Shotgun Proteomics and Quantitative Pathway Analysis of the Mechanisms of Action of Dehydroeffusol, a Bioactive Phytochemical with Anticancer Activity from Juncus effusus.

Dehydroeffusol (DHE) is a phenanthrene isolated from the Chinese medicinal plant Juncus effusus. Biological evaluation of DHE reveals in vitro and in vivo anticancer effects. We performed a shotgun proteomic analysis using liquid chromatography-tandem mass spectrometry to investigate the changes in the protein profiles in cancer cells upon DHE treatment. DHE affected cancer-associated signaling pathways, including NF-κB, ß-catenin, and endoplasmic reticulum stress. Through quantitative pathway and key node analysis of the proteomics data, activating transcription factor 2 (ATF-2) and c-Jun kinase (JNK) were found to be the key components in DHE's modulated biological pathways. Based on the pathway analysis as well as chemical similarity to estradiol, DHE is proposed to be a phytoestrogen. The proteomic, bioinformatic, and chemoinformatic analyses were further verified with individual cell-based experiments. Our study demonstrates a workflow for identifying the mechanisms of action of DHE through shotgun proteomic analysis.

Identification of "sarsasapogenin-aglyconed" timosaponins as novel Aß-lowering modulators of amyloid precursor protein processing.

The inhibition of amyloid ß (Aß) peptide production is a key approach in the development of therapeutics for the treatment of Alzheimer's disease (AD). We have identified that timosaponins consisting of sarsasapogenin (SSG) as the aglycone can effectively lower the production of Aß peptides and stimulate neurite outgrowth in neuronal cell cultures. Structure-activity relationship studies revealed that the cis-fused AB ring, 3ß-configuration, spiroketal F-ring and 25S-configuration of SSG are the essential structural features responsible for the Aß-lowering effects and neurite-stimulatory activity. New synthetic derivatives that retain the SSG scaffold also exhibited an Aß lowering effect. Treatment of cells with timosaponins led to modulation of amyloid precursor protein (APP) processing through the suppression of ß-cleavage and preferential lowering of the production of the 42-amino acid Aß species (Aß42) without affecting another γ-secretase substrate. The SSG and "SSG-aglyconed" timosaponins also penetrated brain tissue and lowered brain Aß42 levels in mice. Our studies demonstrate that timosaponins represent a unique class of steroidal saponins that may be useful for the development of AD therapeutics.

Activation of autophagy of aggregation-prone ubiquitinated proteins by timosaponin A-III.

Chemical modulators of autophagy provide useful pharmacological tools for examination of autophagic processes, and also may lead to new therapeutic agents for diseases in which control of cellular sequestration and degradation capacity are beneficial. We have identified that timosaponin A-III (TAIII), a medicinal saponin reported to exhibit anticancer properties and improve brain function, is a pronounced activator of autophagy. In this work, the salient features and functional role of TAIII-induced autophagy were investigated. In TAIII-treated cells, autophagic flux with increased formation of autophagosomes and conversion into autolysosomes is induced in association with inhibition of mammalian target of rapamycin activity and elevation of cytosolic free calcium. The TAIII-induced autophagy is distinct from conventional induction by rapamycin, exhibiting large autophagic vacuoles that appear to contain significant contents of endosomal membranes and multivesicular bodies. Furthermore, TAIII stimulates biosynthesis of cholesterol, which is incorporated to the autophagic vacuole membranes. The TAIII-induced autophagic vacuoles capture ubiquitinated proteins, and in proteasome-inhibited cells TAIII promotes autophagy of aggregation-prone ubiquitinated proteins. Our studies demonstrate that TAIII induced a distinct form of autophagy, and one of its pharmacological actions is likely to enhance the cellular quality control capacity via autophagic clearance of otherwise accumulated ubiquitinated protein aggregates.

Timosaponin A-III induces autophagy preceding mitochondria-mediated apoptosis in HeLa cancer cells.

Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. Here, we provide evidence that TAIII induces autophagy in HeLa cells followed by apoptotic cell death. TAIII-induced autophagy was morphologically characterized by the formation of membrane-bound autophagic vacuoles recognizable at the ultrastructural level. TAIII-treated cells expressing green fluorescent protein (GFP)-labeled microtubule-associated protein 1 light chain 3 (LC3) displayed punctate fluorescence indicative of LC3 recruitment to the autophagosome. This was associated with the conversion of LC3-I (the cytosolic form) into LC3-II (the lipidated form located on the autophagosome membrane). TAIII treatment also induced mitochondrial dysfunction involving overproduction of reactive oxygen species and reduction of mitochondrial membrane potential accompanied by induction of mitochondrial permeability transition. Prolonged exposure to TAIII resulted in cytochrome c release and caspase-3 activation, events that signified the onset of apoptotic cell death. TAIII-induced autophagy preceded apoptosis, as evidenced by early autophagic vacuole formation, GFP-LC3 translocation, and LC3-II increase in the absence of caspase-3 cleavage. Notably, TAIII-mediated apoptotic cell death was potentiated by treatment with autophagy inhibitor 3-methyladenine or small interfering RNA against the autophagic gene beclin 1. These findings suggest that TAIII-elicited autophagic response plays a protective role that impedes the eventual cell death. In terms of structure-activity relationship, the sugar chain in TAIII is indispensable to the drug action, as the sugar-lacking aglycone sarsasapogenin did not induce autophagy and exhibited weaker cytotoxicity.

Terpenoids from the seeds of Artemisia annua.

Fourteen sesquiterpenes, three monoterpenes and one diterpene natural product have been isolated from the seeds of Artemisia annua. The possible biogenesis of some of these natural products are discussed by reference to recently reported experimental results for the autoxidation of dihydroartemisinic acid and other terpenoids from Artemisia annua.

A prezizaane sesquiterpene from Illicium angustisepalum.

The prezizaane sesquiterpene angustisepalin has been isolated from the aerial parts of Illicium angustisepalum, and its structure determined by 2D-NMR spectroscopy. Angustisepalin is formally the 10-benzoyl ester of neomajucin, previously reported from Illicium majus.

Determination of the absolute stereochemistry of the epoxide group in alpinia epoxide by NMR.

The absolute stereochemistry of the epoxide group in alpinia epoxide [14,15-epoxylabda-8(17),12-dien-16-al (E)] has been determined by simultaneous reduction of the aldehyde and epoxide functional groups in this molecule to primary and secondary alcohols, followed by selective protection of the primary alcohol and derivitization of the secondary alcohol with S(+) and R(-) MTPCl as Mosher esters. Changes in (1)HNMR chemical shifts for all positions in these two esters were determined by 2D-NMR and used to infer the absolute stereochemistry of the epoxide group in the natural product alpinia epoxide.