A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity.

Indirubin-based compounds affect diverse biological processes, such as inflammation and angiogenesis. In this study, we tested a novel indirubin derivative, LDD-1819 (2-((((2Z,3E)-5-hydroxy-5'-nitro-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethan-1-aminium chloride) for two major biological activities: cell plasticity and anti-cancer activity. Biological assays indicated that LDD-1819 induced somatic cell plasticity. LDD-1819 potentiated myoblast reprogramming into osteogenic cells and fibroblast reprogramming into adipogenic cells. Interestingly, in an assay of skeletal muscle dedifferentiation, LDD-1819 induced human muscle cellularization and blocked residual proliferative activity to produce a population of mononuclear refractory cells, which is also observed in the early stages of limb regeneration in urodele amphibians. In cancer cell lines, LDD-1819 treatment inhibited cell invasion and selectively induced apoptosis compared to normal cells. In an animal tumor xenograft model, LDD-1819 reduced human cancer cell metastasis in vivo at doses that did not produce toxicity. Biochemical assays showed that LDD-1819 possessed inhibitory activity against glycogen synthase kinase-3ß, which is linked to cell plasticity, and aurora kinase, which regulates carcinogenesis. These results indicate that novel indirubin derivative LDD-1819 is a dual inhibitor of glycogen synthase kinase-3ß and aurora A kinase, and has potential for development as an anti-cancer drug or as a reprogramming agent for cell-therapy based approaches to treat degenerative diseases.

Biotransformed Metabolites of the Hop Prenylflavanone Isoxanthohumol.

A metabolic conversion study on microbes is known as one of the most useful tools to predict the xenobiotic metabolism of organic compounds in mammalian systems. The microbial biotransformation of isoxanthohumol (1), a major hop prenylflavanone in beer, has resulted in the production of three diastereomeric pairs of oxygenated metabolites (2⁻7). The microbial metabolites of 1 were formed by epoxidation or hydroxylation of the prenyl group, and HPLC, NMR, and CD analyses revealed that all of the products were diastereomeric pairs composed of (2S)- and (2R)- isomers. The structures of these metabolic compounds were elucidated to be (2S,2"S)- and (2R,2"S)-4'-hydroxy-5-methoxy-7,8-(2,2-dimethyl-3-hydroxy-2,3-dihydro-4H-pyrano)-flavanones (2 and 3), (2S)- and (2R)-7,4'-dihydroxy-5-methoxy-8-(2,3-dihydroxy-3-methylbutyl)-flavanones (4 and 5) which were new oxygenated derivatives, along with (2R)- and (2S)-4'-hydroxy-5-methoxy-2"-(1-hydroxy-1-methylethyl)dihydrofuro[2,3-h]flavanones (6 and 7) on the basis of spectroscopic data. These results could contribute to understanding the metabolic fates of the major beer prenylflavanone isoxanthohumol that occur in mammalian system.

Anthraquinones from Morinda longissima and their insulin mimetic activities via AMP-activated protein kinase (AMPK) activation.

AMP-activated protein kinase (AMPK) activators are known to increase energy metabolism and to reduce body weight, as well as to improve glucose uptake. During for searching AMPK activators, a new anthraquinone, modasima A (10), along with eighteen known analogues (1-9 and 11-19) were isolated from an ethanol extract of the roots of Morinda longissima Y. Z. Ruan (Rubiaceae). Using the fluorescent tagged glucose analogues, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose (2-NBDG), insulin mimetics were screened with compounds 1-19 in 3T3-L1 adipocytes. Among them, compounds 2, 8 and 10 enhanced significantly glucose uptake into adipocytes and up-regulated the phosphorylated AMPK (Thr172) whereas the glucose uptake enhancing activities of compounds 2, 8 and 10 were abrogated by treatment of compound C, an AMPK inhibitor. Taken together, these anthraquinones showed the potential action as insulin mimetic to improve glucose uptake via activation of AMPK.

A distinct role for interleukin-6 as a major mediator of cellular adjustment to an altered culture condition.

Tissue microenvironment adjusts biological properties of different cells by modulating signaling pathways and cell to cell interactions. This study showed that epithelial-mesenchymal transition (EMT)/ mesenchymal-epithelial transition (MET) can be modulated by altering culture conditions. HPV E6/E7-transfected immortalized oral keratinocytes (IHOK) cultured in different media displayed reversible EMT/MET accompanied by changes in cell phenotype, proliferation, gene expression at transcriptional, and translational level, and migratory and invasive activities. Cholera toxin, a major supplement to culture medium, was responsible for inducing the morphological and biological changes of IHOK. Cholera toxin per se induced EMT by triggering the secretion of interleukin 6 (IL-6) from IHOK. We found IL-6 to be a central molecule that modulates the reversibility of EMT based not only on the mRNA level but also on the level of secretion. Taken together, our results demonstrate that IL-6, a cytokine whose transcription is activated by alterations in culture conditions, is a key molecule for regulating reversible EMT/MET. This study will contribute to understand one way of cellular adjustment for surviving in unfamiliar conditions.

Chemical genetics and its application to moonlighting in glycolytic enzymes.

Glycolysis is an ancient biochemical pathway that breaks down glucose into pyruvate to produce ATP. The structural and catalytic properties of glycolytic enzymes are well-characterized. However, there is growing appreciation that these enzymes participate in numerous moonlighting functions that are unrelated to glycolysis. Recently, chemical genetics has been used to discover novel moonlighting functions in glycolytic enzymes. In the present mini-review, we introduce chemical genetics and discuss how it can be applied to the discovery of protein moonlighting. Specifically, we describe the application of chemical genetics to uncover moonlighting in two glycolytic enzymes, enolase and glyceraldehyde dehydrogenase. This led to the discovery of moonlighting roles in glucose homoeostasis, cancer progression and diabetes-related complications. Finally, we also provide a brief overview of the latest progress in unravelling the myriad moonlighting roles for these enzymes.

Delineation of the role of glycosylation in the cytotoxic properties of quercetin using novel assays in living vertebrates.

Quercetin is a plant-derived flavonoid and its cytotoxic properties have been widely reported. However, in nature, quercetin predominantly occurs as various glycosides. Thus far the cytotoxic activity of these glycosides has not been investigated to the same extent as quercetin, especially in animal models. In this study, the cytotoxic properties of quercetin (1), hyperoside (quercetin 3-O-galactoside, 2), isoquercitrin (quercetin 3-O-glucoside, 3), quercitrin (quercetin 3-O-rhamnoside, 4), and spiraeoside (quercetin 4'-O-glucoside, 5) were directly compared in vitro using assays of cancer cell viability. To further characterize the influence of glycosylation in vivo, a novel zebrafish-based assay was developed that allows the rapid and experimentally convenient visualization of glycoside cleavage in the digestive tract. This assay was correlated with a novel human tumor xenograft assay in the same animal model. The results showed that 3 is as effective as 1 at inhibiting cancer cell proliferation in vivo. Moreover, it was observed that 3 can be effectively deglycosylated in the digestive tract. Collectively, these results indicate that 3 is a very promising drug candidate for cancer therapy, because glycosylation confers advantageous pharmacological changes compared with the aglycone, 1. Importantly, the development of a novel and convenient fluorescence-based assay for monitoring deglycosylation in living vertebrates provides a valuable platform for determining the metabolic fate of naturally occurring glycosides.

5-Nitro-5'hydroxy-indirubin-3'oxime is a novel inducer of somatic cell transdifferentiation.

Patient-derived cell transplantation is an attractive therapy for regenerative medicine. However, this requires effective strategies to reliably differentiate patient cells into clinically useful cell types. Herein, we report the discovery that 5-nitro-5'hydroxy-indirubin-3'oxime (5'-HNIO) is a novel inducer of cell transdifferentiation. 5'-HNIO induced muscle transdifferentiation into adipogenic and osteogenic cells. 5'-HNIO was shown to inhibit aurora kinase A, which is a known cell fate regulator. 5'-HNIO produced a favorable level of transdifferentiation compared to other aurora kinase inhibitors and induced transdifferentiation across cell lineage boundaries. Significantly, 5'-HNIO treatment produced direct transdifferentiation without up-regulating potentially oncogenic induced pluripotent stem cell (iPSC) reprogramming factors. Thus, our results demonstrate that 5'-HNIO is an attractive molecular tool for cell transdifferentiation and cell fate research.

ZebIAT, an image analysis tool for registering zebrafish embryos and quantifying cancer metastasis.

BACKGROUND: Zebrafish embryos have recently been established as a xenotransplantation model of the metastatic behaviour of primary human tumours. Current tools for automated data extraction from the microscope images are restrictive concerning the developmental stage of the embryos, usually require laborious manual image preprocessing, and, in general, cannot characterize the metastasis as a function of the internal organs. METHODS: We present a tool, ZebIAT, that allows both automatic or semi-automatic registration of the outer contour and inner organs of zebrafish embryos. ZebIAT provides a registration at different stages of development and an automatic analysis of cancer metastasis per organ, thus allowing to study cancer progression. The semi-automation relies on a graphical user interface. RESULTS: We quantified the performance of the registration method, and found it to be accurate, except in some of the smallest organs. Our results show that the accuracy of registering small organs can be improved by introducing few manual corrections. We also demonstrate the applicability of the tool to studies of cancer progression. CONCLUSIONS: ZebIAT offers major improvement relative to previous tools by allowing for an analysis on a per-organ or region basis. It should be of use in high-throughput studies of cancer metastasis in zebrafish embryos.

Protein tyrosine phosphatase 1B (PTP1B) inhibitors from Morinda citrifolia (Noni) and their insulin mimetic activity.

As part of our ongoing search for new antidiabetic agents from medicinal plants, we found that a methanol extract of Morinda citrifolia showed potential stimulatory effects on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation of this active extract yielded two new lignans (1 and 2) and three new neolignans (9, 10, and 14), as well as 10 known compounds (3-8, 11-13, and 15). The absolute configurations of compounds 9, 10, and 14 were determined by ECD spectra analysis. Compounds 3, 6, 7, and 15 showed inhibitory effects on PTP1B enzyme with IC50 values of 21.86 ± 0.48, 15.01 ± 0.20, 16.82 ± 0.42, and 4.12 ± 0.09 µM, respectively. Furthermore, compounds 3, 6, 7, and 15 showed strong stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. This study indicated the potential of compounds 3, 6, 7, and 15 as lead molecules for antidiabetic agents.

Visualizing sweetness: increasingly diverse applications for fluorescent-tagged glucose bioprobes and their recent structural modifications.

Glucose homeostasis is a fundamental aspect of life and its dysregulation is associated with important diseases, such as cancer and diabetes. Traditionally, glucose radioisotopes have been used to monitor glucose utilization in biological systems. Fluorescent-tagged glucose analogues were initially developed in the 1980s, but it is only in the past decade that their use as a glucose sensor has increased significantly. These analogues were developed for monitoring glucose uptake in blood cells, but their recent applications include tracking glucose uptake by tumor cells and imaging brain cell metabolism. This review outlines the development of fluorescent-tagged glucose analogues, describes their recent structural modifications and discusses their increasingly diverse biological applications.

Inhibiting 5-lipoxygenase prevents skeletal muscle atrophy by targeting organogenesis signalling and insulin-like growth factor-1.

BACKGROUND: Skeletal muscle atrophy can occur in response to numerous factors, such as ageing and certain medications, and produces a major socio-economic burden. At present, there are no approved drugs for treating skeletal muscle atrophy. Arachidonate 5-lipoxygenase (Alox5) is a drug target for a number of diseases. However, pharmacological targeting of Alox5, and its role in skeletal muscle atrophy, is unclear. METHODS: The potential effects of gene knockdown and pharmacological targeting of Alox5 on skeletal muscle atrophy were investigated using cell-based models, animal models and human skeletal muscle primary cells. Malotilate, a clinically safe drug developed for enhancing liver regeneration and Alox5 inhibitor, was investigated as a repurposing candidate. Mechanism(s) of action in skeletal muscle atrophy was assessed by measuring the expression level or activation status of key regulatory pathways and validated using gene knockdown and RNA sequencing. RESULTS: Myotubes treated with the atrophy-inducing glucocorticoid, dexamethasone, were protected from catabolic responses by treatment with malotilate (+41.29%, P < 0.01). Similar anti-atrophy effects were achieved by gene knockdown of Alox5 (+30.4%, P < 0.05). Malotilate produced anti-atrophy effects without affecting the myogenic differentiation programme. In an in vivo model of skeletal muscle atrophy, malotilate treatment preserved muscle force/strength (grip strength: +35.72%, latency to fall: +553.1%, P < 0.05), increased mass and fibre cross-sectional area (quadriceps: +23.72%, soleus: +33.3%, P < 0.01) and down-regulated atrogene expression (Atrogin-1: -61.58%, Murf-1: -66.06%, P < 0.01). Similar, beneficial effects of malotilate treatment were observed in an ageing muscle model, which also showed the preservation of fast-twitch fibres (Type 2a: +56.48%, Type 2b: +37.32%, P < 0.01). Leukotriene B4, a product of Alox5 activity with inflammatory and catabolic functions, was found to be elevated in skeletal muscle undergoing atrophy (quadriceps: +224.4%, P < 0.001). Cellular transcriptome analysis showed that targeting Alox5 up-regulated biological processes regulating organogenesis and increased the expression of insulin-like growth factor-1, a key anti-atrophy hormone (+226.5%, P < 0.05). Interestingly, these effects were restricted to the atrophy condition and not observed in normal skeletal muscle cultures with Alox5 inhibition. Human myotubes were also protected from atrophy by pharmacological targeting of Alox5 (+23.68%, P < 0.05). CONCLUSIONS: These results shed new light on novel drug targets and mechanisms underpinning skeletal muscle atrophy. Alox5 is a regulator and drug target for muscle atrophy, and malotilate is an attractive compound for repurposing studies to treat this disease.

Genome-Wide Transcriptomic Analysis of Non-Tumorigenic Tissues Reveals Aging-Related Prognostic Markers and Drug Targets in Renal Cell Carcinoma.

The relationship between expression of aging-related genes in normal tissues and cancer patient survival has not been assessed. We developed a genome-wide transcriptomic analysis approach for normal tissues adjacent to the tumor to identify aging-related transcripts associated with survival outcome, and applied it to 12 cancer types. As a result, five aging-related genes (DUSP22, MAPK14, MAPKAPK3, STAT1, and VCP) in normal tissues were found to be significantly associated with a worse survival outcome in patients with renal cell carcinoma (RCC). This computational approach was investigated using nontumorigenic immune cells purified from young and aged mice. Aged immune cells showed upregulated expression of all five aging-related genes and promoted RCC invasion compared to young immune cells. Further studies revealed DUSP22 as a regulator and druggable target of metastasis. DUSP22 gene knockdown reduced RCC invasion and the small molecule inhibitor BML-260 prevented RCC dissemination in a tumor/immune cell xenograft model. Overall, these results demonstrate that deciphering the relationship between aging-related gene expression in normal tissues and cancer patient survival can provide new prognostic markers, regulators of tumorigenesis and novel targets for drug development.

Investigation of niclosamide as a repurposing agent for skeletal muscle atrophy.

Skeletal muscle atrophy is a feature of aging (termed sarcopenia) and various diseases, such as cancer and kidney failure. Effective drug treatment options for muscle atrophy are lacking. The tapeworm medication, niclosamide is being assessed for repurposing to treat numerous diseases, including end-stage cancer metastasis and hepatic steatosis. In this study, we investigated the potential of niclosamide as a repurposing drug for muscle atrophy. In a myotube atrophy model using the glucocorticoid, dexamethasone, niclosamide did not prevent the reduction in myotube diameter or the decreased expression of phosphorylated FOXO3a, which upregulates the ubiquitin-proteasome pathway of muscle catabolism. Treatment of normal myotubes with niclosamide did not activate mTOR, a major regulator of muscle protein synthesis, and increased the expression of atrogin-1, which is induced in catabolic states. Niclosamide treatment also inhibited myogenesis in muscle precursor cells, enhanced the expression of myoblast markers Pax7 and Myf5, and downregulated the expression of differentiation markers MyoD, MyoG and Myh2. In an animal model of muscle atrophy, niclosamide did not improve muscle mass, grip strength or muscle fiber cross-sectional area. Muscle atrophy is also feature of cancer cachexia. IC50 analyses indicated that niclosamide was more cytotoxic for myoblasts than cancer cells. In addition, niclosamide did not suppress the induction of iNOS, a key mediator of atrophy, in an in vitro model of cancer cachexia and did not rescue myotube diameter. Overall, these results suggest that niclosamide may not be a suitable repurposing drug for glucocorticoid-induced skeletal muscle atrophy or cancer cachexia. Nevertheless, niclosamide may be employed as a compound to study mechanisms regulating myogenesis and catabolic pathways in skeletal muscle.

Lithium Chloride Protects against Sepsis-Induced Skeletal Muscle Atrophy and Cancer Cachexia.

Inflammation-mediated skeletal muscle wasting occurs in patients with sepsis and cancer cachexia. Both conditions severely affect patient morbidity and mortality. Lithium chloride has previously been shown to enhance myogenesis and prevent certain forms of muscular dystrophy. However, to our knowledge, the effect of lithium chloride treatment on sepsis-induced muscle atrophy and cancer cachexia has not yet been investigated. In this study, we aimed to examine the effects of lithium chloride using in vitro and in vivo models of cancer cachexia and sepsis. Lithium chloride prevented wasting in myotubes cultured with cancer cell-conditioned media, maintained the expression of the muscle fiber contractile protein, myosin heavy chain 2, and inhibited the upregulation of the E3 ubiquitin ligase, Atrogin-1. In addition, it inhibited the upregulation of inflammation-associated cytokines in macrophages treated with lipopolysaccharide. In the animal model of sepsis, lithium chloride treatment improved body weight, increased muscle mass, preserved the survival of larger fibers, and decreased the expression of muscle-wasting effector genes. In a model of cancer cachexia, lithium chloride increased muscle mass, enhanced muscle strength, and increased fiber cross-sectional area, with no significant effect on tumor mass. These results indicate that lithium chloride exerts therapeutic effects on inflammation-mediated skeletal muscle wasting, such as sepsis-induced muscle atrophy and cancer cachexia.

SRSF9 Regulates Cassette Exon Splicing of Caspase-2 by Interacting with Its Downstream Exon.

Alternative splicing (AS) is an important posttranscriptional regulatory process. Damaged or unnecessary cells need to be removed though apoptosis to maintain physiological processes. Caspase-2 pre-mRNA produces pro-apoptotic long mRNA and anti-apoptotic short mRNA isoforms through AS. How AS of Caspase-2 is regulated remains unclear. In the present study, we identified a novel regulatory protein SRSF9 for AS of Caspase-2 cassette exon 9. Knock-down (KD) of SRSF9 increased inclusion of cassette exon and on the other hand, overexpression of SRSF9 decreased inclusion of this exon. Deletion mutagenesis demonstrated that exon 9, parts of intron 9, exon 8 and exon 10 were not required for the role of SRSF9 in Caspase-2 AS. However, deletion and substitution mutation analysis revealed that AGGAG sequence located at exon 10 provided functional target for SRSF9. In addition, RNA-pulldown mediated immunoblotting analysis showed that SRSF9 interacted with this sequence. Gene ontology analysis of RNA-seq from SRSF9 KD cells demonstrates that SRSF9 could regulate AS of a subset of apoptosis related genes. Collectively, our results reveal a basis for regulation of Caspase-2 AS.

Analyses of Avascular Mutants Reveal Unique Transcriptomic Signature of Non-conventional Endothelial Cells.

Endothelial cells appear to emerge from diverse progenitors. However, to which extent their developmental origin contributes to define their cellular and molecular characteristics remains largely unknown. Here, we report that a subset of endothelial cells that emerge from the tailbud possess unique molecular characteristics that set them apart from stereotypical lateral plate mesoderm (LPM)-derived endothelial cells. Lineage tracing shows that these tailbud-derived endothelial cells arise at mid-somitogenesis stages, and surprisingly do not require Npas4l or Etsrp function, indicating that they have distinct spatiotemporal origins and are regulated by distinct molecular mechanisms. Microarray and single cell RNA-seq analyses reveal that somitogenesis- and neurogenesis-associated transcripts are over-represented in these tailbud-derived endothelial cells, suggesting that they possess a unique transcriptomic signature. Taken together, our results further reveal the diversity of endothelial cells with respect to their developmental origin and molecular properties, and provide compelling evidence that the molecular characteristics of endothelial cells may reflect their distinct developmental history.

Screening ginseng saponins in progenitor cells identifies 20(R)-ginsenoside Rh2 as an enhancer of skeletal and cardiac muscle regeneration.

Aging is associated with increased prevalence of skeletal and cardiac muscle disorders, such as sarcopenia and cardiac infarction. In this study, we constructed a compendium of purified ginsenoside compounds from Panax ginseng C.A. Meyer, which is a traditional Korean medicinal plant used to treat for muscle weakness. Skeletal muscle progenitor cell-based screening identified three compounds that enhance cell viability, of which 20(R)-ginsenoside Rh2 showed the most robust response. 20(R)-ginsenoside Rh2 increased viability in myoblasts and cardiomyocytes, but not fibroblasts or disease-related cells. The cellular mechanism was identified as downregulation of cyclin-dependent kinase inhibitor 1B (p27Kip1) via upregulation of Akt1/PKB phosphorylation at serine 473, with the orientation of the 20 carbon epimer being crucially important for biological activity. In zebrafish and mammalian models, 20(R)-ginsenoside Rh2 enhanced muscle cell proliferation and accelerated recovery from degeneration. Thus, we have identified 20(R)-ginsenoside Rh2 as a p27Kip1 inhibitor that may be developed as a natural therapeutic for muscle degeneration.

12,23-Dione dammarane triterpenes from Gynostemma longipes and their muscle cell proliferation activities via activation of the AMPK pathway.

The aging population is growing rapidly around the world and there is also an increase in sarcopenia, which is characterized by decreased muscle mass, strength and function in the elderly population. AMP-activated protein kinase (AMPK) is an essential sensor and regulator of glucose, lipid and energy metabolism throughout the body. Previous studies have shown that AMPK pathway activation by regular exercise and appropriate dietary control have beneficial effects on skeletal muscle. In the process of searching for new AMPK activators from medicinal plants, we isolated and characterized eight new 12,23-dione dammarane triterpenoids (1-3 and 5-9), as well as one known gypentonoside A from Gynostemma longipes. When all isolates were tested for their AMPK activation activities, seven compounds (1 and 3-8) were significantly activated AMPK phosphorylation in mouse C2C12 skeletal muscle cell lines. Since G. longipes contained a significant amount of active compound 1 (over 2.08% per dried raw plant), it suggested the potential of this plant to be developed as a functional food or botanical drug that enhances muscle proliferation by activating AMPK signaling pathways.