The Oligostilbene Gnetin H Is a Novel Glycolysis Inhibitor That Regulates Thioredoxin Interacting Protein Expression and Synergizes with OXPHOS Inhibitor in Cancer Cells.

Since aerobic glycolysis was first observed in tumors almost a century ago by Otto Warburg, the field of cancer cell metabolism has sparked the interest of scientists around the world as it might offer new avenues of treatment for malignant cells. Our current study claims the discovery of gnetin H (GH) as a novel glycolysis inhibitor that can decrease metabolic activity and lactic acid synthesis and displays a strong cytostatic effect in melanoma and glioblastoma cells. Compared to most of the other glycolysis inhibitors used in combination with the complex-1 mitochondrial inhibitor phenformin (Phen), GH more potently inhibited cell growth. RNA-Seq with the T98G glioblastoma cell line treated with GH showed more than an 80-fold reduction in thioredoxin interacting protein (TXNIP) expression, indicating that GH has a direct effect on regulating a key gene involved in the homeostasis of cellular glucose. GH in combination with phenformin also substantially enhances the levels of p-AMPK, a marker of metabolic catastrophe. These findings suggest that the concurrent use of the glycolytic inhibitor GH with a complex-1 mitochondrial inhibitor could be used as a powerful tool for inducing metabolic catastrophe in cancer cells and reducing their growth.

Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis.

Eupolauridine and liriodenine are plant-derived aporphinoid alkaloids that exhibit potent inhibitory activity against the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans However, the molecular mechanism of this antifungal activity is unknown. In this study, we show that eupolauridine 9591 (E9591), a synthetic analog of eupolauridine, and liriodenine methiodide (LMT), a methiodide salt of liriodenine, mediate their antifungal activities by disrupting mitochondrial iron-sulfur (Fe-S) cluster synthesis. Several lines of evidence supported this conclusion. First, both E9591 and LMT elicited a transcriptional response indicative of iron imbalance, causing the induction of genes that are required for iron uptake and for the maintenance of cellular iron homeostasis. Second, a genome-wide fitness profile analysis showed that yeast mutants with deletions in iron homeostasis-related genes were hypersensitive to E9591 and LMT. Third, treatment of wild-type yeast cells with E9591 or LMT generated cellular defects that mimicked deficiencies in mitochondrial Fe-S cluster synthesis including an increase in mitochondrial iron levels, a decrease in the activities of Fe-S cluster enzymes, a decrease in respiratory function, and an increase in oxidative stress. Collectively, our results demonstrate that E9591 and LMT perturb mitochondrial Fe-S cluster biosynthesis; thus, these two compounds target a cellular pathway that is distinct from the pathways commonly targeted by clinically used antifungal drugs. Therefore, the identification of this pathway as a target for antifungal compounds has potential applications in the development of new antifungal therapies.

Sampangine (a Copyrine Alkaloid) Exerts Biological Activities through Cellular Redox Cycling of Its Quinone and Semiquinone Intermediates.

The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity.

Neurobehavioral and transcriptional effects of acrylamide in juvenile rats.

Acrylamide is a type-2 alkene monomer with established human neurotoxic effects. While the primary source of human exposure to acrylamide is occupational, other exposure sources include food, drinking water, and smoking. In this study, neurobehavioral assays coupled with transcriptional profiling analysis were conducted to assess both behavioral and gene expression effects induced by acrylamide neurotoxicity in juvenile rats. Acrylamide administration in rat pups induced significant characteristic neurotoxic symptoms including increased heel splay, decrease in grip strength, and decrease in locomotor activity. Transcriptome analysis with the Affymetrix Rat Genome 230 2.0 array indicated that acrylamide treatment caused a significant alteration in the expression of a few genes that are involved in muscle contraction, pain, and dopaminergic neuronal pathways. First, expression of the Mylpf gene involved in muscle contraction was downregulated in the spinal cord in response to acrylamide. Second, in sciatic nerves, acrylamide repressed the expression of the opioid receptor gene Oprk1 that is known to play a role in neuropathic pain regulation. Finally, in the cerebellum, acrylamide treatment caused a decrease in the expression of the nuclear receptor gene Nr4a2 that is required for development of dopaminergic neurons. Thus, our work examining the effect of acrylamide at the whole-genome level on a developmental mammalian model has identified a few genes previously not implicated in acrylamide neurotoxicity that might be further developed into biomarkers for assessing the risk of adverse health effects induced by acrylamide exposure.

Antifungal cyclic peptides from the marine sponge Microscleroderma herdmani.

Screening natural product extracts from the National Cancer Institute Open Repository for antifungal discovery afforded hits for bioassay-guided fractionation. Using LC-MS analysis to generate chemical structure information on potentially active compounds, two new cyclic hexapeptides, microsclerodermins J (1) and K (2), were isolated from the deep-water sponge Microscleroderma herdmani, along with microsclerodermins A (3) and B (4), previously isolated from an unidentified Microscleroderma species. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. In vitro antifungal testing showed that the four compounds possessed strong activities against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and Aspergillus fumigatus.

Sampangine inhibits heme biosynthesis in both yeast and human.

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.

Identification of molecular pathways affected by pterostilbene, a natural dimethylether analog of resveratrol.

BACKGROUND: Pterostilbene, a naturally occurring phenolic compound produced by agronomically important plant genera such as Vitis and Vacciunium, is a phytoalexin exhibiting potent antifungal activity. Additionally, recent studies have demonstrated several important pharmacological properties associated with pterostilbene. Despite this, a systematic study of the effects of pterostilbene on eukaryotic cells at the molecular level has not been previously reported. Thus, the aim of the present study was to identify the cellular pathways affected by pterostilbene by performing transcript profiling studies, employing the model yeast Saccharomyces cerevisiae. METHODS: S. cerevisiae strain S288C was exposed to pterostilbene at the IC50 concentration (70 muM) for one generation (3 h). Transcript profiling experiments were performed on three biological replicate samples using the Affymetrix GeneChip Yeast Genome S98 Array. The data were analyzed using the statistical methods available in the GeneSifter microarray data analysis system. To validate the results, eleven differentially expressed genes were further examined by quantitative real-time RT-PCR, and S. cerevisiae mutant strains with deletions in these genes were analyzed for altered sensitivity to pterostilbene. RESULTS: Transcript profiling studies revealed that pterostilbene exposure significantly down-regulated the expression of genes involved in methionine metabolism, while the expression of genes involved in mitochondrial functions, drug detoxification, and transcription factor activity were significantly up-regulated. Additional analyses revealed that a large number of genes involved in lipid metabolism were also affected by pterostilbene treatment. CONCLUSION: Using transcript profiling, we have identified the cellular pathways targeted by pterostilbene, an analog of resveratrol. The observed response in lipid metabolism genes is consistent with its known hypolipidemic properties, and the induction of mitochondrial genes is consistent with its demonstrated role in apoptosis in human cancer cell lines. Furthermore, our data show that pterostilbene has a significant effect on methionine metabolism, a previously unreported effect for this compound.

Capisterones A and B, which enhance fluconazole activity in Saccharomyces cerevisiae, from the marine green alga Penicillus capitatus.

A whole-cell-based assay using Saccharomyces cerevisiae strains that overexpress Candida albicans CDR1 and MDR1 efflux pumps has been employed to screen natural product extracts for reversal of fluconazole resistance. The tropical green alga Penicillus capitatus was selected for bioassay-guided isolation, leading to the identification of capisterones A and B (1 and 2), which were recently isolated from this alga and shown to possess antifungal activity against the marine pathogen Lindra thallasiae. Current work has assigned their absolute configurations using electronic circular dichroism and determined their preferred conformations in solution based on detailed NOE analysis. Compounds 1 and 2 significantly enhanced fluconazole activity in S. cerevisiae, but did not show inherent antifungal activity when tested against several opportunistic pathogens or cytotoxicity to several human cancer and noncancerous cell lines (up to 35 microM). These compounds may have a potential for combination therapy of fungal infections caused by clinically relevant azole-resistant strains.

Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1.

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

Hypoxia-inducible factor-1 activation by (-)-epicatechin gallate: potential adverse effects of cancer chemoprevention with high-dose green tea extracts.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that induces oxygen-regulated genes in response to reduced oxygen conditions (hypoxia). Expression of the oxygen-regulated HIF-1alpha subunit correlates positively with advanced disease stages and poor prognosis in cancer patients. Green tea catechins are believed to be responsible for the cancer chemopreventive activities of green tea. We found that (-)-epicatechin-3-gallate (ECG, 1), one of the major green tea catechins, strongly activates HIF-1 in T47D human breast carcinoma cells. Among the green tea catechins tested, 1 demonstrated the strongest HIF-1-inducing activity, while (-)-epigallocatechin-3-gallate (EGCG, 2) was significantly less active. However, 2 is relatively unstable in the in vitro system studied. Compound 1 also increases the expression of HIF-1 target genes including GLUT-1, VEGF, and CDKN1A. In T47D cells, 1 induces nuclear HIF-1alpha protein without affecting HIF-1alpha mRNA. Both the induction of HIF-1alpha protein and activation of HIF-1 by 1 can be blocked by iron and ascorbate, indicating that 1 may activate HIF-1 through the chelation of iron. These results suggest that intended cancer chemoprevention with high-dose green tea extracts may be compromised, by the ability of tea catechins to promote tumor cell survival pathways associated with HIF-1 activation.

Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae.

Antifungal compounds exert their activity through a variety of mechanisms, some of which are poorly understood. Novel approaches to characterize the mechanism of action of antifungal agents will be of great use in the antifungal drug development process. The aim of the present study was to investigate the changes in the gene expression profile of Saccharomyces cerevisiae following exposure to representatives of the four currently available classes of antifungal agents used in the management of systemic fungal infections. Microarray analysis indicated differential expression of 0.8, 4.1, 3.0, and 2.6% of the genes represented on the Affymetrix S98 yeast gene array in response to ketoconazole, amphotericin B, caspofungin, and 5-fluorocytosine (5-FC), respectively. Quantitative real time reverse transcriptase-PCR was used to confirm the microarray analyses. Genes responsive to ketoconazole, caspofungin, and 5-FC were indicative of the drug-specific effects. Ketoconazole exposure primarily affected genes involved in ergosterol biosynthesis and sterol uptake; caspofungin exposure affected genes involved in cell wall integrity; and 5-FC affected genes involved in DNA and protein synthesis, DNA damage repair, and cell cycle control. In contrast, amphotericin B elicited changes in gene expression reflecting cell stress, membrane reconstruction, transport, phosphate uptake, and cell wall integrity. Genes with the greatest specificity for a particular drug were grouped together as drug-specific genes, whereas genes with a lack of drug specificity were also identified. Taken together, these data shed new light on the mechanisms of action of these classes of antifungal agents and demonstrate the potential utility of gene expression profiling in antifungal drug development.