RESUMEN
Toxoplasmosis, an infectious zoonotic disease caused by the apicomplexan parasite Toxoplasma gondii (T. gondii), is a major worldwide health problem. However, there are currently no effective options (chemotherapeutic drugs or prophylactic vaccines) for treating chronic latent toxoplasmosis infection. Accordingly, seeking more effective and safer chemotherapeutics for combating this disease remains a long-term and challenging objective. In this paper, we summarize possible molecular biotargets, with an emphasis on those that are druggable and promising, including, without limitation, calcium-dependent protein kinase 1, bifunctional thymidylate synthase-dihydrofolate reductase, and farnesyl diphosphate synthase. Meanwhile, as important components of medicinal chemistry, the binding modes and structure-activity relationship profiles of the corresponding inhibitors were also illuminated. We anticipate that this information will be helpful for further identification of more effective chemotherapeutic interventions to prevent and treat zoonotic infections caused by T. gondii.
Asunto(s)
Antiprotozoarios/uso terapéutico , Toxoplasmosis/tratamiento farmacológico , Animales , Inhibidores Enzimáticos/farmacología , Geraniltranstransferasa/efectos de los fármacos , Geraniltranstransferasa/metabolismo , Humanos , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/efectos de los fármacos , Timidilato Sintasa/metabolismo , Toxoplasma/enzimologíaRESUMEN
The ketogenic diet (KD), a high-lipid and low-carbohydrate diet, has been used in the treatment of epilepsy, neurodegenerative disorders, inborn errors of metabolism and cancer; however, the exact mechanism/s of its therapeutic effect is not completely known. We hypothesized that sirtuins (SIRT)-a group of seven NAD-dependent enzymes and important regulators of energy metabolism may be altered under KD treatment. HT22 hippocampal murine neurons were incubated with two important KD metabolites-beta-hydroxybutyrate (BHB) (the predominant ketone body) and decanoic acid (C10), both accumulating under KD. Enzyme activity, protein, and gene expressions of SIRT 1-4, enzyme capacities of the mitochondrial respiratory chain complexes (MRC), citrate synthase (CS) and gene expression of monocarboxylate transporters were measured in control (untreated) and KD-treated cells. Incubation with both-BHB and C10 resulted in significant elevation of SIRT1 enzyme activity and an overall upregulation of the MRC. C10 incubation showed prominent increases in maximal activities of complexes I + III and complex IV of the MRC and ratios of their activities to that of CS, pointing towards a more efficient functioning of the mitochondria in C10-treated cells.
Asunto(s)
Ácido 3-Hidroxibutírico/farmacología , Ácidos Decanoicos/farmacología , Dieta Cetogénica , Metabolismo Energético/efectos de los fármacos , Hipocampo/efectos de los fármacos , Animales , Citrato (si)-Sintasa/metabolismo , Expresión Génica/efectos de los fármacos , Ratones , Mitocondrias/enzimología , Complejos Multienzimáticos/efectos de los fármacos , Neuronas/efectos de los fármacos , Sirtuinas/efectos de los fármacosRESUMEN
Exposure to arsenic is a risk factor for nonalcoholic steatohepatitis (NASH). Ferroptosis is a form of regulated cell death defined by the accumulation of lipid peroxidation. In the current study, we observed the occurrence of ferroptosis in arsenic-induced NASH by assessing ferroptosis related hallmarks. In vitro, we found that ferrostatin-1 effectively attenuated the executing of ferroptosis and NASH. Simultaneously, the expression of ACSL4 (acyl-CoA synthetase long-chain family member 4) was upregulated in rat's liver and L-02 cells exposed to arsenic. While, suppression of ACSL4 with rosiglitazone or ACSL4 siRNA remarkably alleviated arsenic-induced NASH and ferroptosis through diminishing 5-hydroxyeicosatetraenoic acid (5-HETE) content. Additionally, Mitofusin 2 (Mfn2), a physical tether between endoplasmic reticulum and mitochondria, has rarely been explored in the ferroptosis. Using Mfn2 siRNA or inositol-requiring enzyme 1 alpha (IRE1α) inhibitor, we found NASH and ferroptosis were obviously mitigated through reducing 5-HETE content. Importantly, Co-IP assay indicated that Mfn2 could interact with IRE1α and promoted the production of 5-HETE, ultimately led to ferroptosis and NASH. Collectively, our data showed that ferroptosis is involved in arsenic-induced NASH. These data provide insightful viewpoints into the mechanism of arsenic-induced NASH.
Asunto(s)
Arsénico , Enfermedad del Hígado Graso no Alcohólico , Animales , Arsénico/toxicidad , Coenzima A Ligasas , Endorribonucleasas/efectos de los fármacos , Endorribonucleasas/fisiología , Ferroptosis , GTP Fosfohidrolasas/efectos de los fármacos , GTP Fosfohidrolasas/fisiología , Proteínas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/fisiología , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/fisiología , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , RatasRESUMEN
Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of human NTDs. However, why folate reduces NTDs is largely unknown. The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1) The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5-10-fold higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.
Asunto(s)
Ácido Fólico/uso terapéutico , Defectos del Tubo Neural/prevención & control , Aminoácido Oxidorreductasas/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Femenino , Deficiencia de Ácido Fólico/prevención & control , Humanos , Modelos Biológicos , Complejos Multienzimáticos/efectos de los fármacos , Tubo Neural/embriología , Tubo Neural/metabolismo , Células Neuroepiteliales/citología , Células Neuroepiteliales/efectos de los fármacos , Células Neuroepiteliales/metabolismo , Ácidos Nucleicos/metabolismo , Necesidades Nutricionales , Embarazo , Tetrahidrofolatos/metabolismo , Transferasas/efectos de los fármacosRESUMEN
Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPß pathway.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Hormona del Crecimiento/farmacología , Hígado/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , ARN Mensajero/efectos de los fármacos , Proteína 1 de Unión a la X-Box/efectos de los fármacos , Animales , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/efectos de los fármacos , Endorribonucleasas/metabolismo , Proteínas del Choque Térmico HSP40/efectos de los fármacos , Proteínas del Choque Térmico HSP40/genética , Hipofisectomía , Hígado/metabolismo , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Factor de Transcripción STAT5/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Caracteres Sexuales , Transducción de Señal , Proteína 1 de Unión a la X-Box/genética , eIF-2 Quinasa/efectos de los fármacos , eIF-2 Quinasa/metabolismoRESUMEN
Severe hemorrhagic shock induces cognitive dysfunction by promoting cell death mediated by activating endoplasmic reticulum (ER) stress. Sevoflurane postconditioning prevents neuronal apoptosis against cerebral ischemia/reperfusion injury. It is unknown if this protective effect on hemorrhagic shock and resuscitation rats (HSR) is associated with ER stress attenuation. Male adult Sprague-Dawley rats were subjected HSR by removing 40% blood volume within 30 min, and 60 min later the animals were resuscitated with infusion of the removing blood in 30 min. Sevoflurane postconditioning was performed by inhaling sevoflurane at three different concentrations (0.5, 1.0, 1.5 MAC) at the onset of resuscitation for 30 min. Severe hypotension (mean arterial pressure 40-45 mmHg) occurred in the shock session for 60 min accompanying with significantly elevated lactate, decreased BE and pH values in arterial blood gas analysis. There were impaired spatial learning and memory following HSR indicated by persistently longer escape latency and lower correct rate, as well as less duration and crossing in the target quadrant by using Morris water maze and Y-maze tests. In the hippocampal CA1 region, there was significantly higher activity of caspase-3 induced by HSR. HSR also elevated the expression of inositol-requiring enzyme 1α (IRE1α) and caspase-12 in the hippocampus by western blot analysis. Sevoflurane postconditioning at 1.0 and 1.5 MAC significantly reversed these changes. These findings suggested that sevoflurane postconditioning could improve spatial learning and memory deficits induced by severe hemorrhagic shock and subsequent resuscitation. The suppression of endoplasmic reticulum stress provided critical contribution in neural apoptosis mediated by IRE1α-caspase-12 pathway.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/efectos de los fármacos , Memoria/efectos de los fármacos , Complejos Multienzimáticos/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Sevoflurano/farmacología , Aprendizaje Espacial/efectos de los fármacos , Animales , Caspasa 12/efectos de los fármacos , Poscondicionamiento Isquémico/métodos , Masculino , Éteres Metílicos/farmacología , Fármacos Neuroprotectores/farmacología , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Choque Hemorrágico/tratamiento farmacológicoRESUMEN
3ßHSD1 enzymatic activity is essential for synthesis of potent androgens from adrenal precursor steroids in prostate cancer. A germline variant in HSD3B1, the gene that encodes 3ßHSD1, encodes for a stable enzyme, regulates adrenal androgen dependence, and is a predictive biomarker of poor clinical outcomes after gonadal testosterone deprivation therapy. However, little is known about HSD3B1 transcriptional regulation. Generally, it is thought that intratumoral androgen synthesis is upregulated after gonadal testosterone deprivation, enabling development of castration-resistant prostate cancer. Given its critical role in extragonadal androgen synthesis, we sought to directly interrogate the transcriptional regulation of HSD3B1 in multiple metastatic prostate cancer cell models. Surprisingly, we found that VCaP, CWR22Rv1, LNCaP, and LAPC4 models demonstrate induction of HSD3B1 upon androgen stimulation for approximately 72 hours, followed by attenuation around 120 hours. 3ßHSD1 protein levels mirrored transcriptional changes in models harboring variant (LNCaP) and wild-type (LAPC4) HSD3B1, and in these models androgen induction of HSD3B1 is abrogated via enzalutamide treatment. Androgen treatment increased flux from [3H]-dehydroepiandrosterone to androstenedione and other downstream metabolites. HSD3B1 expression was reduced 72 hours after castration in the VCaP xenograft mouse model, suggesting androgen receptor (AR) regulation of HSD3B1 also occurs in vivo. Overall, these data suggest that HSD3B1 is unexpectedly positively regulated by androgens and ARs. These data may have implications for the development of treatment strategies tailored to HSD3B1 genotype status.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Complejos Multienzimáticos/genética , Progesterona Reductasa/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/metabolismo , Esteroide Isomerasas/genética , Andrógenos/metabolismo , Andrógenos/farmacología , Androstenodiona/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Benzamidas , Línea Celular Tumoral , Deshidroepiandrosterona/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Metribolona/farmacología , Ratones , Complejos Multienzimáticos/efectos de los fármacos , Trasplante de Neoplasias , Nitrilos , Orquiectomía , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Progesterona Reductasa/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/terapia , Receptores Androgénicos/efectos de los fármacos , Esteroide Isomerasas/efectos de los fármacos , Testosterona , Congéneres de la Testosterona/farmacología , Regulación hacia ArribaRESUMEN
OBJECTIVE: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite's DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. RESULTS: A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácido Fólico/análogos & derivados , Leishmania donovani/enzimología , Metotrexato/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Complejos Multienzimáticos/efectos de los fármacos , Extractos Vegetales/farmacología , Tetrahidrofolato Deshidrogenasa/efectos de los fármacos , Timidilato Sintasa/efectos de los fármacos , Withania , Witanólidos/farmacología , Ácido Fólico/farmacología , HumanosRESUMEN
Autotaxin is a dual-function ecto-enzyme, encoded by the gene ENPP2, which is the primary source of the bioactive signaling lipid, lysophosphatidic acid. Aberrations in autotaxin/lysophosphatidic acid signaling have been associated with a number of neurological, psychiatric, neoplastic, and neurodevelopmental conditions, such as pain, pruritus, glioblastoma multiforme, multiple sclerosis, Alzheimer's disease, hydrocephalus, and schizophrenia. This Viewpoint offers a brief overview of the likely indications for therapeutic targeting of autotaxin, in disorders affecting the nervous system.
Asunto(s)
Lisofosfolípidos/farmacología , Complejos Multienzimáticos/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Animales , Glioblastoma/tratamiento farmacológico , Humanos , Lisofosfolípidos/química , Transducción de Señal/efectos de los fármacosRESUMEN
Bacteria sense and respond to osmolarity through the EnvZ-OmpR two-component system. The structure of the periplasmic sensor domain of EnvZ (EnvZ-PD) is not available yet. Here, we present the crystal structure of EnvZ-PD in the presence of CHAPS detergent. The structure of EnvZ-PD shows similar folding topology to the PDC domains of PhoQ, DcuS, and CitA, but distinct orientations of helices and ß-hairpin structures. The CD and NMR spectra of EnvZ-PD in the presence of cholate, a major component of bile salts, are similar to those with CHAPS. Chemical cross-linking shows that the dimerization of EnvZ-PD is significantly inhibited by the CHAPS and cholate. Together with ß-galactosidase assay, these results suggest that bile salts may affect the EnvZ structure and function in Escherichia coli.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Colatos/farmacología , Ácidos Cólicos/farmacología , Detergentes/farmacología , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Complejos Multienzimáticos/química , Proteínas de la Membrana Bacteriana Externa/efectos de los fármacos , Dicroismo Circular , Cristalografía por Rayos X , Proteínas de Escherichia coli/efectos de los fármacos , Modelos Moleculares , Complejos Multienzimáticos/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacosRESUMEN
Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, WalK, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coli. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.
Asunto(s)
Antibacterianos/farmacología , Histidina Quinasa/antagonistas & inhibidores , Histidina Quinasa/química , Quinonas/farmacología , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia Conservada , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Histidina Quinasa/genética , Modelos Estructurales , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , FosforilaciónRESUMEN
In this paper, we present different strategies to vectorize HldE kinase inhibitors with the goal to improve their gram-negative intracellular concentration. Syntheses and biological effects of siderophoric, aminoglycosidic, amphoteric, and polycationic vectors are discussed. While siderophoric and amphoteric vectorization efforts proved to be disappointing in this series, aminoglycosidic and polycationic vectors were able for the first time to achieve synergistic effects of our inhibitors with erythromycin. Although these effects proved to be nonspecific, this study provides information about the required stereoelectronic arrangement of the polycationic amines and their basicity requirements to fulfill outer membrane destabilization resulting in better erythromycin synergies.
Asunto(s)
Eritromicina/metabolismo , Escherichia coli/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Nucleotidiltransferasas/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Aminoglicósidos/farmacología , Antibacterianos/metabolismo , Eritromicina/química , Eritromicina/farmacología , Escherichia coli/efectos de los fármacos , Lipopolisacáridos/biosíntesis , Pruebas de Sensibilidad Microbiana , Complejos Multienzimáticos/efectos de los fármacos , Nucleotidiltransferasas/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Poliaminas/farmacología , PolielectrolitosRESUMEN
OBJECTIVE: To investigate the possible effect of artesunate (ART) on schistosome thioredoxin glutathione reductase (TGR) and cytochrome c peroxidase (CcP) in Schistosoma mansoni-infected mice. METHODS: A total of 200 laboratory bred male Swiss albino mice were divided into 4 groups (50 mice in each group). Group I: infected untreated group (Control group) received a vehicle of 1% sodium carbonyl methylcellulose (CMC-Na); Group II: infected then treated with artesunate; Group III: infected then treated with praziquantel, and group IV: infected then treated with artesunate then praziquantel. Adult S. mansoni worms were collected by Animal Perfusion Method, tissue egg counted, TGR, and CcP mRNA Expression were estimated of in S. mansoni adult worms by semi-quantitative rt-PCR. RESULTS: Semi-quantitative rt-PCR values revealed that treatment with artesunate caused significant decrease in expression of schistosome TGR and CcP in comparison to the untreated group. In contrast, the treatment with praziquantel did not cause significant change in expression of these genes. The results showed more reduction in total worm and female worm count in combined ART-PZQ treated group than in monotherapy treated groups by either ART or PZQ. Moreover, complete disappearance (100%) of tissue eggs was recorded in ART-PZQ treated group with a respective reduction rate of 95.9% and 68.4% in ART- and PZQ-treated groups. CONCLUSION: The current study elucidated for the first time that anti-schistosomal mechanisms of artesunate is mediated via reduction in expression of schistosome TGR and CcP. Linking these findings, addition of artesunate to praziquantel could achieve complete cure outcome in treatment of schistosomiasis.
Asunto(s)
Artemisininas/farmacología , Citocromo-c Peroxidasa/efectos de los fármacos , Complejos Multienzimáticos/efectos de los fármacos , NADH NADPH Oxidorreductasas/efectos de los fármacos , Schistosoma/efectos de los fármacos , Animales , Artesunato , Citocromo-c Peroxidasa/genética , Masculino , Ratones , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Schistosoma/enzimologíaRESUMEN
Impairment of the electron transport chain (ETC) is implicated in cadmium (Cd)- and calcium (Ca)-induced mitochondrial dysfunction. To localize the sites of the impairment, effects of 0-50µM Cd and Ca, singly and in combination, on complex I- to IV-driven respirations were investigated using isolated rainbow trout liver mitochondria. Mitochondrial Cd/Ca accumulation and respiration rates were measured following sequential inhibition and activation of complexes I, II, III, and IV. Mitochondrial adenosine triphosphate (ATP) synthesis was measured on exposure to (micromolar) 20 Cd and 50 Ca, singly and combined, whereas malondialdehyde (MDA) was measured on incubation with 0-1µM Cd and/or Ca. We show that mitochondrial accumulation of Cd and Ca and the states 3 and 4 rates of respiration depended on the active ETC complex. Although complex IV was highly recalcitrant to Cd and/or Ca, dose-dependent inhibitions of complex I-, II-, and III-driven state 3 respiration rates were observed with half maximal inhibitory concentrations (IC(50)) of (micromolar) 12.4, 12, and 13.7 (Cd); 57.1, 46.1, and 26.2 (Ca); and 8.3, 13.5, and 5.1 (Cd + Ca), respectively. The lower IC(50) values for complex I- and III-mediated respirations in the Cd + Ca treatment suggests that these complexes are the sites of cooperative actions of Cd and Ca. State 4 respiration rates were unaffected by Cd and/or Ca exposure but reduced mitochondrial coupling was apparent from the lower respiratory control and adenosine diphosphate/O ratios except in mitochondria oxidizing complex IV substrate. Additionally, there was reduced ATP synthesis in complex I substrates-energized mitochondria and increased MDA concentrations symptomatic of membrane lipid peroxidation.
Asunto(s)
Cloruro de Cadmio/toxicidad , Cloruro de Calcio/toxicidad , Mitocondrias Hepáticas/efectos de los fármacos , Complejos Multienzimáticos/efectos de los fármacos , Oncorhynchus mykiss , Contaminantes Químicos del Agua/toxicidad , Adenosina Trifosfatasas/metabolismo , Animales , Cloruro de Cadmio/metabolismo , Cloruro de Calcio/metabolismo , Respiración de la Célula/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Mitocondrias Hepáticas/enzimología , Complejos Multienzimáticos/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
AIMS: We investigated the in vitro effects of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in tissues of patients affected by mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies, on various parameters of energy homeostasis in mitochondrial preparations from brain of young rats. MAIN METHODS: We measured the respiratory parameters state 4, state 3, respiratory control ratio (RCR) and ADP/O ratio by the rate of oxygen consumption, as well as the mitochondrial membrane potential and the matrix NAD(P)H levels in the presence of the fatty acids. KEY FINDINGS: We found that 3HDA, 3HTA and 3HPA markedly increased state 4 respiration and diminished the RCR using glutamate plus malate or succinate as substrates. 3HTA and 3HPA also diminished the mitochondrial membrane potential and the matrix NAD(P)H levels. In addition, 3HTA decreased state 3 respiration using glutamate/malate, but not pyruvate/malate or succinate as substrates. Our data indicate that the long-chain 3-hydroxy fatty acids that accumulate in LCHAD/MTP deficiencies act as uncouplers of oxidative phosphorylation, while 3HTA also behaves as a metabolic inhibitor. SIGNIFICANCE: It is presumed that impairment of brain energy homeostasis caused by these endogenous accumulating compounds may contribute at least in part to the neuropathology of LCHAD/MTP deficiencies.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/deficiencia , Encéfalo/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Ácidos Láuricos/farmacología , Mitocondrias/efectos de los fármacos , Complejos Multienzimáticos/deficiencia , Ácidos Mirísticos/farmacología , 3-Hidroxiacil-CoA Deshidrogenasas/efectos de los fármacos , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Encéfalo/metabolismo , Homeostasis/efectos de los fármacos , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Proteína Trifuncional Mitocondrial , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , NADP/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Important roles for vascular endothelial growth factor (VEGF) and autotaxin (ATX) have been established for embryonic vasculogenesis and cancer progression. We examined whether these two angiogenic factors cooperate in regulation of endothelial cell migratory responses. VEGF stimulated expression of ATX and LPA1, a receptor for the ATX enzymatic product lysophosphatidic acid (LPA), in human umbilical vein endothelial cells. Knockdown of ATX expression significantly decreased mRNA levels for the receptors LPA1, LPA2, S1P1, S1P2, S1P3, and VEGFR2 and abolished cell migration to lysophosphatidylcholine, LPA, recombinant ATX, and VEGF. Migration to sphingosylphosphorylcholine and sphinogosine-1-phosphate was also reduced in ATX knockdown cells, whereas migration to serum remained unchanged. Furthermore, ATX knockdown decreased Akt2 mRNA levels, whereas LPA treatment strongly stimulated Akt2 expression. We propose that VEGF stimulates LPA production by inducing ATX expression. VEGF also increases LPA1 signaling, which in turn increases Akt2 expression. Akt2 is strongly associated with cancer progression, cellular migration, and promotion of epithelial-mesenchymal transition. These data show a role for ATX in maintaining expression of receptors required for VEGF and lysophospholipids to accelerate angiogenesis. Because VEGF and ATX are upregulated in many cancers, the regulatory mechanism proposed in these studies could apply to cancer-related angiogenesis and cancer progression. These data further suggest that ATX could be a prognostic factor or a target for therapeutic intervention in several cancers.
Asunto(s)
Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfodiesterasa I/metabolismo , Pirofosfatasas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/fisiopatología , Fosfodiesterasa I/efectos de los fármacos , Fosfodiesterasa I/genética , Hidrolasas Diéster Fosfóricas , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirofosfatasas/efectos de los fármacos , Pirofosfatasas/genética , Interferencia de ARN/fisiología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores del Ácido Lisofosfatídico/efectos de los fármacos , Receptores de Lisoesfingolípidos/efectos de los fármacos , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Autotaxin is a protein of approximately 900 amino acids discovered in the early 1990s. Over the past 15 years, a strong association between cancer cells and autotaxin production has been observed. Recent publications indicate that autotaxin and the capacity of cancer to metastasise are intimately linked. The discovery of new molecular targets in pharmacology is a mixture of pure luck, hard work and industrial strategy. Despite a crucial and desperate need for new therapeutic tools, many targets are approached in oncology, but only a few are validated and end up at the patient bed. Outside the busy domain of kinases, few targets have been discovered that can be useful in treating cancer, particularly metastatic processes. The fortuitous relationship between autotaxin and lysophosphatidic acid renders the results of observations made in the diabetes/obesity context considerably important. The literature provides observations that may aid in redesigning experiments to validate autotaxin as a potential oncology target.
Asunto(s)
Complejos Multienzimáticos/fisiología , Neoplasias/tratamiento farmacológico , Fosfodiesterasa I/fisiología , Pirofosfatasas/fisiología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Lisofosfolípidos , Complejos Multienzimáticos/efectos de los fármacos , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias/patología , Obesidad/tratamiento farmacológico , Fosfodiesterasa I/efectos de los fármacos , Hidrolasas Diéster Fosfóricas , Pirofosfatasas/efectos de los fármacosRESUMEN
Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC(50) of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases alpha, delta and epsilon is 15, 45 and 125 muM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC(50)=88 muM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.
Asunto(s)
Cromo/toxicidad , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/efectos de los fármacos , ADN/biosíntesis , Contaminantes Ambientales/toxicidad , Complejos Multienzimáticos/efectos de los fármacos , Ciclo Celular , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Factores de TiempoRESUMEN
Breast cancer is characterized, among others, by the concurrence of lipophilic xenobiotica such as 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) with hypoxic tissue conditions. This condition activates the transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and aryl hydrocarbon receptor (AhR) that are known to promote tumor progression. An interrelation between these transcription factors and nuclear factor of activated T-cells (NFAT) was implied by gene array analysis. In the present study, the interplay of the three transcription factors was studied and correlated with the migration of MCF-7 cells in response to TCDD and/or hypoxia. An AhR-activation by 10nM TCDD and HIF-1alpha activation by 5% oxygen induced activation of NFATc1. The effects were inhibited by cyclosporine A (CsA), suggesting that the activation of NFAT by AhR or HIF-1alpha signaling is calcineurin-dependent. The expression/activity of the NFAT target gene autotaxin (ATX) was increased. ATX is known to stimulate migration of tumor cells. The hydrolysis product of ATX, lysophosphatidic acid (LPA), increased the migration of MCF-7 cells under normoxia but not under hypoxia. This effect correlated with increased migration observed after TCDD treatment. Hypoxia did not promote migration of MCF-7 cells, suggesting that ATX down-stream signaling was inhibited by hypoxia. In conclusion, the TCDD-mediated activation of NFATc1 is suggested to promote cell migration via ATX/LPA-signaling.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Hipoxia/metabolismo , Complejos Multienzimáticos/efectos de los fármacos , Factores de Transcripción NFATC/efectos de los fármacos , Fosfodiesterasa I/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Pirofosfatasas/efectos de los fármacos , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Cultivadas , Ciclosporina/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunosupresores/farmacología , Técnicas In Vitro , Complejos Multienzimáticos/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosfodiesterasa I/metabolismo , Hidrolasas Diéster Fosfóricas , Pirofosfatasas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Several studies strongly suggest that DC differentiated in vitro in the presence of type I IFN acquire more potent immune stimulatory properties, compared with DC differentiated in vitro with IL-4. However, little is known about the molecular mechanisms underlying this phenomenon. To address this question, we compared the Ag-processing machinery (APM) profile in human DC grown in the presence of IFN-alpha ((IFN)DC) or IL-4 ((IL-4)DC). Using a panel of APM component-specific mAb in Western blot experiments, we found that (IFN)DC preferentially express inducible proteasome subunits (LMP2, LMP7, and MECL1) both at immature and mature stages. In contrast, immature (IL-4)DC co-express both constitutive (beta1, beta2, and beta5) and inducible subunits, as shown by Western blotting analysis. In addition, immature (IFN)DC express higher levels of TAP1, TAP2, calnexin, calreticulin, tapasin, and HLA class I molecules than (IL-4)DC. The different proteasome profiles of (IFN)DC and (IL-4)DC were associated with a greater ability of (IFN)DC to present an immunodominant epitope that requires LMP7 expression for its processing. In general, these data show the impact of cytokines on APM component expression and hence the Ag-processing ability of DC.