RÉSUMÉ
Since the dawn of molecular biology, cancer therapy has focused on druggable targets. Despite some remarkable successes, cell-level evolution remains a potent antagonist to this approach. We suggest that a deeper understanding of the breakdown of cooperation can synergize the evolutionary and druggable-targets approaches. Complexity requires cooperation, whether between cells of different species (symbiosis) or between cells of the same organism (multicellularity). Both forms of cooperation may be associated with nutrient scarcity, which in turn may be associated with a chemiosmotic metabolism. A variety of examples from modern organisms supports these generalities. Indeed, mammalian cancers-unicellular, glycolytic, and fast-replicating-parallel these examples. Nutrient scarcity, chemiosmosis, and associated signaling may favor cooperation, while under conditions of nutrient abundance a fermentative metabolism may signal the breakdown of cooperation. Manipulating this metabolic milieu may potentiate the effects of targeted therapeutics. Specific opportunities are discussed in this regard, including avicins, a novel plant product.
Sujet(s)
Évolution biologique , Tumeurs , Animaux , Humains , Tumeurs/traitement médicamenteux , Nutriments , Sélection génétique , SymbioseRÉSUMÉ
K-Ras must interact primarily with the plasma membrane (PM) for its biological activity. Therefore, disrupting K-Ras PM interaction is a tractable approach to block oncogenic K-Ras activity. Here, we found that avicin G, a family of natural plant-derived triterpenoid saponins from Acacia victoriae, mislocalizes K-Ras from the PM and disrupts PM spatial organization of oncogenic K-Ras and H-Ras by depleting phosphatidylserine (PtdSer) and cholesterol contents, respectively, at the inner PM leaflet. Avicin G also inhibits oncogenic K- and H-Ras signal output and the growth of K-Ras-addicted pancreatic and non-small cell lung cancer cells. We further identified that avicin G perturbs lysosomal activity, and disrupts cellular localization and activity of neutral and acid sphingomyelinases (SMases), resulting in elevated cellular sphingomyelin (SM) levels and altered SM distribution. Moreover, we show that neutral SMase inhibitors disrupt the PM localization of K-Ras and PtdSer and oncogenic K-Ras signaling. In sum, this study identifies avicin G as a new potent anti-Ras inhibitor, and suggests that neutral SMase can be a tractable target for developing anti-K-Ras therapeutics.
Sujet(s)
Saponines/composition chimique , Sphingomyeline phosphodiesterase/antagonistes et inhibiteurs , Protéines G ras/métabolisme , Animaux , Lignée cellulaire , Membrane cellulaire/métabolisme , Cholestérol/métabolisme , Cricetinae , Chiens , Endocytose/effets des médicaments et des substances chimiques , Humains , Saponines/métabolisme , Saponines/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Sphingomyeline phosphodiesterase/métabolisme , Sphingomyéline/métabolismeRÉSUMÉ
Stratum corneum, the outermost layer of skin, allows transport of only low-molecular weight (<500) lipophilic solutes. Here, we report a surprising finding that avicins (Avs), a family of naturally occurring glycosylated triterpenes with a molecular weight > 2,000, exhibit skin permeabilities comparable to those of small hydrophobic molecules, such as estradiol. Systematic fragmentation of the Av molecule shows that deletion of the outer monoterpene results in a 62% reduction in permeability, suggesting an important role for this motif in skin permeation. Further removal of the tetrasaccharide residue results in a further reduction of permeability by 79%. These results, taken in sum, imply that synergistic effects involving both hydrophobic and hydrophilic residues may hold the key in facilitating translocation of Avs across skin lipids. In addition to exhibiting high permeability, Avs provided moderate enhancements of skin permeability of estradiol and polysaccharides, including dextran and inulin but not polyethylene glycol.
Sujet(s)
Derme/métabolisme , Structures macromoléculaires/métabolisme , Adsorption , Animaux , Transport biologique , Calorimétrie différentielle à balayage , Oestradiol/métabolisme , Femelle , Glycosylation , Humains , Techniques in vitro , Modèles moléculaires , Octanols/composition chimique , Saponines/composition chimique , Saponines/métabolisme , Absorption cutanée , Sus scrofa , Thermodynamique , Eau/composition chimiqueRÉSUMÉ
Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from "ancient hopanoids," avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation. In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin's ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D's transrepressive effects. Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use.
Sujet(s)
Antinéoplasiques d'origine végétale/pharmacologie , Cellules/métabolisme , Récepteurs aux glucocorticoïdes/métabolisme , Saponines/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Triterpènes/pharmacologie , Antinéoplasiques d'origine végétale/composition chimique , Antinéoplasiques d'origine végétale/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Noyau de la cellule/effets des médicaments et des substances chimiques , Noyau de la cellule/métabolisme , Cellules/effets des médicaments et des substances chimiques , ADN/métabolisme , Fatty acid synthase type I/métabolisme , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Humains , Modèles biologiques , Facteur de transcription NF-kappa B/antagonistes et inhibiteurs , Facteur de transcription NF-kappa B/métabolisme , Phosphoenolpyruvate carboxykinase (GTP)/métabolisme , Liaison aux protéines/effets des médicaments et des substances chimiques , Structure tertiaire des protéines , Transport des protéines/effets des médicaments et des substances chimiques , Récepteurs aux glucocorticoïdes/composition chimique , Éléments de réponse/génétique , Saponines/composition chimique , Saponines/métabolisme , Activation de la transcription/effets des médicaments et des substances chimiques , Triterpènes/composition chimique , Triterpènes/métabolisme , Facteur de nécrose tumorale alpha/pharmacologieRÉSUMÉ
Avicins, a family of plant-derived triterpenoids, have been shown to possess pro-apoptotic, anti-mutagenic and anti-inflammatory properties in mammalian cells. Through thiol binding, avicins can also mediate antioxidant defense. Accumulating evidence uncovered during the past several years suggests that avicins induce tumor cell death via multiple mechanisms. This review will focus on recent studies that provide insights into the cellular and molecular processes and pathways by which avicins induce tumor cell death, including the canonical intrinsic mitochondrial and the Fas-mediated apoptosis cascades as well as autophagy-associated non-apoptotic programmed cell death.
RÉSUMÉ
Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the "osmotic protection" method with 100mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.
Sujet(s)
Antinéoplasiques/pharmacologie , Membrane érythrocytaire/métabolisme , Tumeurs de la prostate/traitement médicamenteux , Saponines/pharmacologie , Animaux , Lignée cellulaire tumorale , Perméabilité des membranes cellulaires/effets des médicaments et des substances chimiques , Tests de criblage d'agents antitumoraux/méthodes , Hémolyse/effets des médicaments et des substances chimiques , Humains , Solution hypotonique , Mâle , Pression osmotique/effets des médicaments et des substances chimiques , Polyéthylène glycols/pharmacologie , Tumeurs de la prostate/métabolisme , Rats , Rat Sprague-Dawley , Saponines/pharmacocinétiqueRÉSUMÉ
Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a) the cross-talk that occurs between redox and phosphorylation processes, and (b) the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.
Sujet(s)
Régulation de l'expression des gènes codant pour des enzymes , Facteur de transcription STAT-3/métabolisme , Saponines/pharmacologie , 7,12-Diméthyl-benzo[a]anthracène/pharmacologie , Animaux , Apoptose/effets des médicaments et des substances chimiques , Apoptose/physiologie , Transport biologique/effets des médicaments et des substances chimiques , Technique de Western , Cancérogènes/pharmacologie , Lignée cellulaire , Lignée cellulaire tumorale , Activation enzymatique/effets des médicaments et des substances chimiques , Humains , Immunohistochimie , Janus kinases/métabolisme , Souris , Microscopie de fluorescence , Myélome multiple/métabolisme , Myélome multiple/anatomopathologie , Phosphoprotein Phosphatases/métabolisme , Phosphorylation/effets des médicaments et des substances chimiquesRÉSUMÉ
Avicins, a family of triterpene electrophiles originally identified as potent inhibitors of tumor cell growth, have been shown to be pleiotropic compounds that also possess antioxidant, anti-mutagenic, and anti-inflammatory activities. We previously showed that Jurkat cells, which express a high level of Fas, are very sensitive to treatment with avicins. Thus, we hypothesized that avicins may induce cell apoptosis by activation of the Fas pathway. By using a series of cell lines deficient in cell death receptors, we demonstrated that upon avicin D treatment, Fas translocates to the cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. In the lipid rafts, Fas interacts with Fas-associated death domain (FADD) and Caspase-8 to form death-inducing signaling complex (DISC) and thus mediates cell apoptosis. Interfering with lipid raft organization by using a cholesterol-depleting compound, methyl-beta-cyclodextrin, not only prevents the clustering of Fas and its DISC complex but also reduces the sensitivity of the cells to avicin D. Avicin D activates Fas pathways independent of the association between extracellular Fas ligands and Fas receptors. A deficiency in Fas and its downstream signaling molecules leads to the resistance of the cells to avicin D treatment. Taken together, our results demonstrate that avicin D triggers the redistribution of Fas in the membrane lipid rafts, where Fas activates receptor-mediated cell death.
Sujet(s)
Acacia/composition chimique , Apoptose/effets des médicaments et des substances chimiques , Microdomaines membranaires/métabolisme , Saponines/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Triterpènes/pharmacologie , Antigènes CD95/métabolisme , Caspase 8/métabolisme , Lignée cellulaire tumorale , Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/métabolisme , Tests de criblage d'agents antitumoraux , Activation enzymatique/effets des médicaments et des substances chimiques , Espace extracellulaire/effets des médicaments et des substances chimiques , Espace extracellulaire/métabolisme , HumainsRÉSUMÉ
Avicin D, a natural triterpenoid saponin, inhibits cell growth and induces apoptosis in transformed tumor cell lines in vitro and mouse skin carcinogenesis models in vivo. To investigate the anti-tumor effects of avicin D in cutaneous T-cell lymphomas (CTCL), we compared three CTCL cell lines and Sézary cells from three Sézary syndrome (SS) patients with normal CD4+ and activated CD4+ T cells from three healthy donors. Avicin D at 0.5-5 mug ml(-1) induced apoptosis in a time- and dose-dependent manner in three cell lines: MJ (-0.2 to 13% and 0.6-37%), Hut78 (2-39% and 3-53%), and HH (13-83% and 44-89%) at 24 and 48 hours, respectively. Avicin D at 0.5-5 microg ml(-1) for 48 hours caused more apoptosis in patients' Sézary cells than in healthy donors' CD4+ T cells and activated CD4+ T cells. The general caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Z-DEVD-FMK decreased avicin D-induced apoptosis in CTCL cells. Caspase-3 was activated and poly (ADP-ribose) polymerase was cleaved after avicin D treatment. Avicin D did not change the expression of signal transducer and activator of transcription-3 (STAT-3) but decreased phospho-signal transducer and activator of transcription-3 (p-STAT-3) protein levels in all three cell lines and two patients' Sézary cells. Avicin D also decreased expression of the inhibitor of apoptosis protein survivin, the anti-apoptotic protein bcl-2, but not the pro-apoptotic protein bax in these CTCL cells. In summary, avicin D selectively induced apoptosis, inhibited STAT-3 activation, and decreased apoptosis inhibitors (bcl-2 and survivin) in CTCL cell lines and SS patients' Sézary cells. Our findings underlie the therapeutic potential of avicin D in patients with SS.
Sujet(s)
Antinéoplasiques d'origine végétale/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Lymphome T cutané/métabolisme , Protéines associées aux microtubules/métabolisme , Protéines tumorales/métabolisme , Protéines proto-oncogènes c-bcl-2/métabolisme , Facteur de transcription STAT-3/métabolisme , Saponines/pharmacologie , Tumeurs cutanées/métabolisme , Sujet âgé , Lymphocytes T CD4+/effets des médicaments et des substances chimiques , Lymphocytes T CD4+/métabolisme , Lymphocytes T CD4+/anatomopathologie , Études cas-témoins , Caspase-3/métabolisme , Inhibiteurs des caspases , Lignée cellulaire tumorale , Relation dose-effet des médicaments , Régulation négative/effets des médicaments et des substances chimiques , Antienzymes/pharmacologie , Femelle , Humains , Protéines IAP , Lymphome T cutané/anatomopathologie , Protéines associées aux microtubules/effets des médicaments et des substances chimiques , Protéines associées aux microtubules/génétique , Adulte d'âge moyen , Protéines tumorales/effets des médicaments et des substances chimiques , Protéines tumorales/génétique , Protéines proto-oncogènes c-bcl-2/effets des médicaments et des substances chimiques , Protéines proto-oncogènes c-bcl-2/génétique , Facteur de transcription STAT-3/effets des médicaments et des substances chimiques , Facteur de transcription STAT-3/génétique , Syndrome de Sézary/métabolisme , Syndrome de Sézary/anatomopathologie , Tumeurs cutanées/anatomopathologie , Survivine , Facteurs temps , Protéine Bax/génétique , Protéine Bax/métabolismeRÉSUMÉ
Avicins are pro-apoptotic, anti-inflammatory molecules with antioxidant effects both in vitro and in vivo. Based on their ability to perturb mitochondrial functions and initiate apoptosis in tumor cells, we chose to study the bioenergetic effects of avicins on tumor cell mitochondria. Avicin-treated Jurkat cells, showed a decrease in the levels of cellular ATP as well as the rate of oxygen consumption. These effects on cellular metabolism appear to be a result of avicin's actions on the outer mitochondrial membrane (OMM). We speculate that avicins might initially inhibit the exchange of metabolites across the OMM leading to its subsequent permeabilization to cytochrome c. This speculation is supported by biophysical studies using lipid bilayers, which suggest that upstream of these effects, avicins target and close the voltage dependent anion channel (VDAC). Closure of VDAC would lead to an overall lowering of the cell energy metabolism, subsequently pushing these cells towards the apoptotic pathway by permeabilization of the OMM and release of cyt-c. Avicins therefore not only represent a novel pharmacological tool for treatment of cancers, but also highlight the influence ancient plant metabolites could have on human health.
Sujet(s)
Acacia/métabolisme , Adénosine triphosphate/métabolisme , Mitochondries/métabolisme , Membranes mitochondriales/métabolisme , Saponines/usage thérapeutique , Animaux , Métabolisme énergétique , Humains , Cellules Jurkat , Mitochondries/effets des médicaments et des substances chimiques , Mitochondries du foie/effets des médicaments et des substances chimiques , Mitochondries du foie/métabolisme , Consommation d'oxygène/effets des médicaments et des substances chimiques , Rats , Canaux anioniques voltage-dépendants/effets des médicaments et des substances chimiques , Canaux anioniques voltage-dépendants/métabolismeRÉSUMÉ
Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27(Kip1), is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein-AMP-activated protein kinase (LKB1-AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27(T198A)) is sufficient to induce autophagy. Under stress conditions that activate the LKB1-AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1-AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.
Sujet(s)
Apoptose/physiologie , Autophagie/physiologie , Inhibiteur p27 de kinase cycline-dépendante/métabolisme , Métabolisme énergétique , Complexes multienzymatiques/métabolisme , Protein-Serine-Threonine Kinases/métabolisme , AMP-activated protein kinase kinases , AMP-Activated Protein Kinases , Lignée cellulaire tumorale , Humains , Phosphorylation , Transduction du signal/physiologieRÉSUMÉ
Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the influence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a significant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c. On the other hand, avicins increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, long before high amplitude swelling of mitochondria was observed. The increase in external NADH oxidation was cyclosporin A-insensitive. Avicin G significantly accelerated hydroperoxide-induced oxidation of mitochondrial endogenous NAD(P)H, the drop of the inner membrane potential and the high amplitude swelling of mitochondria. The obtained data might explain selective induction of apoptosis in tumor cells by avicins. Based on other studies showing that tumor cells generate hydroperoxides with a very high rate, avicins could provide a new strategy of anticancer therapy by sensitizing cells with high levels of reactive oxygen species to apoptosis.
Sujet(s)
Antinéoplasiques/pharmacologie , Mitochondries du foie/effets des médicaments et des substances chimiques , Membranes mitochondriales/effets des médicaments et des substances chimiques , Consommation d'oxygène/effets des médicaments et des substances chimiques , Saponines/pharmacologie , Animaux , Ciclosporine/pharmacologie , Cytochromes c/métabolisme , Peroxyde d'hydrogène/métabolisme , Techniques in vitro , Mâle , Potentiel de membrane mitochondriale , Mitochondries du foie/métabolisme , Membranes mitochondriales/métabolisme , Gonflement mitochondrial/effets des médicaments et des substances chimiques , NAD/métabolisme , NADP/métabolisme , Phosphorylation oxydative , Perméabilité , Rats , Rat Sprague-Dawley , Roténone/pharmacologieRÉSUMÉ
Avicins comprise a class of triterpenoid compounds that exhibit tumor inhibitory activity. Here we show that avicin G is inhibitory to growth of the fission yeast Schizosaccharomyces pombe. S. pombe cells treated with a lethal concentration of avicin G (20 microM) exhibited a shrunken morphology, indicating that avicin G adversely affects cell integrity. Cells treated with a sublethal concentration of avicin G (6.5 microM) exhibited a strong cytokinesis-defective phenotype (multiseptated cells), as well as cell morphology defects. These phenotypes bear resemblance to those resulting from loss of Rho1 GTPase function in S. pombe. Indeed, Rho1-deficient S. pombe cells were strongly hypersensitive to avicin G, suggesting that the compound may perturb Rho1-dependent processes. Consistent with previously observed effects in human Jurkat T cells, avicin G treatment resulted in hyperaccumulation of ubiquitinated proteins in S. pombe cells. Interestingly, proteasome-defective S. pombe mutants were not markedly hypersensitive to avicin G, whereas an anaphase-promoting complex (mitotic ubiquitin ligase) mutant exhibited avicin G resistance, suggesting that the increase in levels of ubiquitinated proteins resulting from avicin G treatment may be due to increased protein ubiquitination, rather than inhibition of 26S proteasome activity. Mutants defective in the cAMP/PKA pathway also exhibited resistance to avicin G. Our results suggest that S. pombe will be a useful model organism for elucidating molecular targets of avicin G and serve as a guide to clinical application where dysfunctional aspects of Rho and/or ubiquitination function have been demonstrated as in cancer, fibrosis, and inflammation.
Sujet(s)
Antinéoplasiques/pharmacologie , Cytocinèse/effets des médicaments et des substances chimiques , Saponines/pharmacologie , Schizosaccharomyces/cytologie , Schizosaccharomyces/effets des médicaments et des substances chimiques , Terpènes/pharmacologie , Ubiquitine/métabolisme , Complexe promoteur de l'anaphase , Perméabilité des membranes cellulaires/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Résistance des champignons aux médicaments , Mutation/génétique , Liaison aux protéines , Schizosaccharomyces/génétique , Schizosaccharomyces/métabolisme , Protéines de Schizosaccharomyces pombe , Ubiquitin-protein ligase complexes/génétique , Ubiquitin-protein ligase complexes/métabolisme , Régulation positive , Protéines G rho/déficit , Protéines G rho/génétique , Protéines G rho/métabolismeRÉSUMÉ
Avicins are a recently discovered family of plant-derived terpenoid molecules that possess proapoptotic, antiinflammatory, and cytoprotective properties in mammalian cells. Previous work demonstrating that avicins can exert their effects by suppressing or activating the redox-sensitive transcription factors NF-kappaB and nuclear factor-erythroid 2 p45-related factor (Nrf2), respectively, has raised the idea that they may react with critical cysteine residues. To understand the molecular mechanism through which avicins regulate protein function, we examined their effects on the paradigmatic redox-responsive transcriptional activator, OxyR of Escherichia coli, which protects bacterial cells against oxidative and nitrosative stresses. In vitro transcription assays demonstrated that avicins activate OxyR and its target genes katG and oxyS in a DTT-reversible manner. In addition, katG-dependent hydroperoxidase I activity was enhanced in avicin-treated bacteria. Mass spectrometric analysis of activated OxyR revealed thioesterification of the critical regulatory cysteine, Cys-199, to an avicin fragment comprising the outer monoterpene side chain. Our results indicate that avicinylation can induce adaptive responses that protect cells against oxidative or nitrosative stress. More generally, transesterification may represent a previously undescribed thiol-directed posttranslational modification, which extends the code for redox regulation of protein function.
Sujet(s)
Cystéine/métabolisme , Protéines de liaison à l'ADN/métabolisme , Régulation de l'expression des gènes bactériens/physiologie , Modèles moléculaires , Stress oxydatif/physiologie , Protéines de répression/métabolisme , Saponines/métabolisme , Facteurs de transcription/métabolisme , Catalase/métabolisme , Escherichia coli , Protéines Escherichia coli/métabolisme , Spectrométrie de masse , Oxydoréduction , Spectrométrie de masse MALDIRÉSUMÉ
Mitochondria have the capacity to integrate environmental signals and, in animals with active stem cell populations, trigger responses in terms of growth and growth form. Colonial hydroids, which consists of feeding polyps connected by tube-like stolons, were treated with avicis, triterpenoid electrophiles whose anti-cancer properties in human cells are mediated in part by mitochondria. In treated hydroids, both oxygen uptake and mitochondrial reactive oxygen species were diminished relative to controls, similar to that observed in human cells exposed to avicins. While untreated colonies exhibit more stolon branches and connections in the centre of the colony than at the periphery, treated colonies exhibit the opposite: fewer stolon branches in the centre of the colony than at the periphery. The resulting growth form suggest an inversion of the normal pattern of colony development mediated by mitochondrial and redox-related perturbations. An as-yet-uncharacterized gradient within the colony may determine the ultimate phenotypic effects of avicin perturbation.
Sujet(s)
Hydrozoa/effets des médicaments et des substances chimiques , Hydrozoa/croissance et développement , Mitochondries/physiologie , Saponines/pharmacologie , Transduction du signal/physiologie , Analyse de variance , Animaux , Diméthylsulfoxyde , Fluorescéines/métabolisme , Fluorescence , Peroxyde d'hydrogène/métabolisme , Mitochondries/effets des médicaments et des substances chimiques , Consommation d'oxygène/effets des médicaments et des substances chimiques , Espèces réactives de l'oxygène/métabolisme , Transduction du signal/effets des médicaments et des substances chimiquesRÉSUMÉ
Avicins, a family of triterpenoid saponins from Acacia victoriae, can regulate the innate stress response in human cells. Their ability to induce apoptosis in transformed cells makes them potential anticancer agents. We report that avicins can form channels in membranes. The conductance reached a steady state after each addition, indicating a dynamic equilibrium between avicin in solution and in the membrane. The high power dependence (up to 10) of the membrane conductance on the avicin concentration indicates the formation of multimeric channels, consistent with the estimated pore radius of 1.1 nm. This radius is too small to allow protein flux across the mitochondrial outer membrane, a process known to initiate apoptosis. Channel formation is lost when avicin's amphipathic side chain is removed, implicating this as the channel-forming region. A small difference in this side chain results in strong cholesterol dependence of channel formation in avicin G that is not found in avicin D. In neutral membranes, avicin channels are nonselective, but negatively-charged lipids confer cation selectivity (5:1, K(+):Cl(-)), indicating that phospholipids form part of the permeation pathway. Avicin channels in the mitochondrial outer membrane may favor apoptosis by altering the potential across this membrane and the intermembrane space pH.
Sujet(s)
Apoptose , Membrane cellulaire/métabolisme , Cholestérol/composition chimique , Saponines/composition chimique , Acacia/métabolisme , Animaux , Cholestérol/métabolisme , Relation dose-effet des médicaments , Érythrocytes/métabolisme , Humains , Concentration en ions d'hydrogène , Hydrolyse , Mâle , Protéines membranaires/composition chimique , Mitochondries/métabolisme , Modèles chimiques , Modèles moléculaires , Oxydoréduction , Phosphatidylcholines , Phospholipides/composition chimique , Phospholipides/métabolisme , Potassium/composition chimique , Rats , Rat Sprague-Dawley , Eau/composition chimiqueRÉSUMÉ
Avicins are proapoptotic and anti-inflammatory triterpene electrophiles isolated from an Australian desert tree, Acacia victoriae. The presence of two alpha,beta unsaturated carbonyl groups (Michael reaction sites) in the side chain of the avicin molecule prompted us to study its effects on NF-E2-related factor 2 (Nrf2), a redox-regulated transcription factor that controls the expression of a battery of detoxification and antioxidant proteins via its binding to antioxidant response element (ARE). Avicin D-treated Hep G2 cells showed translocation of Nrf2 into the nucleus and a time-dependent increase in ARE activity. These properties were sensitive to DTT, suggesting that avicins affect one or more critical cysteine residues, probably on the Keap1 molecule. Downstream of ARE, an activation of a battery of stress-induced proteins occurred. The implications of these findings were evaluated in vivo in mouse skin exposed to an ancient stressor, UV light. Avicins inhibited epidermal hyperplasia, reduced p53 mutation, enhanced apoptosis, decreased generation of 8-hydroxy-2'-deoxyguanosine, and enhanced expression of NADPH:quinone oxidoreductase 1 and heme oxygenase-1. These data, combined with our earlier published work, demonstrate that avicins represent a new class of plant stress metabolites capable of activating stress adaptation and suppressing proinflammatory components of the innate immune system in human cells by redox regulation. The relevance for treatment of clinical diseases in which stress responses are dysfunctional or deficient is discussed.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Saponines/pharmacologie , Triterpènes/pharmacologie , Carcinome hépatocellulaire , Dithiothréitol/pharmacologie , Régulation de l'expression des gènes tumoraux/génétique , Glutathion/métabolisme , Glutathione peroxidase/métabolisme , Glutathione transferase/métabolisme , Humains , Cinétique , Tumeurs du foie , Protéines recombinantes/métabolisme , Transfection , Cellules cancéreuses en cultureRÉSUMÉ
Two new saponins named avicins D (1) and G (2) were isolated from the seed pods of the desert legume plant Acacia victoriae. The structures, elucidated by 1D and 2D NMR studies and by chemical means, were characterized as acacic acid-bearing oligosaccharides at C-3 and C-28 with a side chain linked to C-21 comprised of two monoterpene carboxylic acids and a quinovose moiety. Both compounds exhibited potent cytotoxicity (apoptosis) against human T-cell leukemia (Jurkat cells) in vitro.
Sujet(s)
Acacia/composition chimique , Plantes médicinales/composition chimique , Saponines/isolement et purification , Terpènes/isolement et purification , Arizona , Tests de criblage d'agents antitumoraux , Humains , Cellules Jurkat , Structure moléculaire , Résonance magnétique nucléaire biomoléculaire , Oligosaccharides/composition chimique , Oligosaccharides/isolement et purification , Oligosaccharides/pharmacologie , Saponines/composition chimique , Saponines/pharmacologie , Terpènes/composition chimique , Terpènes/pharmacologie , Cellules cancéreuses en culture/effets des médicaments et des substances chimiquesRÉSUMÉ
One of the goals of cancer chemotherapy and prevention is the discovery of compounds that are relatively selective to tumor cells and, therefore, have reduced effects on normal cell growth. In previously published studies, it was shown that certain triterpene saponins (called avicins) from a desert tree, Acacia victoriae, are selectively toxic to tumor cells at very low doses (IC(50): 0.2 microg/mL for Jurkat cells). To extend this research to human clinical studies, we needed to find a reliable supply of avicins and have developed a transformed "hairy root" culture as a means of biomass production. Protocols were optimized for A. victoriae micropropagation; after a boiling water treatment, A. victoriae seeds were maintained under in vitro conditions on defined media. Embryo-axis explants from shoot tips were removed and infected with Agrobacterium rhizogenes Conn (R 1000) for hairy root induction. Plasmid integration was confirmed by PCR analysis with a primer set for a segment of the rol B gene. Culture conditions have been optimized for root biomass production, and various inducers have been investigated for enhancement of avicin production. Hairy root cultures were compared with intact pod tissue from field-grown sources for avicin content following partial purification of triterpene glycosides and HPLC separation of the secondary metabolites. From bioassays of the collected HPLC fractions, we have identified putative triterpene "metabolic clusters" with enhanced activity against tumor cells. This now provides a system for both production of clinical trial lots of active samples, but also a means to correlate structure of individual triterpene glycosides with specific cellular target activity in mammalian cells.