Small Natural Compound Research Articles

A natural compound jaceosidin ameliorates endoplasmic reticulum stress and insulin resistance via upregulation of SERCA2b

Increased endoplasmic reticulum (ER) stress has emerged as a vital contributor to dysregulated glucose homeostasis, and impaired function of sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) is one of the central mechanisms underlying ER stress. In this study, we reported that SERCA2b upregulation contributed to the amelioration of ER stress and insulin resistance by a small natural compound jaceosidin. In a model of differentiated C2C12 myotubes, jaceosidin-triggered SERCA2b upregulation enhanced insulin sensitivity and decreased ER stress. Moreover, the activity of Ca2+-ATPase in thapsigargin-treated myotubes was also augmented by jaceosidin. Furthermore, jaceosidin significantly suppressed blood glucose levels, improved glucose tolerance and lowered body weight, but did not alter food intake in insulin-resistant obese mice. In addition, this compound markedly reduced lipid accumulation, suppressed the expression of lipogenic genes in liver and ameliorated liver injury. The ameliorative effects of jaceosidin were due to its ability to reduce ER stress via increasing the expression of SERCA2b in the muscles of obese mice. Taken together, jaceosidin could improve ER stress and attenuate insulin resistance via SERCA2b upregulation in mice skeletal muscles.

Polydatin Prevents Methylglyoxal-Induced Apoptosis through Reducing Oxidative Stress and Improving Mitochondrial Function in Human Umbilical Vein Endothelial Cells.

Methylglyoxal (MGO), an active metabolite of glucose, has been reported to induce vascular cell apoptosis in diabetic complication. Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions, such as antioxidative, anti-inflammatory, and nephroprotective properties. However, the protective effects of PD on MGO-induced apoptosis in endothelial cells remain to be elucidated. In this study, human umbilical vein endothelial cells (HUVECs) were used to explore the effects of PD on MGO-induced cell apoptosis and the possible mechanism involved. HUVECs were pretreated with PD for 2 h, followed by stimulation with MGO. Then cell apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) impairment, mitochondrial morphology alterations, and Akt phosphorylation were assessed. The results demonstrated that PD significantly prevented MGO-induced HUVEC apoptosis. PD pretreatment also significantly inhibited MGO-induced ROS production, MMP impairment, mitochondrial morphology changes, and Akt dephosphorylation. These results and the experiments involving N-acetyl cysteine (antioxidant), Cyclosporin A (mitochondrial protector), and LY294002 (Akt inhibitor) suggest that PD prevents MGO-induced HUVEC apoptosis, at least in part, through inhibiting oxidative stress, maintaining mitochondrial function, and activating Akt pathway. All of these data indicate the potential application of PD for the treatment of diabetic vascular complication.

A Novel, Multi-Target Natural Drug Candidate, Matrine, Improves Cognitive Deficits in Alzheimer's Disease Transgenic Mice by Inhibiting Aβ Aggregation and Blocking the RAGE/Aβ Axis.

The treatment of AD is a topic that has puzzled researchers for many years. Current mainstream theories still consider Aβ to be the most important target for the cure of AD. In this study, we attempted to explore multiple targets for AD treatments with the aim of identifying a qualified compound that could both inhibit the aggregation of Aβ and block the RAGE/Aβ axis. We believed that a compound that targets both Aβ and RAGE may be a feasible strategy for AD treatment. A novel and small natural compound, Matrine (Mat), was identified by high-throughput screening of the main components of traditional Chinese herbs used to treat dementia. Various experimental techniques were used to evaluate the effect of Mat on these two targets both in vitro and in AD mouse model. Mat could inhibit Aβ42-induced cytotoxicity and suppress the Aβ/RAGE signaling pathway in vitro. Additionally, the results of in vivo evaluations of the effects of Mat on the two targets were consistent with the results of our in vitro studies. Furthermore, Mat reduced proinflammatory cytokines and Aβ deposition and attenuated the memory deficits of AD transgenic mice. We believe that this novel, multi-target strategy to inhibit both Aβ and RAGE, is worthy of further exploration. Therefore, our future studies will focus on identifying even more effective multi-target compounds for the treatment of AD based on the molecular structure of Mat.

Embelin binds to human neuroserpin and impairs its polymerisation.

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.

Cuminaldehyde as the Major Component of Cuminum cyminum, a Natural Aldehyde with Inhibitory Effect on Alpha-Synuclein Fibrillation and Cytotoxicity.

Fibrillation of alpha-synuclein (α-SN) is a critical process in the pathophysiology of several neurodegenerative diseases, especially Parkinson's disease. Application of bioactive inhibitory compounds from herbal extracts is a potential therapeutic approach for this cytotoxic process. Here, we investigated the inhibitory effects of the Iranian Cuminum cyminum essential oil on the fibrillation of α-SN. Analysis of different fractions from the total extract identified cuminaldehyde as the active compound involved in the antifibrillation activity. In comparison with baicalein, a well-known inhibitor of α-SN fibrillation, cuminaldehyde showed the same activity in some aspects and a different activity on other parameters influencing α-SN fibrillation. The presence of spermidine, an α-SN fibrillation inducer, dominantly enforced the inhibitory effects of cuminaldehyde even more intensively than baicalein. Furthermore, the results from experiments using preformed fibrils and monobromobimane-labeled monomeric protein also suggest that cuminaldehyde prevents α-SN fibrillation even in the presence of seeds, having no disaggregating impact on the preformed fibrils. Structural studies showed that cuminaldehyde stalls protein assembly into β-structural fibrils, which might be achieved by the interaction with amine groups through its aldehyde group as a Schiff base reaction. This assumption was supported by FITC labeling efficiency assay. In addition, cytotoxicity assays on PC12 cells showed that cuminaldehyde is a nontoxic compound, treatment with cuminaldehyde throughout α-SN fibrillation showed no toxic effects on the cells. Taken together, these results show for the first time that the small abundant natural compound, cuminaldehyde, can modulate α-SN fibrillation. Hence, suggesting that such natural active aldehyde could have potential therapeutic applications.

Upregulation of cyclin-dependent kinase inhibitors CDKN1B and CDKN1C in hepatocellular carcinoma-derived cells via goniothalamin-mediated protein stabilization and epigenetic modifications

Cell cycle deregulation is common in human hepatocellular carcinoma (HCC). To ensure proper cell cycle controlling, cyclin/cyclin-dependent kinases (CDK) complexes are tightly regulated by CDK inhibitors (CKIs) in normal cells. However, insufficient cyclin-dependent kinase inhibitor 1B (CDKN1B, also known as p27Kip1) and CDKN1C (p57Kip2) proteins are characteristics of high-risk HCC. In two HCC-derived cell lines with distinct genetic backgrounds, we identified a small natural compound, goniothalamin (GTN), serving as an inducer of CKIs. In TP53-mutated (Y220C) and retinoblastoma 1 (RB1)-positive Huh-7 cells, GTN stabilized CDKN1B protein levels by targeting the degradation of its specific E3 ubiquitin ligase (S-phase kinase-associated protein 2). Alternatively, in TP53- and RB1-negative Hep-3B cells, GTN increased CDKN1C transcription and its subsequent translation by acting as a histone deacetylase inhibitor. In both cell lines, GTN induced G0/G1 cell cycle arrest, delayed S phase entry of cells and inhibited anchorage-independent cell growth which might be attributed to the upregulation of CKIs and downregulation of several positive cell cycle regulators, including CDC28 protein kinase regulator subunit 1B, cyclin E1 and D1, cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, E2F transcription factor 1 and/or transcription factor Dp-1. Therefore, GTN might represent a novel class of anticancer drug that induces CKIs through post-translational and epigenetic modifications.

Polydatin inhibits growth of lung cancer cells by inducing apoptosis and causing cell cycle arrest

Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions. However, the anticancer activity of PD has been poorly investigated. In the present study, thiazolyl blue tetrazolium bromide assay was used to evaluate the inhibitory effect of PD on cell growth. Cell cycle distribution and apoptosis were investigated by flow cytometry. In addition, the expression of several proteins associated with apoptosis and cell cycle were analyzed by western blot analysis. The results demonstrated that PD significantly inhibits the proliferation of A549 and NCI-H1975 lung cancer cell lines and causes dose-dependent apoptosis. Cell cycle analysis revealed that PD induces S phase cell cycle arrest. Western blot analysis showed that the expression of Bcl-2 decreased as that of Bax increased, and the expression of cyclin D1 was also suppressed. The results suggest that PD has potential therapeutic applications in the treatment of lung cancer.

Transient exposure to echinacoside is sufficient to activate Trk signaling and protect neuronal cells from rotenone

Neurotrophins exert their physiological functions mainly through Trk receptors, and the neurotrophic signaling network is critical to the survival of neurons. However, therapeutic use of neurotrophins in treating neurodegenerative diseases is hampered by a number of pharmacological challenges, and the most significant challenge is their delivery into the central nervous system. Here, we reported that echinacoside, a small natural compound, elicits neuroprotection by activating Trk receptors and their downstream signal pathways. Echinacoside is the major active component of Cistanches Herba, a widely used Chinese herb with neuroprotective effects. We showed in this study that transient exposure to echinacoside is sufficient to protect neuronal cells and non-neuronal cells over-expressed with TrkA or TrkB against rotenone injury. Additional investigations on the mechanisms underlying suggested that transient treatment with echinacoside inhibits cytochrome c release and caspase-3 activation caused by ensuing rotenone exposure via activating Trk-extracellular signal-regulated kinase (ERK) pathway in neuronal cells. As echinacoside is able to cross the blood-brain barrier freely, it may have a promising potential in neurodegenerative diseases treatment.

Extensive In vivo Metabolite‐Protein Interactions Revealed by Large‐Scale Systematic Analyses

Extensive effort has been made to systematically characterize protein‐protein and DNA‐protein interactions. Natural small compounds comprise the majority of the cellular molecules and bind proteins as substrates, products, cofactors and ligands. However, a large scale investigation of in vivo protein‐small metabolite interactions has not been performed. We have developed a mass spectrometry assay for the large scale identification of in vivo protein‐hydrophobic small metabolite interactions in yeast and used it to analyze compounds that bind ergosterol biosynthetic proteins and protein kinases. 17–20% of these proteins bind small metabolites; a few interactions are known, but the vast majority are novel. Importantly, many key regulatory proteins such as protein kinases bind small metabolites. Ergosterol was found to bind many proteins and may function as a general regulator. It is required for the full activity of Ypk1, a homolog of mammalian AKT/SGK1 kinase family. Overall, our study defines potential key regulatory steps in lipid biosynthetic pathways and suggests small metabolites may play a hitherto underappreciated role as general regulators of protein activity and function.

Alantolactone Induces Apoptosis in Imatinib-Sensitive and -Resistant CML Cells by Inhibiting NF-KappaB Signaling and Degrading Bcr/Abl Protein

Abstract 4419Chronic myelogenous leukemia (CML) is characterized by the t(9;22) chromosomal translocation, which results in the generation of Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Despite the great success of imatinib therapy in CML, not all patients, especially those in the accelerated or blastic phase, experience an optimal response to this drug or even to the more potent, second-generation TKIs. The emergence of resistance to TKIs has also become a significant problem implying that physiopathologically relevant cellular targets should be validated, such as NF-kappaB (NF-kB).Here we describe the activity of alantolactone, a small natural compound and see its effect on K562-sensitive (K562s, sensitive to imatinib) and K562-resistant (K562r, resistant to imatinib) cells. Results:Firstly, we used CCK-8 assay to determine the cell viability and found that K562s were inhibited up to 64.5% after 48 hours of incubation of 10uM concentration of alantolactone, Annexin V+ cells were up to 68% which were confirmed by morphological observation, and apoptosis-related proteins were activated during the treatment, suggesting its inducing-apoptosis ability. Secondly, incubation of alantolactone could inhibit NF-kB signaling activity, proved by inhibition of IkBa phosphorylation and blockade of nuclear translocation of p65, while the luciferase assay further revealed the inhibited transcription of NF-kB dependent reporter gene, which was confirmed by immunofluorescence staining. Of note, Bcr-Abl protein was specifically and rapidly down-regulated as early as 6 hours after 10uM of alantolactone treatment. And alantolactone-induced Bcr-Abl down-regulation could be partially reversed by the autophagy inhibitor chloroquine but not by the proteasome inhibitor MG132 or bortezomib, indicating that autophagy may contributes to Bcr-Abl reduction. Furthermore, similar concentration of alantolactone could also induce apoptosis in K562r cells and inhibited the growth of primary CD34+ leukemic cells from CML patients.Collectively, our results primarily demonstrated that alantolactone could induce apoptosis in CML cells, regardless of imatinib-sensitive or -resistant, by inhibiting NF-kB signaling and depleting Bcr/Abl through a unique mechanism. Alantolactone could be used as a probe for deciphering the mechanism of Bcr-Abl destruction and NF-kB inhibition, and this agent may be a promising candidate for treating CML patients. Disclosures:No relevant conflicts of interest to declare.

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to polydatin-induced apoptosis in human nasopharyngeal carcinoma CNE cells

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti-cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose- and time-dependent manner. The compound also dose-dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt in CNE cells, while knock-down of CCAAT/enhancer-binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti-tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin.

Extensive In Vivo Metabolite-Protein Interactions Revealed by Large-Scale Systematic Analyses

Natural small compounds comprise most cellular molecules and bind proteins as substrates, products, cofactors, and ligands. However, a large-scale investigation of invivo protein-small metabolite interactions has not been performed. We developed a mass spectrometry assay for the large-scale identification of invivo protein-hydrophobic small metabolite interactions in yeast and analyzed compounds that bind ergosterol biosynthetic proteins and protein kinases. Many of these proteins bind small metabolites; a few interactions were previously known, but the vast majority are new. Importantly, many key regulatory proteins such as protein kinases bind metabolites. Ergosterol was found to bind many proteins and may function as a general regulator. It is required for the activity of Ypk1, a mammalian AKT/SGK kinase homolog. Our study defines potential key regulatory steps in lipid biosynthetic pathways and suggests that small metabolites may play a more general role as regulators of protein activity and function than previously appreciated.

Novel immunomodulatory properties of cirsilineol through selective inhibition of IFN-γ signaling in a murine model of inflammatory bowel disease

Regulation of signal transducer and activator of transcription (STAT) 1 signaling is being explored as a new approach to the treatment of inflammatory bowel diseases. However, few chemicals have been reported to inhibit IFN-γ/STAT1 signaling for Crohn's disease therapy. In the present study, we found that cirsilineol, a small natural compound isolated from Artemisia vestita, significantly ameliorated trinitro-benzene sulfonic acid (TNBS)-induced T-cell-mediated experimental colitis in mice, which was closely associated with reduced autoreactive T-cell proliferation and activation. Moreover, the regulatory action of pro-inflammatory and anti-inflammatory cytokine by cirsilineol treatment was found to decrease the activity of effector Th1 cells but increase the activity of regulatory T cells as characterized by down-regulation of IFN-γ and corresponding up-regulation of IL-10 and TGF-β. The therapeutic effect of cirsilineol was attributable to a novel regulatory mechanism with selective inhibiting IFN-γ signaling in colonic lamina propria CD4+ T cells, which was mediated through down-regulating STAT1 activation and T-bet expression. Furthermore, cirsilineol was found to down-regulate the activation of JAK2, a critical kinase for IFN-γ/STAT1 signaling, and abrogate the expression of T-bet, resulting in markedly decreased proliferation and activation of T cells in vitro. Importantly, the inhibition of IFN-γ/STAT1 signaling by cirsilineol was reversible in the presence of high level of IFN-γ. These results strongly suggest that cirsilineol might be potentially useful for treating T-cell-mediated human inflammatory bowel diseases.

Selective triggering of apoptosis of concanavalin A-activated T cells by fraxinellone for the treatment of T-cell-dependent hepatitis in mice

Selectively inducing apoptosis of activated T cells is essential for the clearance of pathogenic injurious cells and subsequent efficient resolution of inflammation. However, few chemicals have been reported to trigger apoptosis of activated T cells in the treatment of hepatitis without affecting quiescent T cells. In the present study, we found that fraxinellone, a small natural compound isolated from the root bark of Dictamnus dasycarpus, selectively facilitated apoptosis of concanavalin A (Con A)-activated CD4 + T cells rather than those non-activated, by disrupting the mitochondrial transmembrane potential, decreasing the ratio of Bcl-2/Bax, and increasing cytochrome c release from the mitochondria to the cytosol. The enhancement in Fas expression and caspase-8 activity, truncation of Bid, and down-regulation of anti-apoptotic cellular FLICE-inhibitory protein expression by fraxinellone also suggested the participation of an extrinsic apoptosis pathway. Furthermore, fraxinellone significantly alleviated Con A-induced T-cell-dependent hepatitis in mice, which was closely associated with reduced serum transaminases, pro-inflammatory cytokines, and pathologic parameters. Consistent with the in vitro results, fraxinellone dramatically induced apoptosis of activated peripheral CD4 + T cells in vivo, consequently resulting in less CD4 + T-cell activation and infiltration to the liver. These results strongly suggest fraxinellone might be a potential leading compound useful in treating T-cell-mediated liver disorders in humans.

Structure-based design of novel pyrazoles as aurora kinase A inhibitors.

AbstractInhibition of Aurora kinase, a member of serine/threonine kinase involved in the regulation of cell division is emerging as a new molecular targeted cancer treatment option. Three isoforms of Aurora kinase, A, B and C are known. Both Aurora A and B are over expressed in many human cancers and are linked to chromosome instability, oncogenic transformation, tumour progression and development of chemoresistance. Inhibitors of Aurora kinase, regardless of their kinase specificity spectrum have shown to promote cancer cell death by induction of apoptosis and mitotic catastrophe. Based on the current success of Aurora kinase inhibitors in the development of kinase-based cancer therapy, we have initiated a structure-based virtual screening for the identification of Aurora kinase inhibitors by using published aurora protein structure (PDB code:1MQ4). Small molecule and natural product compound libraries were docked into the active site/ATP binding site using the programs Gold 3.0 and Glide 4.5. A total of six compounds were identified with high priority scores through this method to possess Aurora A inhibition. One of the virtual screen (VS) hit, BPR1K0025S0 was synthesised in 3 steps and found to have Aurora A IC50~15 M and x-ray co-crystal structure in complex with the aurora protein was solved for this compound. Based on the binding mode of this compound to Aurora A protein, further modification in the ester part was envisaged and a total of around 40 compounds were synthesised. Many of them possessed enhanced activity level compared to the original hit. Of particular interest is the aniline series of compounds, which showed submicromolar activity in the enzyme based Aurora inhibition assay. Thus a forty-fold improvement in activity for BPR1K0269S0 was achieved from the initial virtual screen hit BPR1K0025S0.