RESUMO
Tamoxifen has been widely used in the treatment of estrogen receptor (ER)-positive breast cancer, whereas it also exhibits ER-independent anticancer effects in various cancer cell types. As one of the convincing mechanisms underlying the ER-independent effects, induction of apoptosis through mitochondrial dysfunction has been advocated. However, the mechanism of action of tamoxifen even at the isolated mitochondrial level is not fully understood and remains controversial. Here, we attempted to comprehensively understand tamoxifen's multiple actions in isolated rat liver mitochondria through not only revisiting the actions hitherto reported but also conducting originally designed experiments. Using submitochondrial particles, we found that tamoxifen has potential as an inhibitor of both respiratory complex I and ATP synthase. However, these inhibitory effects were not elicited in intact mitochondria, likely because penetration of tamoxifen across the inner mitochondrial membrane is highly restricted owing to its localized positive charge (-N+H(CH3)2). This restricted penetration may also explain why tamoxifen is unable to function as a protonophore-type uncoupler in mitochondria. Moreover, tamoxifen suppressed opening of the mitochondrial permeability transition pore induced by Ca2+ overload through enhancing phosphate uptake into the matrix. The photoaffinity labeling experiments using a photolabile tamoxifen derivative (pTAM1) indicated that pTAM1 specifically binds to voltage-dependent anion channels (VDACs) 1 and 3, which regulate transport of various substances into mitochondria. The binding of tamoxifen to VDAC1 and/or VDAC3 could be responsible for the enhancement of phosphate uptake. Taking all the results together, we consider the principal impairment of mitochondrial functions caused by tamoxifen.
Assuntos
TamoxifenoRESUMO
The mitochondrial machineries presiding over ATP synthesis via oxidative phosphorylation are promising druggable targets. Fusaramin, a 3-acyl tetramic acid isolated from Fusarium concentricum FKI-7550, is an inhibitor of oxidative phosphorylation in Saccharomyces cerevisiae mitochondria, although its target has yet to be identified. Fusaramin significantly interfered with [3H]ADP uptake by yeast mitochondria at the concentration range inhibiting oxidative phosphorylation. A photoreactive fusaramin derivative (pFS-5) specifically labeled voltage-dependent anion channel 1 (VDAC1), which facilitates trafficking of ADP/ATP across the outer mitochondrial membrane. These results strongly suggest that the inhibition of oxidative phosphorylation by fusaramin is predominantly attributable to the impairment of VDAC1 functions. Fusaramin also inhibited FoF1-ATP synthase and ubiquinol-cytochrome c oxidoreductase (complex III) at concentrations higher than those required for the VDAC inhibition. Considering that other tetramic acid derivatives are reported to inhibit FoF1-ATP synthase and complex III, natural tetramic acids were found to elicit multiple inhibitory actions against mitochondrial machineries.
Assuntos
Fosforilação OxidativaRESUMO
Two new tetramic acid derivatives, traminines A (1) and B (2), were isolated from a culture broth of Fusarium concentricum FKI-7550 by bioassay-guided fractionation using multidrug-sensitive Saccharomyces cerevisiae 12geneΔ0HSR-iERG6. The chemical structures of 1 and 2 were elucidated by NMR studies. Compounds 1 and 2 inhibited the growth of the multidrug-sensitive yeast strain on nonfermentable medium containing glycerol, but not on fermentable medium containing glucose. These results strongly suggest that they target mitochondrial machineries presiding over ATP production via oxidative phosphorylation. Throughout the assay monitoring overall ADP-uptake/ATP-release in yeast mitochondria, 1 and 2 were shown to inhibit one or more enzymes involving oxidative phosphorylation. Based on biochemical characterization, we found that the interference with oxidative phosphorylation by 1 is attributable to the dual inhibition of complex III and FoF1-ATPase, whereas that by 2 is solely due to the inhibition of complex III.
Assuntos
Fusarium , Saccharomyces cerevisiae , Mitocôndrias/metabolismo , Fosforilação OxidativaRESUMO
A new compound, fusaramin (1), along with three known compounds, sambutoxin (2), N-demethylsambutoxin (3) and (-)-6-deoxyoxysporidinone (4), was isolated from a culture broth of Fusarium sp. FKI-7550 by bioassay-guided fractionation using multidrug-sensitive Saccharomyces cerevisiae 12geneΔ0HSR-iERG6. The chemical structure of 1 was elucidated by NMR studies and electronic circular dichroism spectrum. Compound 1 showed antibacterial activity against some Gram-positive and Gram-negative bacteria and inhibited the growth of S. cerevisiae 12geneΔ0HSR-iERG6 grown on glycerol-containing medium. The MICs of 1 against wild-type and multidrug-sensitive yeasts grown on glycerol-containing medium were >128 µg ml-1 and 0.64 µg ml-1, respectively. However, MICs of 1 against both yeast strains grown on glucose-containing medium were >128 µg ml-1. All compounds showed inhibition of ATP synthesis via oxidative phosphorylation using isolated S. cerevisiae mitochondria.
Assuntos
Anti-Infecciosos/isolamento & purificação , Fusarium/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Anti-Infecciosos/química , Dicroísmo Circular , Fusarium/crescimento & desenvolvimento , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura MolecularRESUMO
Voltage-dependent anion channel 1 (VDAC1) situated in the outer mitochondrial membrane regulates the transfer of various metabolites and is a key player in mitochondria-mediated apoptosis. Although many small chemicals that modulate the functions of VDAC1 have been reported to date, most, if not all, of them cannot be regarded as specific reagents due to their interactions with other transporters or enzymes. By screening our chemical libraries using isolated Saccharomyces cerevisiae mitochondria, we found pentenediol (PTD)-type compounds (e.g., PTD-023) as new specific inhibitors of VDAC1. PTD-023 inhibited overall ADP-uptake/ATP-release reactions in isolated mitochondria at a single digit µM level. To identify the binding position of PTDs in VDAC1 by visualizing PTD-bound peptides, we conducted ligand-directed tosyl (LDT) chemistry using the synthetic LDT reagent t-PTD-023 derived from the parent PTD-023 in combination with mutagenesis experiments. t-PTD-023 made a covalent bond predominantly and subsidiarily with nucleophilic Cys210 and Cys130, respectively, indicating that PTDs bind to the region interactive with both residues. Site-directed mutations of hydrogen bond-acceptable Asp139 and Glu152 to Ala, which were selected as potential interactive partners of the critical pentenediol moiety based on the presumed binding model of PTDs in VDAC1, resulted in a decrease in susceptibility against PTD-023. This result strongly suggests that PTDs bind to VDAC1 through a specific hydrogen bond with the two residues. The present study is the first to demonstrate the binding position of specific inhibitors of VDAC1 at the amino acid level.