RESUMEN
The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR's pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR-HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern-Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR-HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA's binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR-HSA complexes, reaffirming that AICAR binds to both site I and site II.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Simulación del Acoplamiento Molecular , Ribonucleótidos/metabolismo , Albúmina Sérica Humana/metabolismo , Análisis Espectral , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Transferencia de Energía , Humanos , Cinética , Unión Proteica , Ribonucleótidos/química , Albúmina Sérica Humana/química , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
We present a new highly interdisciplinary project-based course in computer aided drug discovery (CADD). This course was developed in response to a call for alternative pedagogical approaches during the COVID-19 pandemic, which caused the cancellation of a face-to-face summer research program sponsored by the Louisiana Biomedical Research Network (LBRN). The course integrates guided research and educational experiences for chemistry, biology, and computer science students. We implement research-based methods with publicly available tools in bioinformatics and molecular modeling to identify and prioritize promising antiviral drug candidates for COVID-19. The purpose of this course is three-fold: I. Implement an active learning and inclusive pedagogy that fosters student engagement and research mindset; II. Develop student interdisciplinary research skills that are highly beneficial in a broader scientific context; III. Demonstrate that pedagogical shifts (initially incurred during the COVID-19 pandemic) can furnish longer-term instructional benefits. The course, which has now been successfully taught a total of five times, incorporates four modules, including lectures/discussions, live demos, inquiry-based assignments, and science communication.
Asunto(s)
COVID-19 , Descubrimiento de Drogas , SARS-CoV-2 , Estudiantes , Humanos , Estudiantes/psicología , COVID-19/epidemiología , Descubrimiento de Drogas/educación , Pandemias , Curriculum , Biología Computacional/educación , Investigación Biomédica/educación , Aprendizaje Basado en Problemas/métodos , AntiviralesRESUMEN
Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 µM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 µM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.
Asunto(s)
Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Complejo II de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Vitamina E/administración & dosificación , Animales , Antineoplásicos/farmacología , Bovinos , Transporte de Electrón , Humanos , Concentración 50 Inhibidora , Células Jurkat , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa , Vitamina E/farmacologíaRESUMEN
Fusarochromanone is an experimental drug with unique and potent anti-cancer activity. Current cancer therapies often incorporate a combination of drugs to increase efficacy and decrease the development of drug resistance. In this study, we used drug combinations and cellular phenotypic screens to address important questions about FC101's mode of action and its potential therapeutic synergies in triple negative breast cancer (TNBC). We hypothesized that FC101's activity against TNBC is similar to the mTOR inhibitor, everolimus, because FC101 downregulates the phosphorylation of two mTOR substrates, S6K and S6. Since everolimus synergistically enhances the anti-cancer activities of two known EGFR inhibitors (erlotinib or lapatinib) in TNBC, we performed analogous studies with FC101. Phenotypic cellular assays helped assess whether FC101 acts similarly to everolimus, in both single and combination treatments with the two inhibitors. FC101 outperformed all other single treatments in both cell proliferation and viability assays. However, unlike everolimus, FC101 produced a sustained decrease in cell viability in drug washout studies. None of the other drugs were able to maintain comparable effects upon removal. Although we observed slightly additive effects when the TNBC cells were treated with FC101 and the two EGFR inhibitors, those effects were not truly synergistic in the manner displayed with everolimus.
RESUMEN
The vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of alpha-tocopheryl maleamide (alpha-TAM), an esterase-resistant analogue of alpha-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of alpha-TAM towards cancer cells (MCF-7, B16F10) compared to alpha-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that alpha-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both alpha-TOS and alpha-TAM to solve the problem with cytotoxicity of free alpha-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal alpha-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of alpha-TAM and alpha-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of alpha-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of alpha-TOS. Thus, the liposomal formulation of alpha-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/administración & dosificación , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Química Farmacéutica , Femenino , Humanos , Liposomas , Maleimidas/administración & dosificación , Maleimidas/farmacología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacología , Vitamina E/administración & dosificación , Vitamina E/análogos & derivados , Vitamina E/farmacología , alfa-Tocoferol/farmacologíaRESUMEN
In spite of unrelenting effort, the net incidence of neoplastic diseases appears not to have been curbed. While some types of cancer have been suppressed significantly, others are either stagnating or on the increase. Therefore, the need for a cure is imperative, in particularly a drug or combination of drugs that would be selective for malignant cells, i.e. with as low secondary toxicity as possible. Recent data strongly suggest that analogues of vitamin E, epitomised by the most studied alpha-tocopheryl succinate (alpha-TOS), may meet the need for the coveted drugs with a selective anti-neoplastic effect. The reasons for this optimism are reviewed in this article.
Asunto(s)
Antineoplásicos/uso terapéutico , Antioxidantes/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Vitamina E/análogos & derivados , Vitamina E/uso terapéutico , HumanosRESUMEN
PURPOSE: For 30 years nature has provided a plethora of natural products with potential meaningful anti-cancer activity. Fusarochromanone (FC101a) is a small molecule fungal metabolite exhibiting potent in-vitro growth inhibitory effects and is capable of inducing apoptosis, suppressing angiogenesis and tumorigenesis, and inhibiting endothelial cell growth in multiple cancer cell lines. Despite all we know regarding FC101a, the mechanism of action and molecular target(s) of this compound have remained an enigma. Furthermore, modest in-vivo activity has been documented and requires addressing. METHOD: Early stage pharmacokinetics (PK) assessment is vital to successful drug development. Herein, we aimed to use in-silico assays to i) characterize an in-depth ADMET profile of FC101a and ii) to probe for possible therapeutic targets. Two-dimensional SDF files of FC101a and 13 analogs were introduced into ADMET Predictor Version 7.1 that parses the structures in order to calculate molecular descriptors, which are used to estimate ADMET properties. Calculated ADMET values were analyzed and subjected to multiple drug-like indices, delivering a PK profile of each analog. To probe for possible targets, a total of 49 proteins were introduced into SYBYL-X Version 2.0 platform and the deepest binding pocket of each protein was virtually docked with parent compound, FC101a; with the negative control, FC101b; and with the model compound, kynurenine. RESULTS: Each analog showed promising ADMET qualities, although FC101 Oxazole was identified as the most optimized analog. Despite FC101a having a desirable ADME and toxicity profile, areas of concern were identified and must be addressed in-vitro. These include potential mutagenic properties and estrogen receptor toxicity. We provide potential avenues medicinal chemists could use to achieve higher effective permeation, higher blood brain barrier (BBB) penetration, and higher aqueous solubility in FC101a. Molecular docking assays revealed procaspase-8 - cFLIP(L) complex as a potential biological target and led to proposed mechanisms of action by which FC101a facilitates procaspase-8 heterodimerization, thereby increasing proteolytic activity and up regulating extrinsic apoptosis. CONCLUSION: Our data revealed both potential mechanisms of action and a promising ADMET profile of FC101a. These attributes render FC101a a promising lead candidate for development into a low toxic anti-cancer agent effective against a broad range of cancers.
RESUMEN
Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death.
Asunto(s)
Cromonas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Antracenos/farmacología , Western Blotting , Células COS , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Células HEK293 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2/metabolismoRESUMEN
Fungal metabolites continue to show promise as a viable class of anticancer agents. In the present study, we investigated the efficacy of the fungal metabolite, fusarochromanone (FC101), for its antitumor activities in glioblastomas, which have a median survival of less than two years and a poor clinical response to surgical resection, radiation therapy and chemotherapy. Using clinically applicable doses, we demonstrated that FC101 induced glioblastoma apoptotic cell death via caspase dependent signaling, as indicated by the cleavage of poly(ADP-ribose) polymerase, glioblastoma (PARP). FC101 also induced differential reactive oxygen species (ROS) levels in glioblastoma cells, contrasting a defined role of oxidative stress in apoptotic cell death observed with other fungal metabolites. Furthermore, the antitumorigenic effects of FC101 on tumor cell migration were assessed. Cell migration assays revealed that FC101 significantly reduced the migratory capacity of glioblastomas, which are incredibly invasive tumors. Taken together, the present study establishes FC101 as a candidate anticancer agent for the cooperative treatment of glioblastomas.
Asunto(s)
Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Caspasas/metabolismo , Cromonas/farmacología , Glioblastoma/enzimología , Glioblastoma/patología , Transducción de Señal/efectos de los fármacos , Actinina/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
Fusarochromanone (FC101), a mycotoxin produced by the fungus Fusarium equiseti, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G1 cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death.
Asunto(s)
Cromonas/farmacología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis , Células COS , Inhibidores de Caspasas/farmacología , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Células HEK293 , HumanosRESUMEN
BACKGROUND: Fusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity. RESULTS: Herein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 µM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells. CONCLUSIONS: Our data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101's molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery.
Asunto(s)
Antineoplásicos/farmacología , Cromonas/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity.
Asunto(s)
Sistemas de Liberación de Medicamentos , Mitocondrias/metabolismo , Compuestos Organofosforados , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tocoferoles , Animales , Apoptosis/efectos de los fármacos , Quimioterapia/tendencias , Humanos , Células Jurkat , Modelos Animales , Terapia Molecular Dirigida , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Especies Reactivas de Oxígeno/metabolismo , Tocoferoles/química , Tocoferoles/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
The search for a selective and efficient anticancer agent for treating all neoplastic disease has yet to deliver a universally suitable compound(s). The majority of established anticancer drugs either are nonselective or lose their efficacy because of the constant mutational changes of malignant cells. Until recently, a largely neglected target for potential anticancer agents was the mitochondrion, showing a considerable promise for future clinical applications. Vitamin E (VE) analogs, epitomized by alpha-tocopheryl succinate, belong to the group of "mitocans" (mitochondrially targeted anticancer drugs). They are selective for malignant cells, cause destabilization of their mitochondria, and suppress cancer in preclinical models. This review focuses on our current understanding of VE analogs in the context of their proapoptotic/anticancer efficacy and suggests that their effect on mitochondria may be amplified by modulation of alternative pathways operating in parallel. We show here that the analogs of VE that cause apoptosis (which translates into their anticancer efficacy) generally do not possess antioxidant (redox) activity and are prototypical of the mitocan group of anticancer compounds. Therefore, by analogy to Oscar Wilde's play The Importance of Being Earnest, we use the motto in the title "the importance of being redox-silent" to emphasize an essentially novel paradigm for cancer therapy, in which redox-silence is a prerequisite property for most of the anticancer activities described in this communication.
Asunto(s)
Antineoplásicos/farmacología , Mitocondrias/efectos de los fármacos , Vitamina E/análogos & derivados , Vitamina E/farmacología , Animales , Apoptosis/efectos de los fármacos , Humanos , Oxidación-Reducción/efectos de los fármacos , Relación Estructura-Actividad , Vitamina E/químicaRESUMEN
Vitamin E (VE) analogues, epitomized by alpha-tocopheryl succinate (alpha-TOS), are proapoptotic agents with selective antineoplastic activity. The molecule of alpha-TOS comprises several structurally and functionally distinct moieties that can be modified in order to yield analogues with higher activity. In order to find analogues with higher apoptogenic efficacy, we prepared novel compounds where the ester bond was replaced by an amide bond. All of these analogues were significantly more proapoptotic than their ester counterparts, with alpha-tocopheryl maleyl amide being the most effective. Importantly, methylation of the free carboxylic group completely obliterated apoptogenic activity of the compounds. Similarly as shown for the ester analogues, the amides induced apoptosis by mitochondrial destabilization. Superiority of amides over the ester analogues may be due to their higher partitioning into the lipid phase, as suggested by the log p-values that were lower for the amides than the corresponding esters. In conclusion, we present evidence that modification of the ester bond of agents such as alpha-TOS can be used as a basis for generating novel analogues with higher efficacy of killing malignant cells, an activity that suggests anticancer effect of the agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Vitamina E/análogos & derivados , Vitamina E/farmacología , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Células Jurkat , Cinética , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Estructura Molecular , Relación Estructura-Actividad , TocoferolesRESUMEN
Vitamin E (VE) analogues, epitomized by alpha-tocopheryl succinate (alpha-TOS), are potent inducers of apoptosis and anti-cancer agents. Here, we tested their effect on the highly malignant N-type neuroblastoma (Nb) cells and their differentiated, neurone-like counterparts. Nb cells were highly susceptible to several VE analogues, while differentiated Nb cells were relatively resistant to alpha-TOS. The importance of caspase-9 rather than caspase-8, as judged by specific siRNAs studies, together with the loss of the inner mitochondrial potential, suggests that alpha-TOS triggers apoptosis in Nb cells via the mitochondrial pathway. Cultured Nb cells were sensitized to alpha-TOS by pre-treatment with Bcl-2, Bcl-xL or Mcl-1 siRNAs, while the malignant cell line was more resistant to the vitamin E analogue when Bax was knocked down. In contrast, overexpression of Bcl-2 in Nb cells rendered them more resistant to alpha-TOS-induced apoptosis. The resistance of differentiated Nb cells to alpha-TOS-mediated apoptosis occurred via two modes: first, by up-regulation of the anti-apoptotic Bcl-2 family proteins and second, by accumulation of decreased levels of reactive oxygen species when challenged with alpha-TOS. We conclude that alpha-TOS is highly selective in killing malignant brain cancer cells while relatively inert toward differentiated neuronal cells, and that vitamin E analogues may be novel therapeutics for the treatment of tumours such as neuroblastomas.
Asunto(s)
Apoptosis/efectos de los fármacos , Neuroblastoma/fisiopatología , Vitamina E/análogos & derivados , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Humanos , Mitocondrias , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Neoplasias del Sistema Nervioso/tratamiento farmacológico , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Tocoferoles , Regulación hacia Arriba , Vitamina E/farmacología , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Alpha-tocopheryl succinate (alpha-TOS) is a redox-silent vitamin E (VE) analog with high pro-apoptotic and anti-neoplastic activity. Here we investigated whether alpha-TOS and several novel VE analogs kill breast cancer cells over-expressing the anti-apoptotic receptor protein HER2/erbB2. The agents induced apoptosis at comparable levels in both erbB2-low and -high cells. Generation of reactive oxygen species (ROS) preceded mitochondrial destabilization and execution of apoptosis, as evidenced by the anti-apoptotic effects of exogenous superoxide dismutase and mitochondrially targeted coenzyme Q. Dissipation of DeltaPsi(m) was followed by cytochrome c and Smac/Diablo re-localization and caspase-dependent cleavage of death substrate. A resistance to apoptosis for the corresponding rho(0) counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in breast cancer cells mediated by VE analogs and linked apoptosis to generation of radicals as judged by the delayed accumulation of ROS in the cybrid cell types. We conclude that alpha-TOS causes efficient apoptosis in breast cancer cells independent of their erbB2 status. Since erbB2 is frequently over-expressed in breast cancers and renders the neoplastic disease resistant to established treatment, our findings are of clinical interest.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Mitocondrias/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Vitamina E/análogos & derivados , Vitamina E/farmacología , Animales , Neoplasias de la Mama/metabolismo , Línea Celular , ADN Mitocondrial/genética , Regulación Neoplásica de la Expresión Génica , Concentración 50 Inhibidora , Ratones , Mitocondrias/genética , Estructura Molecular , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Receptor ErbB-2/genéticaRESUMEN
The discovery that structurally unique "filamentous integumentary appendages" are associated with several different non-avian dinosaurs continues to stimulate the development of models to explain the evolutionary origin of feathers. Taking the phylogenetic relationships of the non-avian dinosaurs into consideration, some models propose that the "filamentous integumentary appendages" represent intermediate stages in the sequential evolution of feathers. Here we present observations on a unique integumentary structure, the bristle of the wild turkey beard, and suggest that this non-feather appendage provides another explanation for some of the "filamentous integumentary appendages." Unlike feathers, beard bristles grow continuously from finger-like outgrows of the integument lacking follicles. We find that these beard bristles, which show simple branching, are hollow, distally, and express the feather-type beta keratins. The significance of these observations to explanations for the evolution of archosaurian integumentary appendages is discussed.
Asunto(s)
Dinosaurios/anatomía & histología , Queratinas/genética , Piel/ultraestructura , Pavos/anatomía & histología , Pavos/genética , Animales , Western Blotting , Plumas , Microscopía Electrónica de RastreoRESUMEN
The feathers of birds develop from embryonic epidermal lineages that differentiate during outgrowth of the feather germ. Independent cell populations also form an embryonic epidermis on scutate scales, which consists of peridermal layers, a subperiderm, and an alpha stratum. Using an antiserum (anti-FbetaK) developed to react specifically with the beta (beta) keratins of feathers, we find that the feather-type beta keratins are expressed in the subperiderm cells of embryonic scutate scales, as well as the barb ridge lineages of the feather. However, unlike the subperiderm of scales, which is lost at hatching, the cells of barb ridges, in conjunction with adjacent cell populations, give rise to the structural elements of the feather. The observation that an embryonic epidermis, consisting of peridermal and subperidermal layers, also characterizes alligator scales (Thompson, 2001. J Anat 198:265-282) suggests that the epidermal populations of the scales and feathers of avian embryos are homologous with those forming the embryonic epidermis of alligators. While the embryonic epidermal populations of archosaurian scales are discarded at hatching, those of the feather germ differentiate into the periderm, sheath, barb ridges, axial plates, barbules, and marginal plates of the embryonic feather filament. We propose that the development of the embryonic feather filament provides a model for the evolution of the first protofeather. Furthermore, we hypothesize that invagination of the epidermal lineages of the feather filament, namely the barb ridges, initiated the formation of the follicle, which then allowed continuous renewal of the feather epidermal lineages, and the evolution of diverse feather forms.