Characterization of ecdysteroid biosynthesis in the pond wolf spider, Pardosa pseudoannulata.

Ecdysteroids, as the key growth hormones, regulate moulting, metamorphosis and reproduction in arthropods. Ecdysteroid biosynthesis is catalysed by a series of cytochrome P450 monooxygenases (CYP450s) encoded by Halloween genes, including spook (spo), phantom (phm), disembodied (dib), shadow (sad) and shade (shd). The ecdysteroid biosynthesis in insects is clear with 20-hydroxyecdysone (20E) as the main ecdysteroid. However, the information on the major ecdysteroids in arachnids is limited. In this study, Halloween genes spo, dib, sad and shd, but not phm, were identified in the pond wolf spider, Pardosa pseudoannulata. Phylogenetic analysis grouped arachnid and insect Halloween gene products into two CYP450 clades, the CYP2 clan (spo and phm) and the mitochondrial clan (dib, sad, and shd). In P. pseudoannulata, the temporal expression profile of the four Halloween genes in concurrence with spiderling moulting with steady increase in the course of the 2nd instar followed by a rapid dropdown once moulting was completed. Spatially, the four Halloween genes were highly expressed in spiderling abdomen and in the ovaries of female adults. In parallel, ponasterone A (PA), but not 20E, was detected by LC-MS/MS analysis in P. pseudoannulata, and it was demonstrated as a functional ecdysteroid in the spider by accelerating of moulting with PA addition. The present study revealed the different ecdysteroid biosynthesis pathways in spiders and insects.

Heat shock promotes inclusion body formation of mutant huntingtin (mHtt) and alleviates mHtt-induced transcription factor dysfunction.

PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in individuals with and models of Huntington's disease (HD). The role of IB versus diffusible mHtt in neurotoxicity remains unclear. Using a ponasterone (PA)-inducible cell model of HD, here we evaluated the effects of heat shock on the appearance and functional outcome of Htt103QExon1-EGFP expression. Quantitative image analysis indicated that 80-90% of this mHtt protein initially appears as "diffuse" signals in the cytosol, with IBs forming at high mHtt expression. A 2-h heat shock during the PA induction reduced the diffuse signal, but greatly increased mHtt IB formation in both cytosol and nucleus. Dose- and time-dependent mHtt expression suggested that nucleated polymerization drives IB formation. RNA-mediated knockdown of heat shock protein 70 (HSP70) and heat shock cognate 70 protein (HSC70) provided evidence for their involvement in promoting diffuse mHtt to form IBs. Reporter gene assays assessing the impacts of diffuse versus IB mHtt showed concordance of diffuse mHtt expression with the repression of heat shock factor 1, cAMP-responsive element-binding protein (CREB), and NF-κB activity. CREB repression was reversed by heat shock coinciding with mHtt IB formation. In an embryonic striatal neuron-derived HD model, the chemical chaperone sorbitol similarly promoted the structuring of diffuse mHtt into IBs and supported cell survival under stress. Our results provide evidence that mHtt IB formation is a chaperone-supported cellular coping mechanism that depletes diffusible mHtt conformers, alleviates transcription factor dysfunction, and promotes neuron survival.

Ponasterone A and F, Ecdysteroids from the Arctic Bryozoan Alcyonidium gelatinosum.

A new ecdysteroid, ponasterone F (1) and the previously reported compound ponasterone A (2) were isolated from specimens of the Arctic marine bryozoan Alcyonidium gelatinosum collected at Hopenbanken, off the coast of Edgeøya, Svalbard. The structure of 1 was elucidated, and the structure of 2 confirmed by spectroscopic methods including 1D and 2D NMR and analysis of HR-MS data. The compounds were evaluated for their ability to affect bacterial survival and cell viability, as well as their agonistic activities towards the estrogen receptors α and ß. The compounds were not active in these assays. Compound 2 is an arthropod hormone controlling molting and are known to act as an allelochemical when produced by plants. Even though its structure has been previously reported, this is the first time a ponasterone has been isolated from a bryozoan. A. gelatinosum produced 1 and 2 in concentrations surpassing those expected of hormonal molecules, indicating their function as defence molecules against molting predators. This work adds to the chemical diversity reported from marine bryozoans and expanded our knowledge of the chemical modifications of the ponasterones.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

New septanoside and 20-hydroxyecdysone septanoside derivative from Atriplex portulacoides roots with preliminary biological activities.

The phytochemical investigation of a Tunisian plant Atriplex portulacoides (Chenopodiaceae) led to the isolation of two new compounds designated as portulasoid (2) and septanoecdysone (3) along with the known 20-hydroxyecdysone (20HE) (1). Their chemical structures were elucidated on the basis of extensive spectroscopic methods including ES-HRMS, 1D and 2D-NMR. The isolated compounds were finally tested for their antioxidant activity by using DPPH, ABTS(+), Fe(3+) and catalase assays and also for their antibacterial and anticholinesterase activities.

The minor ecdysteroids from Ajuga turkestanica.

INTRODUCTION: Ajuga turkestanica is a plant used in traditional medicine for its high ecdysteroid content, including the presence of the particularly active turkesterone, which possess efficient anabolic activity. OBJECTIVES: To isolate and identify minor ecdysteroids present in a semi-purified plant fraction containing ca. 70% turkesterone. MATERIAL AND METHODS: Multi-step preparative HPLC (combining RP- and NP-HPLC systems) was used to purify the different components present in the turkesterone fraction. Isolated compounds were identified by high-resolution mass spectrometry and 2D-NMR. RESULTS: Fourteen ecdysteroids (including turkesterone and 20-hydroxyecdysone) were isolated. Seven of these, all bearing an 11α-hydroxy group, were previously unreported. CONCLUSION: Ajuga turkestanica ecdysteroids are characterised by the abundance of 11α-hydroxylated compounds and by the simultaneous presence of 24C, 27C, 28C and 29C ecdysteroids. It is expected that even more ecdysteroids are to be found in this plant since the starting material for this study lacked the less polar ecdysteroids. The simultaneous presence of 20-hydroxyecdysone and turkesterone (its 11α-hydroxy analogue) as the two major ecdysteroids suggests that every ecdysteroid is probably present in both 11α-hydroxy and 11-deoxy forms.

1H and 13C NMR investigation of 20-hydroxyecdysone dioxolane derivatives, a novel group of MDR modulator agents.

The synthesis, structure elucidation and the complete (1)H and (13)C signal assignment of a series of dioxolane derivatives of 20-hydroxyecdysone, synthesized as novel modulators of multidrug resistance, are presented. The structures and NMR signal assignment were established by comprehensive one-dimensional and two-dimensional NMR spectroscopy supported by mass spectrometry.

Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella.

BACKGROUND: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. RESULT: In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM). In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. CONCLUSIONS: Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists) to their targets (ecdysone receptors) leads to an adaptive response (resistance), is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

Proliferation of cancer cells despite CDK2 inhibition.

We have investigated the contribution of CDK4 and CDK2 inhibition to G1 arrest in colon cancers following inhibition of the MEK/MAP kinase pathway. CDK4 inhibition is sufficient to cause arrest, but inhibition of CDK2 by p27 Kip1 redistribution or ectopic expression has no effect on proliferation. Likewise, inhibition of CDK2 through expression of dominant-negative (DN) CDK2 or antisense oligonucleotides did not prevent cell proliferation in these cells. We therefore tested whether CDK2 activity is dispensable in other cells. Surprisingly, osteosarcomas and Rb-negative cervical cancers continued to proliferate after depletion of CDK2 through antisense oligonucleotides or small interfering (si) RNA. Here we report of sustained cell proliferation in the absence of CDK2, and we suggest that CDK2 is not a suitable target for cancer therapy.

Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects.

In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.

Novel ecdysteroids from Serratula wolffii.

Two new and one known ecdysteroids were identified in the methanolic extract of the roots of Serratula wolffii. The new compounds isolated were ponasterone A-22-apioside (1) and 3-epi-shidasterone (3), together with the known 3-epi-22-deoxy-20-hydroxyecdysone (2). The structures of compounds 1-3 were determined by extensive spectroscopic techniques, including one- and two-dimensional NMR methods.

[Estimation of the hypoglycemic effect of phytoecdysteroids].

A series of phytoecodysteroids, including alpha-ecdysone, 2-deoxy-alpha-ecdysone, and 2-deoxyecdysterone isolated from Silene praemixta, integristerone A and ecdysterone isolated from Rhaponticum carthamoides and 22-acetylcyasterone and turkesterone isolated from Ajuga turkestanica, exhibit a pronounced hypoglycemic effect in experiments on intact male rats. The most active compounds--ecdysteron and turkesterone--also produce an expressed hypoglycemic effect in animals with model hyperglycemia induced by the administration of glucose, adrenalin and alloxan. Phytoecdysteroids are substances possessing protein-anabolic activity and are somewhat similar to steranobols in this aspect. Phytoecdysteroids exhibit unidirectional effect and are well comparable with steranabol actionon the carbohydrate metabolism.

Inhibition of secretion of interleukin (IL)-12/IL-23 family cytokines by 4-trifluoromethyl-celecoxib is coupled to degradation via the endoplasmic reticulum stress protein HERP.

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RT-PCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate inappropriate inflammation in autoimmune conditions.

Genome-wide examination of the transcriptional response to ecdysteroids 20-hydroxyecdysone and ponasterone A in Drosophila melanogaster.

BACKGROUND: The 20-hydroxyecdysone (20E) hierarchy of gene activation serves as an attractive model system for studying the mode of steroid hormone regulated gene expression and development. Many structural analogs of 20E exist in nature and among them the plant-derived ponasterone A (PoA) is the most potent. PoA has a higher affinity for the 20E nuclear receptor, composed of the ecysone receptor (EcR) and Ultraspiracle proteins, than 20E and a comparison of the genes regulated by these hormones has not been performed. Furthermore, in Drosophila different cell types elicit different morphological responses to 20E yet the cell type specificity of the 20E transcriptional response has not been examined on a genome-wide scale. We aim to characterize the transcriptional response to 20E and PoA in Drosophila Kc cells and to 20E in salivary glands and provide a robust comparison of genes involved in each response. RESULTS: Our genome-wide microarray analysis of Kc167 cells treated with 20E or PoA revealed that far more genes are regulated by PoA than by 20E (256 vs 148 respectively) and that there is very little overlap between the transcriptional responses to each hormone. Interestingly, genes induced by 20E relative to PoA are enriched in functions related to development. We also find that many genes regulated by 20E in Kc167 cells are not regulated by 20E in salivary glands of wandering 3rd instar larvae and we show that 20E-induced levels of EcR isoforms EcR-RA, ER-RC, and EcR-RD/E differ between Kc cells and salivary glands suggesting a possible cause for the observed differences in 20E-regulated gene transcription between the two cell types. CONCLUSIONS: We report significant differences in the transcriptional responses of 20E and PoA, two steroid hormones that differ by only a single hydroxyl group. We also provide evidence that suggests that PoA induced death of non-adapted insects may be related to PoA regulating different set of genes when compared to 20E. In addition, we reveal large differences between Kc cells and salivary glands with regard to their genome-wide transcriptional response to 20E and show that the level of induction of certain EcR isoforms differ between Kc cells and salivary glands. We hypothesize that the differences in the transcriptional response may in part be due to differences in the EcR isoforms present in different cell types.

Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents.

Nitric-oxide synthase (NOS) activity has been detected in many human tumours, although its function is unclear. Here we show that exposure of cells to nitric oxide (NO) results in a 4-5-fold increase in expression of the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs), one of the key enzymes involved in repairing double-stranded DNA breaks. This NO-mediated increase in enzymatically active DNA-PK not only protects cells from the toxic effects of NO, but also provides crossprotection against clinically important DNA-damaging agents, such as X-ray radiation, adriamycin, bleomycin and cisplatin. The NO-mediated increase in DNA-PKcs described here demonstrates the presence of a new and highly effective NO-mediated mechanism for DNA repair.

Virtual screening for ligands of the insect molting hormone receptor.

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.

Ecdysteroid receptor from the American lobster Homarus americanus: EcR/RXR isoform cloning and ligand-binding properties.

In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.

Proapoptotic and proautophagic activity of 20-hydroxyecdysone in breast cancer cells in vitro.

The present study was designed to identify the biological activity of three ecdysones, i.e., 20-hydroxyecdysone (20-HE), ajugasterone C, and polypodine B isolated from Serratula coronata. The main objective was to investigate the molecular mechanism of the biological activity of those compounds and to assess their impact on breast cancer cell survival and cell cycle. Cell lines were selected according to their hormone receptor status since this factor is perceived as a crucial one in the cancer prognosis as well as cancer cell response to therapy. Consequently, MCF7 (ER/PR+, HER2-), T-47D (ER/PR+, HER2-/+), and MDA-MB-231 (ER/PR-, HER2-) were enrolled in the study. Additionally, a non-tumorigenic, MCF10A cells were selected to verify any potential specificity to cancer cells. Interestingly, none of the studied compounds affected the viability of MCF10A cells while cancer cells were altered, albeit in different ways. Polypodine B did not affect the viability or cell cycle distribution of studied breast cancer cells. By contrast, 20-HE and ajugasterone C significantly inhibited the viability of triple-negative cell line, MDA-MB-231. Interestingly, 20-HE revealed proapoptotic activity in MDA-MB-231 and T-47D cells that was manifested by alterations in PARP, Bax, and Bcl-2 levels as well as caspase-3 activation. Moreover, 20-HE induced autophagy that was mediated by modification of autophagy-associated proteins, i.e., LC3, p62, and mTOR, but only in MDA-MB-231 cells. This study is the first to report diverse biological activity of phytoecdysones in different breast cancer cells, that suggests association with molecular characteristics including receptor status but also other biological properties and genetic markers.

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

Ferroportin is a manganese-responsive protein that decreases manganese cytotoxicity and accumulation.

Although manganese (Mn) is an essential trace element for human development and growth, chronic exposure to excessive Mn levels can result in psychiatric and motor disturbances, referred to as manganism. However, there are no known mechanism(s) for efflux of excess Mn from mammalian cells. Here, we test the hypothesis that the cytoplasmic iron (Fe) exporter ferroportin (Fpn) may also function as a Mn exporter to attenuate Mn toxicity. Using an inducible human embryonic kidney (HEK293T) cell model, we examined the influence of Fpn expression on Mn-induced cytotoxicity and intracellular Mn concentrations. We found that induction of an Fpn-green fluorescent protein fusion protein in HEK293T cells was cytoprotective against several measures of Mn toxicity, including Mn-induced cell membrane leakage and Mn-induced reductions in glutamate uptake. Fpn-green fluorescent protein mediated cytoprotection correlated with decreased Mn accumulation following Mn exposure. Thus, Fpn expression reduces Mn toxicity concomitant with reduced Mn accumulation. To determine if mammalian cells may utilize Fpn in response to increased intracellular Mn concentrations and toxicity, we assessed endogenous Fpn levels in Mn-exposed HEK293T cells and in mouse brain in vivo. We find that 6 h of Mn exposure in HEK293T cells is associated with a significant increase in Fpn levels. Furthermore, mice exposed to Mn showed an increase in Fpn levels in both the cerebellum and cortex. Collectively, these results indicate that (i) Mn exposure promotes Fpn protein expression, (ii) Fpn expression reduces net Mn accumulation, and (iii) reduces cytotoxicity associated with exposure to this metal.