Extract of Ganoderma sinensis spores induces cell cycle arrest of hepatoma cell via endoplasmic reticulum stress.

CONTEXT: Ganoderma sinensis Zhao, Xu et Zhang (Ganodermataceae) has been used for the prevention or treatment of a variety of diseases, including cancer. OBJECTIVE: We investigated the antitumor activity and mechanism of an extract from G. sinensis against hepatocellular carcinoma. MATERIALS AND METHODS: A G. sinensis extract (GSE) was obtained from sporoderm-broken G. sinensis spores by supercritical fluid carbon dioxide extraction. Hepatoma cells, HepG2 cells, were treated with emulsified sample of GSE at 12.5, 25, 50, 100 and 150 µg/mL for 24 h. The Alamar Blue assay was used to examine growth inhibitory effects. Changes in cell structure and morphology were assessed via transmission electron microscopy and confocal laser scanning microscope. Cell cycle distribution was analysed by flow cytometry. RESULTS: GSE suppressed the proliferation of HepG2 cells (IC50=70.14 µg/mL). Extensive cytoplasmic vacuolation originating from dilation of the endoplasmic reticulum (ER) was shown in GSE-treated HepG2 cells. GSE treatment also upregulated the expression of ER stress-related proteins in HepG2 cells. Cells tended to be arrested at the G2/M cell cycle stage after GSE treatment (30.8 ± 1.4% and 42.2 ± 2.6% at GSE with 50 µg/mL and 100 µg/mL vs. 21.03 ± 1.10%, control). Pre-treatment with salubrinal, an inhibitor of ER stress, effectively attenuated cell cycle arrest induced by GSE. DISCUSSION AND CONCLUSIONS: Our findings provide new evidence that GSE suppresses growth of cancer cells in vitro through activating the ER stress pathway. The GSE may be clinically applied in the prevention and/or treatment of cancer.

Time-scale dynamics of proteome predicts the central carbon metabolism involved in triterpenoid accumulation responsive to nitrogen limitation in Ganoderma lucidum.

Central carbon metabolism describes the integration of transport pathway of main carbon sources inside the cell. Nitrogen (N) limitation is a favorable approach to stimulate ganoderic triterpenoid (GT) accumulation in Ganoderma lucidum. In this study, the dynamic regulation of metabolism reassignment towards GT biosynthesis responsive to N limitation was investigated by iTRAQ-based proteome. Physiological data suggested that N limitation slightly affected cell growth but significantly enhanced GT contents in the initial 20 days. From day 10, the protein contents were halted by prolonged N limitation duration. Proteomics-based investigations revealed that the carbon skeletons integrated into GT precursors were regenerated by glycolysis and the tricarboxylic acid (TCA) cycle. Cells strategically reserved nitrogen by barely incorporating it into TCA cycle intermediates to form amino acids, and enzymes involved in protein degradation were up regulated. Furthermore, regulation of proteins in response to abiotic stress and oxidation- reduction processes played a critical role in maintaining cellular homeostasis. These findings indicated that the flux of carbon into GT following N deficiency was a consequence of the remodeling of intermediate metabolism in TCA cycle and glycolysis reactions. This study provides a rationale for genetic engineering of G. lucidum, which may enable synchronized biomass and GT synthesis.

Echinacoside­induced nitric oxide production in endothelial cells: Roles of androgen receptor and the PI3K­Akt pathway.

Echinacoside (ECH) is a natural compound with an endothelium­dependent vasodilatory effect. Nitric oxide (NO) is an important vasorelaxant released from endothelial cells. In order to examine the molecular mechanism of ECH­induced NO production in endothelial cells, the present study investigated the involvement of androgen receptor (AR) and the phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt) pathway in the phosphorylation of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs). Using the fluorescent probe DAF­FM, the production of NO was found to be significantly increased, and eNOS was phosphorylated at Ser1177 in a concentration­â€‹dependent manner under 0.01­10 µM ECH treatment in HUVECs. In addition, NO production and eNOS phosphorylation induced by ECH were diminished when pretreated with the AR antagonist nilutamide, or when transfected with AR small interfering RNAs. Furthermore, the ECH­induced phosphorylation of the Akt at Ser473 was abrogated by 5 µM wortmannin (a PI3K inhibitor). These data indicated that ECH stimulated NO production via the AR­dependent activation of eNOS in HUVECs, and that the PI3K/Akt pathway may be involved in eNOS phosphorylation induced by ECH.

1H NMR spectroscopy-based metabolic profiling of Ophiocordyceps sinensis and Cordyceps militaris in water-boiled and 50% ethanol-soaked extracts.

Ophiocordyceps sinensis, a well-known Chinese complementary herb, is a rare and valuable therapeutic resource. Cordyceps militaris (C. militaris) is a commonly used substitute for O. sinensis. A metabolomic-based approach for exploring the similarities and differences in the metabolites of O. sinensis and C. militaris in water-boiled and 50% ethanol-soaked extracts is of great significance. To distinguish between the global metabolite profiles of O. sinensis and C. militaris extracts obtained from either the water-boiled or 50% ethanol-soaked methods, we investigated the herb samples using 1HNMR-based metabolic fingerprints combined with multivariate statistical analysis. This study revealed that a total of 52 primary metabolites were identified and quantified from O. sinensis and C. militaris samples. Forty-three (83% of 52) metabolites were detectable in both O. sinensis and C. militaris. According to the variable importance in projection (VIP) value and p-value from the Mann-Whitney test, 7 metabolites (alanine, aspartate, glutamate, mannitol, ornithine, serine, and trehalose) differed between O. sinensis and C. militaris. Arginine, glucose, putrescine, pyroglutamate, betaine, O-phosphocholine, and xylose differed significantly between the water-boiled and 50% ethanol-soaked methods used to prepare the herb extracts. This study demonstrated that water-boiled extraction was a much faster method (30 min. vs 360 days) that resulted in a 30% higher number of extracted metabolites (compared to 50% for the ethanol-soaked method) for both O. sinensis and C. militaris.

Profile of Ophiocordyceps sinensis transcriptome and differentially expressed genes in three different mycelia, sclerotium and fruiting body developmental stages.

Ophiocordyceps sinensis, a Chinese complementary and alternative medicine (CAM), is an entomopathogenic, fungus, parasitizing larvae of the moth genus Thitarodes. It has three stages of the life cycle, i.e., the anamorph mycelia prior to infection (Cm_Os), the mycelia sclerotium forming in the caterpillar (Te_Ca), and the fruiting bodies or stromata (Te_St). Characterization of the O. sinensis transcriptome among these stages could provide a better understanding of the underlying biology processes. Transcriptomics of the O. sinensis asexual mycelia and hyphae in deceased caterpillars and perithecial stroma was assessed by using Illumina HiSeq™ 2000 technology. A total of 14,922 unigenes were identified and categorized into 46 sub-categories under three gene ontology categories ("biological process", "cellular component", and "molecular function"). Of these genes, 5520 were differentially expressed among the libraries of these three groups of samples (P < 0.05), and 391 genes occurred in all three groups. Compared to the anamorph stage, there were 3049 differentially expressed genes (DEGs) in the teleomorph stage, but only 1023 DEGs occurred within the teleomorph groups (Te_St vs. Te_Ca). Collectively, this study provides a novel resource to further investigate O. sinensis and their three different development stages.