), yemenine A (12), and epinorgalanthamine (13)-were isolated from the rhizome and fruits of Crinum asiaticum var. japonicum. The structural elucidation of the isolated compounds was performed by spectroscopic methods including 2D NMR. The isolated compounds were evaluated for cytotoxicity against HeLa and HL-60 cells lines and were tested for acetylcholinesterase inhibition activity.

A new LC-MS assay for the quantitative analysis of vitamin K metabolites in human urine.

Vitamin K (VK), in both its phylloquinone and menaquinone forms, has been hypothesized to undergo ω- and ß-oxidation on its hydrophobic side chain in order to generate the observed urinary metabolites, K acid I and K acid II, which are excreted primarily as glucuronide conjugates. Synthetic standards of K acid I, K acid II, and a putative intermediate metabolite, menaquinone (MK)1 ω-COOH, were used to develop and optimize a new atmospheric pressure negative chemical ionization LC-MS/MS assay for the quantitation of these compounds in urine from untreated individuals and subjects treated with a high dose VK supplement. VK catabolites were extracted from urine, deconjugated, and converted to their methyl ester derivatives using previously reported methodology. The assay showed a high degree of sensitivity, with limits of detection below 10-50 fmol of metabolite per milliliter of urine, as well as an inter-assay precision of 8-12%. Metabolite standards provided unambiguous evidence for MK1 ω-COOH as a new human urinary metabolite of VK. This assay provides a minimally invasive, highly sensitive, and specific alternative for monitoring VK status in humans.

Chemical compounds that suppress hypoxia-induced stress granule formation enhance cancer drug sensitivity of human cervical cancer HeLa cells.

In eukaryotic cells, when exposed to certain types of stress including hypoxia, eIF2α is phosphorylated by several kinases including protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK). Subsequently, protein translation is stopped and stress granules (SGs) are formed. Cancer cells form SGs under hypoxia. SGs accumulate apoptosis-related molecules and play anti-apoptotic roles. Thus, hypoxia-induced SG formation contributes to drug resistance in cancer cells. For this reason, inhibition of SG formation is expected to be beneficial in cancer therapy. To prove this concept, chemical reagents that inhibit SG formation are required as experimental tools. We searched for chemical compounds that suppress SG formation and identified that ß-estradiol, progesterone, and stanolone (hereafter described as EPS) inhibit SG formation in human cervical cancer HeLa cells. As it turned out, EPS block PKR but not PERK, thus fail to suppress SG formation in most cancer cells, where SGs are formed via PERK. Nevertheless, in this study, we used HeLa cells as a model and demonstrated that EPS block hypoxia-induced SG formation in HeLa cells and consequently reduce drug resistance that HeLa cells acquire under hypoxia. Our findings support that inhibition of SG formation is a useful method to control cancers.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy.

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Down-regulation of histone deacetylase 4, -5 and -6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin.

(-)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, -5 and -6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and -5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, -5 and -6 and stimulates apoptotic induction.

Two New Diterpenoids from Salvia przewarskii.

One abietane-type and one dinoricetexane-type diterpenoid, salviskin A (1) and salviskin B (2), respectively, together with fourteen known diterpenoids, were isolated from Salvia przewarskii. Structural elucidation of these compounds was performed by spectroscopic methods including 2D NMR. The compounds isolated were evaluated for their cytotoxicity against HeLa and HL-60 cells.

Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.

Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/Pomc Expression.

Various retinoid X receptor (RXR) agonists have recently been developed, and some of them have shown anti-tumor effects both in vivo and in vitro. However, there has been no report showing the effects of RXR agonists on Cushing's disease, which is caused by excessive ACTH secretion in a corticotroph tumor of the pituitary gland. Therefore, we examined the effects of synthetic RXR pan-agonists HX630 and PA024 on the proliferation, apoptosis, ACTH secretion, and pro-opiomelanocortin (Pomc) gene expression of murine pituitary corticotroph tumor AtT20 cells. We demonstrated that both RXR agonists induced apoptosis dose-dependently in AtT20 cells, and inhibited their proliferation at their higher doses. Microarray analysis identified a significant gene network associated with caspase 3 induced by high dose HX630. On the other hand, HX630, but not PA024, inhibited Pomc transcription, Pomc mRNA expression, and ACTH secretion dose-dependently. Furthermore, we provide new evidence that HX630 negatively regulates the Pomc promoter activity at the transcriptional level due to the suppression of the transcription factor Nur77 and Nurr1 mRNA expression and the reduction of Nur77/Nurr1 heterodimer recruiting to the Pomc promoter region. We also demonstrated that the HX630-mediated suppression of the Pomc gene expression was exerted via RXRα. Furthermore, HX630 inhibited tumor growth and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. Thus, these results indicate that RXR agonists, especially HX630, could be a new therapeutic candidate for Cushing's disease.

A cell-based screening for TAZ activators identifies ethacridine, a widely used antiseptic and abortifacient, as a compound that promotes dephosphorylation of TAZ and inhibits adipogenesis in C3H10T1/2 cells.

Transcriptional co-activator with PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif (TAZ) regulates in cell proliferation and differentiation. In mesenchymal stem cells it promotes osteogenesis and myogenesis, and suppresses adipogenesis. TAZ activators are expected to prevent osteoporosis, obesity and muscle atrophy. TAZ activation induces epithelial-mesenchymal transition, confers stemness to cancer cells and leads to poor clinical prognosis in cancer patients. In this point of view, TAZ inhibitors should contribute to cancer therapy. Thus, TAZ attracts attention as a two-faced drug target. We screened for TAZ modulators by using human lung cancer A549 cells expressing the fluorescent reporter. Through this assay, we obtained TAZ activator candidates. We unexpectedly found that ethacridine, a widely used antiseptic and abortifacient, enhances the interaction of TAZ and protein phosphatases and increases unphosphorylated and nuclear TAZ. Ethacridine inhibits adipogenesis in mesenchymal C3H10T1/2 cells through the activation of TAZ. This finding suggests that ethacridine is a bona fide TAZ activator and supports that our assay is useful to discover TAZ activators.

Screening with a novel cell-based assay for TAZ activators identifies a compound that enhances myogenesis in C2C12 cells and facilitates muscle repair in a muscle injury model.

The transcriptional coactivator with a PDZ-binding motif (TAZ) cooperates with various transcriptional factors and plays various roles. Immortalized human mammalian epithelial MCF10A cells form spheres when TAZ is overexpressed and activated. We developed a cell-based assay using sphere formation by TAZ-expressing MCF10A cells as a readout to screen 18,458 chemical compounds for TAZ activators. Fifty compounds were obtained, and 47 were confirmed to activate the TAZ-dependent TEAD-responsive reporter activity in HEK293 cells. We used the derived subset of compounds as a TAZ activator candidate minilibrary and searched for compounds that promote myogenesis in mouse C2C12 myoblast cells. In this study, we focused on one compound, IBS008738. IBS008738 stabilizes TAZ, increases the unphosphorylated TAZ level, enhances the association of MyoD with the myogenin promoter, upregulates MyoD-dependent gene transcription, and competes with myostatin in C2C12 cells. TAZ knockdown verifies that the effect of IBS008738 depends on endogenous TAZ in C2C12 cells. IBS008738 facilitates muscle repair in cardiotoxin-induced muscle injury and prevents dexamethasone-induced muscle atrophy. Thus, this cell-based assay is useful to identify TAZ activators with a variety of cellular outputs. Our findings also support the idea that TAZ is a potential therapeutic target for muscle atrophy.

The retinoic acid receptor agonist Am80 increases mucosal inflammation in an IL-6 dependent manner during Trichuris muris infection.

PURPOSE: Vitamin A metabolites, such as all-trans-retinoic acid (RA) that act through the nuclear receptor; retinoic acid receptor (RAR), have been shown to polarise T cells towards Th2, and to be important in resistance to helminth infections. Co-incidentally, people harbouring intestinal parasites are often supplemented with vitamin A, as both vitamin A deficiency and parasite infections often occur in the same regions of the globe. However, the impact of vitamin A supplementation on gut inflammation caused by intestinal parasites is not yet completely understood. METHODS: Here, we use Trichuris muris, a helminth parasite that buries into the large intestine of mice causing mucosal inflammation, as a model of both human trichuriasis and IBD, treat with an RARα/ß agonist (Am80) and quantify the ensuing pathological changes in the gut. RESULTS: Critically, we show, for the first time, that rather than playing an anti-inflammatory role, Am80 actually exacerbates helminth-driven inflammation, demonstrated by an increased colonic crypt length and a significant CD4(+) T cell infiltrate. Further, we established that the Am80-driven crypt hyperplasia and CD4(+) T cell infiltrate were dependent on IL-6, as both were absent in Am80-treated IL-6 knock-out mice. CONCLUSIONS: This study presents novel data showing a pro-inflammatory role of RAR ligands in T. muris infection, and implies an undesirable effect for the administration of vitamin A during chronic helminth infection.

The transcription factors Nur77 and retinoid X receptors participate in amphetamine-induced locomotor activities.

INTRODUCTION: The major substrate underlying amphetamine (AMPH)-induced locomotor activity is associated with dopamine forebrain circuits. Brain regions associated with AMPH-induced locomotor activity express high levels of retinoid receptors. However, the role of these transcription factors in dopamine-mediated effects remains poorly understood. Two nuclear receptor families, the retinoic acid receptors (RAR) and the retinoid X receptors (RXR), transduce retinoic acid signal. RARs are specifically involved in retinoid signaling, whereas RXRs also participate in other signaling pathways as partners for other nuclear receptors such as Nur77, an orphan member of the nuclear receptor family expresses in dopamine system. MATERIALS AND METHODS: To explore the role of retinoid receptors and Nur77 in AMPH-induced locomotor activity, we administered selective retinoid receptor drugs in combination with AMPH in adult wild-type and Nur77-deficient mice. At a low dose, AMPH similarly increased ambulatory activity in wild-type and Nur77-deficient mice, while it did not alter non-ambulatory activity. RESULTS AND DISCUSSION: At a high dose, AMPH did not alter ambulatory activity anymore, while non-ambulatory activity strongly increased in wild-type mice. Nur77-deficient mice still displayed a higher ambulatory activity with no change in non-ambulatory activity. HX531, a synthetic RXR antagonist, blocks AMPH-induced ambulatory activity, whereas RAR drugs tested remained without effect. Interestingly, the effect of HX531 was abolished in Nur77-deficient mice, suggesting that this orphan nuclear receptor is essential for the action of the RXR drug. CONCLUSION: This study shows that RXR and Nur77 participate in AMPH-induced locomotor activity and prompts for further investigations on the role of Nur77 and RXR in addiction and reward-related behaviors.

Tamibarotene.

Tamibarotene is a new synthetic retinoid drug recently approved for relapsed or refractory acute promyelocytic leukemia (APL) in Japan. It is a specific agonist for retinoic acid receptor alpha/beta. Compared to all-trans retinoic acid (ATRA), a natural retinoid indicated for a first-line treatment of APL, tamibarotene is chemically more stable and several times more potent as an inducer of differentiation in promyelocytic leukemia cells. In contrast to ATRA, whose plasma concentration declines considerably during daily administration, tamibarotene sustains plasma level probably due to a lower affinity for cellular retinoic acid binding protein. Furthermore, adverse side effects were milder than those of ATRA in clinical trials. Clinical trials held in Japan showed that tamibarotene had efficacy in APL patients who had relapsed from ATRA-induced complete remission. Recently, better understanding of the various mechanisms of action of retinoids has stimulated great interest in its potential use for treatment of various diseases. Tamibarotene is being investigated for treatment of multiple myeloma and Crohn's disease in clinical trials. This review focuses on tamibarotene's mechanisms of action, chemical properties, pharmacokinetics and its use in APL as well as its potential use in various disorders.

Clinically potential subclasses of retinoid synergists revealed by gene expression profiling.

Retinoids have chemopreventive and therapeutic potency in oncology and dermatology, although their application is restricted by many undesirable side effects. For the development of more effective and less toxic retinoids, gene expression analyses using DNA microarrays have the potential to supplement conventional screening methods, which are based on the changes in cell morphology and/or function. In this study, we applied the class prediction algorithm, which was used in the molecular phenotyping of tumors, for the classification of synthetic retinoids (Am80 and Tp80) and retinoid synergists (HX630, TZ335, and PA024) as all-trans retinoic acid-like, 9-cis retinoic acid-like, and control-like classes. By analyzing the effects of all-trans retinoic acid and 9-cis retinoic acid on the gene expressions in a human promyelocytic leukemia cell line, HL60, we successfully selected 50 marker genes whose expression pattern could distinguish these classes. Moreover, the classification revealed the existence of two subclasses among the retinoid synergists used with Am80. Close inspection of the DNA microarray analyses indicated that these two subclasses had different effects on the apoptosis of HL60 cells, and this was confirmed by in vivo experiments. These results indicate that the retinoidal activity of Am80, which has already been used in clinical trials, could be modulated differently by the two classes of retinoid synergists. Thus, these two subclasses of retinoid synergists have the potency to widen the usage of Am80. Our analyses demonstrated that the gene expression profiling could provide important information for developing useful retinoid synergists by compensating conventional screening methods.

Effect of synthetic retinoid, TAC-101, on experimental autoimmune disease.

The effect of 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid (TAC-101), one of the synthetic retinoids, on collagen-induced arthritis (CIA) in mice and experimental autoimmune encephalomyelitis (EAE) in rats was studied. TAC-101 at doses of 5 and 20 mg/kg clearly inhibited the development of CIA in terms of the swelling of fore- and hind-limbs and bone destruction in knee joints. TAC-101 also suppressed the production of anti-type II collagen (CII) IgG antibody and delayed-type hypersensitivity (DTH) against CII. In addition, TAC-101 delayed the onset and development of EAE but did not affect the maximum symptom of EAE in rats. The elevation of serum antimyelin basic protein (MBP) antibody and DTH to MBP on day 13 clearly suppressed by TAC-101 in EAE rats. Moreover, TAC-101 inhibited the IL-1beta-induced PGE(2) production by MG-63 cells, human osteoblast-like cells, through the suppression of cyclooxygenase II mRNA expression. These findings suggest that TAC-101 inhibits CIA in mice and EAE in rats due to the suppression of immune response to auto-antigen and the production of PGE(2).