RESUMO
The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA(Ile) (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA(Ile). Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA(Ile) is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patient's clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA(Ile), while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.
Assuntos
Cardiomiopatia Hipertrófica/enzimologia , Cardiomiopatia Hipertrófica/genética , Isoleucina-tRNA Ligase/metabolismo , Penetrância , Mutação Puntual , RNA de Transferência de Isoleucina/genética , Adolescente , Sequência de Bases , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Isoleucina-tRNA Ligase/genética , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , RNA de Transferência de Isoleucina/metabolismoRESUMO
Leber's hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Leber's hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Leber's hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17ß-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Leber's hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17ß-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17ß-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor ß localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Leber's hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules.
Assuntos
Estradiol/farmacologia , Mitocôndrias/efeitos dos fármacos , Atrofia Óptica Hereditária de Leber/fisiopatologia , Células Ganglionares da Retina/efeitos dos fármacos , Análise de Variância , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Linhagem Celular , DNA Mitocondrial/metabolismo , Estradiol/metabolismo , Estrogênios/metabolismo , Estrogênios/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/fisiologia , Atrofia Óptica Hereditária de Leber/metabolismo , Atrofia Óptica Hereditária de Leber/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/metabolismoRESUMO
To explore the molecular mechanisms by which complexes of Grp94 with IgG, purified from the plasma of diabetic subjects, could drive an inflammatory risk in vascular cells, native Grp94 was co-incubated with human, non-immune IgG to obtain the formation of complexes that were then tested on human umbilical vein endothelial cells (HUVECs). Co-incubation of Grp94 with IgG led to the formation of stable, SDS-resistant complexes that displayed effects partly similar and partly significantly different from those of Grp94 alone. Both Grp94 alone and with IgG stimulated the cell growth and promoted angiogenesis by a mechanism of autocrine/paracrine activation of the expression of heat shock protein (HSP)90 and HSP70. However, the most striking alterations in the cell cytoskeleton, characterized by dramatic rearrangement of actin and increased formation of podosomes, were induced by Grp94 with IgG, and were mediated by the enhanced expression of HSP90. At variance with Grp94 alone, Grp94 with IgG promoted the angiogenic differentiation by activating a signaling pathway apparently independent of the intense stimulation of the ERK1/2 pathway that was instead more directly involved in mediating the proliferative effects on HUVECs. Results show unprecedented cytokine-like effects of Grp94 and a so far undisclosed capacity to bind irreversibly IgG, forming complexes that, with respect to Grp94 alone, display a more intense angiogenic transforming capacity that may predict an increased inflammatory risk in vascular cells in vivo.
Assuntos
Diferenciação Celular , Citocinas/fisiologia , Imunoglobulina G/metabolismo , Glicoproteínas de Membrana/metabolismo , Neovascularização Fisiológica , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Imunoglobulina G/farmacologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Glicoproteínas de Membrana/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Complexos Multiproteicos/farmacologia , Complexos Multiproteicos/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Ligação Proteica/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer's disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1delta inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1delta inhibitors known today (IC(50)=0.3 and 0.6 microM, respectively).
Assuntos
Caseína Quinase I/antagonistas & inibidores , Caseína Quinase I/química , Inibidores Enzimáticos/síntese química , Antraquinonas/química , Química Farmacêutica/métodos , Simulação por Computador , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Conformação Molecular , Fosforilação , Isoformas de Proteínas , Software , Tirosina/químicaRESUMO
In the last years, we have synthesized some new platinum(II), palladium(II), gold(I/III) complexes with dithiocarbamato derivatives as potential anticancer drugs, to obtain compounds with superior chemotherapeutic index in terms of increased bioavailability, higher cytotoxicity, and lower side effects than cisplatin. On the basis of the obtained encouraging results, we have been studying the interaction of CuCl2 with methyl-/ethyl-/tert-butylsarcosine-dithiocarbamato moieties in a 1:2 molar ratio; we also synthesized and studied the N,N-dimethyl- and pyrrolidine-dithiocarbamato copper complexes for comparison purposes. The reported compounds have been successfully isolated, purified, and fully characterized by means of several spectroscopic techniques. Moreover, the electrochemical properties of the designed compounds have been studied through cyclic voltammetry. In addition, the behavior in solution was followed by means of UV-vis technique to check the stability with time in physiological conditions. To evaluate their in vitro cytotoxic properties, preliminary biological assays (MTT test) have been carried out on a panel of human tumor cell lines. The results show that cytotoxicity levels of all of the tested complexes are comparable or even greater than that of the reference drug (cisplatin).
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Cobre/química , Linhagem Celular Tumoral , Humanos , Estrutura MolecularRESUMO
The effects of 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine, and sumatriptan on rat caudal arteries were examined, with the goal of finding experimental conditions useful in enhancing the 'silent' 5-HT(1B) receptor subtype. It was shown that both reserpine treatment and K(+) depolarization increased the vasoconstriction by 5-HT receptor agonists. The role of the 5-HT(2A) receptor in vasoconstriction was examined using ritanserin (50 nmol/l), a selective 5-HT(2A) antagonist, whereas that of the 'silent' 5-HT(1B) receptor was examined using SB-224289 (0.2 micromol/l), a selective 5-HT(1B) receptor antagonist. The influence of age on the 'silent' 5-HT(1B) receptor subtype was also investigated; for this, the effect of sumatriptan, a selective 5-HT(1B/1D )agonist, was tested on arterial tissues of both young and old rats which had been either K(+) depolarized or reserpine treated or both. It was found that aging strongly shifted the concentration-vasoconstriction curve generated by sumatriptan to the left, also increasing the maximum contractile response, mainly in reserpine-treated tissues. RT-PCR was used to study the expression of 5-HT(1B) and 5-HT(2A) receptors in both young and old tissues. The results support the idea that reserpine-treated and K(+)-depolarized caudal arteries from old rats can be a pharmacological model which is useful in highlighting the 'silent' 5-HT(1B) receptor subtype.
Assuntos
Envelhecimento/metabolismo , Artérias Cerebrais/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Resistência Vascular/efeitos dos fármacos , Animais , Artérias Cerebrais/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serotonina/análogos & derivados , Serotonina/farmacologia , Agonistas do Receptor 5-HT1 de Serotonina , Antagonistas do Receptor 5-HT1 de Serotonina , Sumatriptana/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Since alterations of tryptophan metabolism have been reported in diabetes and atherosclerosis, it was thought of interest to investigate any role of cloricromene through the influence on the oxidative metabolism of the amino acid by using diabetic/hyperlipidemic rabbits. Male 4-month-old New Zealand white rabbits, fed a diet enriched with 1% cholesterol and 10% corn oil, were made diabetic with alloxan. During the hyperlipidemic diet, a group of rabbits was treated with cloricromene (10 mg/kg/day subcutaneously plus 1.5 mg/kg/day intravenously, for 5 weeks). The other group received saline. Normometabolic New Zealand rabbits fed standard diet, treated or not with cloricromene, were used as control. The specific activities of liver tryptophan 2,3-dioxygenase and small intestine indole 2,3-dioxygenase were not significantly changed by the drug treatment. Also the specific activities of other enzymes of the kynurenine pathway in the liver and kidneys, specifically kynurenine 3-monooxygenase, kynureninase and kynurenine-oxoglutarate transaminase, did not show any significant difference in both tissues between the two groups of rabbits. On the contrary, 3-hydroxyanthranilate 3,4-dioxygenase activity in the liver of diabetic/hyperlipidemic rabbits and control rabbits treated with cloricromene showed a slight increase in comparison with untreated animals. Conversely, the specific activity of the enzyme in kidneys was not affected by the drug treatment in diabetic/hyperlipidemic animals but was reduced in controls. Aminocarboxymuconate-semialdehyde decarboxylase specific activity remained unchanged in the liver following cloricromene treatment, instead the specific activity of the enzyme in the kidneys of the diabetic/hyperlipidemic rabbits was significantly increased by the drug, with a value more than double in comparison to untreated animals. The activity of the scavenger enzyme Cu/Zn superoxide dismutase (Cu/Zn SOD) in the small intestine was also determined and found significantly increased of about twice as much in the group of diabetic/hyperlipidemic rabbits treated with cloricromene. In conclusion, in diabetic/hyperlipidemic rabbits, cloricromene appeared to influence the enzymes involved in the last steps of tryptophan oxidative metabolism through the kynurenine pathway. This, together with the antioxidant action through the activation of Cu/Zn SOD, might deserve further investigation for evaluating any link between the observed experimental findings at the level of the kynurenine pathway and the clinical effect of the drug.
Assuntos
Cromonar/análogos & derivados , Diabetes Mellitus Experimental/enzimologia , Hiperlipidemias/enzimologia , Niacina/metabolismo , Oxigenases/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Triptofano/metabolismo , Animais , Colesterol na Dieta/administração & dosagem , Cromonar/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Sequestradores de Radicais Livres/metabolismo , Hiperlipidemias/tratamento farmacológico , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Oxirredução , CoelhosRESUMO
Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits. The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits. These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.
Assuntos
Colesterol na Dieta/administração & dosagem , Diabetes Mellitus Experimental/metabolismo , Dioxigenases , Triptofano/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenase , Animais , Carboxiliases/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/enzimologia , Hiperlipidemias/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase , Rim/enzimologia , Cinurenina/biossíntese , Quinurenina 3-Mono-Oxigenase , Fígado/enzimologia , Masculino , Oxigenases de Função Mista/metabolismo , Niacina/metabolismo , Oxigenases/metabolismo , Coelhos , Triptofano Oxigenase/metabolismoRESUMO
The aim of this study was to investigate in vitro the variations with age of the activities of the two antioxidant enzymes Cu/Zn-superoxide dismutase (SOD) and indole 2,3-dioxygenase (IDO) in metabolically active tissues of rats of various ages. In rats aged one week and 2-3 months the highest Cu/Zn-SOD activity was found in the liver and the lowest in the small intestine. At 12 and 18 months of age, the activity was higher in the brain and kidneys, when compared to the small intestine, lungs and liver. Cu/Zn-SOD activity decreased significantly after 2-3 months of age with advancing age in all tissues examined. In newborn rats IDO activity was present only in the small intestine. In the group of rats aged 2-3 months, the highest specific activity was observed in the small intestine and the lowest in the lungs and kidneys, whereas at 12 months of age, the highest IDO activity was found in the brain, with kidneys presenting the lowest activity. At 18 months, IDO returned to be more elevated in the small intestine. At 12 months of age the values of IDO in the tissues varied slightly, while at 18 months similar activities were found between the lungs and brain and between the small intestine and kidneys. In relation to age, IDO specific activity declined in the small intestine, after 2-3 months of age. In the lungs, the activity remained unchanged; in the brain and in the kidneys activity decreased significantly from 2-3 to 18 months of age. In conclusion, this study demonstrates an age-related decline in Cu/Zn-SOD and IDO activities, the two enzymes responsible for scavenging O2*-.
Assuntos
Envelhecimento , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Superóxido Dismutase/metabolismo , Fatores Etários , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Resistance to chemotherapeutic drugs is a major problem in cancer treatment. The search for new interventions able to overcome this resistance may involve compounds of natural origin, such as flavonoids, ubiquitously present in many foods. In the present study, the cytotoxic effects and cell cycle modulation of the flavonoid quercetin were investigated in ovarian carcinoma (SKOV3) and osteosarcoma (U2OS) human cell lines and in their cisplatin (CDDP)-resistant counterparts (SKOV3/CDDP and U2OSPt cells, respectively). Quercetin (10-50 µM) caused evident changes in the distribution of cell cycle phases in the CDDP-resistant SKOV3/CDDP ovarian cell line. The levels of cyclin D1 and cyclin B1 were determined by means of Western blot in all cell lines incubated with quercetin (50 µM) for 48 hours. The cyclin D1 expression was significantly decreased following the treatment with quercetin in SKOV3 and U2OSPt cells, but not in SKOV3/CDDP and U2OS cells. The reduction of cyclin D1 level could be linked to the G1/S phase alteration found in quercetin-treated cells. Although cyclin B1 is required for G2/M phase, and despite our observation that quercetin influenced the G2/M phase of cell cycle, the flavonoid did not affect cyclin B1 levels in all cell lines, indicating the involvement of other possible mechanisms. These results suggest that quercetin, exceeding the resistance to CDDP, might become an interesting tool to evaluate cytotoxic activity in combination with chemotherapy drugs.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclinas/genética , Osteossarcoma/genética , Neoplasias Ovarianas/genética , Quercetina/farmacologia , Linhagem Celular Tumoral , Ciclinas/metabolismo , Feminino , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/fisiopatologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/fisiopatologiaRESUMO
Aminosalicylates, corticosteroids and immunosuppressants are currently the therapeutic choices in inflammatory bowel diseases (IBD), however, with limited remission and often serious side effects. Meanwhile complementary and alternative medicine (CAM) use is increasing, particularly herbal medicine. Boswellia serrata is a traditional Ayurvedic remedy with anti-inflammatory properties, of interest for its usefulness in IBDs. The mechanism of this pharmacological potential of Boswellia serrata was investigated in colonic epithelial cell monolayers exposed to H2O2 or INF-γ+TNF-α, chosen as in vitro experimental model of intestinal inflammation. The barrier function was evaluated by the transepithelial electrical resistance (TEER) and paracellular permeability assay, and by the tight junction proteins (zonula occludens-1, ZO-1 and occludin) immunofluorescence. The expression of phosphorylated NF-κB and reactive oxygen species (ROS) generation were determined by immunoblot and cytofluorimetric assay, respectively. Boswellia serrata oleo-gum extract (BSE) and its pure derivative acetyl-11-keto-ß-boswellic acid (AKBA), were tested at 0.1-10 µg/ml and 0.027 µg/ml, respectively. BSE and AKBA safety was demonstrated by no alteration of intestinal cell viability and barrier function and integrity biomarkers. H2O2 or INF-γ+TNF-α treatment of Caco-2 cell monolayers significantly reduced TEER, increased paracellular permeability and caused the disassembly of tight junction proteins occludin and ZO-1. BSE and AKBA pretreatment significantly prevented functional and morphological alterations and also the NF-κB phosphorylation induced by the inflammatory stimuli. At the same concentrations BSE and AKBA counteracted the increase of ROS caused by H2O2 exposure. Data showed the positive correlation of the antioxidant activity with the mechanism involved in the physiologic maintenance of the integrity and function of the intestinal epithelium. This study elucidates the pharmacological mechanisms mediated by BSE, in protecting intestinal epithelial barrier from inflammatory damage and supports its use as safe adjuvant in patients affected by IBD.
Assuntos
Antioxidantes/administração & dosagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Triterpenos/administração & dosagem , Antioxidantes/química , Boswellia/química , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Peróxido de Hidrogênio/toxicidade , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , NF-kappa B/metabolismo , Extratos Vegetais/química , Proteínas de Junções Íntimas/metabolismoRESUMO
The mechanisms of cisplatin resistance, one of the major limitations of current chemotherapy, has only partially been described. We previously demonstrated that cisplatin-resistant ovarian cancer cells (C13), are characterized by reduced mitochondrial activity and higher glucose-dependency when compared to the cisplatin-sensitive counterpart (2008). In this work we further characterized the role of metabolic transformation in cisplatin resistance. By using transmitochondrial hybrids we show that metabolic reprogramming of cisplatin-resistant cell is not caused by inherent mtDNA mutations. We also found that C13 cells not only present an increased glucose-uptake and consumption, but also exhibit increased expression and enzymatic activity of the Pentose Phosphate pathway (PPP) enzyme Glucose-6-Phosphate Dehydrogenase (G6PDH). Moreover, we show that cisplatin-resistant cells are more sensitive to G6PDH inhibition. Even if the metabolomic fingerprint of ovarian cancer cells remains to be further elucidated, these findings indicate that PPP offers innovative potential targets to overcome cisplatin resistance.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Relação Dose-Resposta a Droga , Feminino , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glutamina/metabolismo , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND: The activity of nicotinic acid in hypercholesterolemia has been poorly understood. In man, nicotinic acid derives for the most part from tryptophan along the tryptophan-nicotinic acid pathway, also called the kynurenine pathway, kynurenine being the key metabolite in this process. In the present paper, we investigated if, in animals with hypercolesterolemia, degradation of tryptophan to nicotinic acid along the kynurenine pathway was perturbated. METHODS: Liver, kidney and intestine enzyme activities of the tryptophan-nicotinic acid pathway in normolipidemic, diet-induced hyperlipidemic New Zealand and heritable hypercholesterolemic Watanabe (WHHL) rabbits were determined. RESULTS: Liver tryptophan 2,3-dioxygenase (TDO) activity was present only as a holoenzyme and was higher in the controls than in the hyperlipidemic and Watanabe rabbits, but no difference was present between the group fed an atherogenic hyperlipidic diet and the WHHL rabbits. Small intestine indole 2,3-dioxygenase (IDO) did not vary significantly among the three groups but was higher in comparison with liver TDO activity. In liver, kynurenine 3-monooxygenase and kynurenine-oxoglutarate transaminase activities did not show any significant difference among the three groups of rabbits. Kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase activities per g of fresh tissue decreased significantly in the group of hyperlipidemic and in WHHL rabbits. In the kidneys, kynurenine 3-monooxygenase and kynureninase activity did not change significantly in the three groups of rabbits; kynurenine-oxoglutarate transaminase activity per g of fresh tissue decreased in both hyperlipidemic groups, but no significant difference was observed between hyperlipidemic and Watanabe rabbits. 3-Hydroxyanthranilate 3,4-dioxygenase activity in kidney was decreased markedly in hyperlipidemic and Watanabe rabbits, but there was no difference between the two hypercholesterolemic groups. Aminocarboxymuconate-semialdehyde decarboxylase activity did not change. Thus 3-hydroxyanthranilate 3,4-dioxygenase may be an important regulatory mechanism in the control of the flow of tryptophan along the kynurenine pathway to NAD in hypercholesterolemic rabbits. CONCLUSIONS: This study first demonstrates that in rabbits, hypercholesterolemia, both diet- or genetically induced, can influence the enzyme activities of the tryptophan-nicotinic acid pathway leading to a decreased formation of nicotinic acid, and thus NAD.
Assuntos
Hipercolesterolemia/metabolismo , Cinurenina/metabolismo , Triptofano/metabolismo , Animais , Carboxiliases/metabolismo , Hipercolesterolemia/enzimologia , Hipercolesterolemia/genética , Indolamina-Pirrol 2,3,-Dioxigenase , Intestino Delgado/enzimologia , Rim/enzimologia , Quinurenina 3-Mono-Oxigenase , Fígado/enzimologia , Oxigenases de Função Mista/metabolismo , Niacina/metabolismo , Coelhos , Transaminases/metabolismo , Triptofano Oxigenase/metabolismoRESUMO
Mannich bases obtained by aminoalkylation of 3H-pyrrolo[3,2-f]quinoline were designed and prepared as potential vasorelaxing agents. Compounds Ia-Va were characterised by IR, 1H-NMR, mass spectral data and elemental analysis; IIb,c-Vb,c were also confirmed by 1H-NMR spectra of reaction mixtures. To estimate their vascular activity, prototypes 1-(N,N-dimethylaminomethyl)- (Ia) and 1-(4-phenyl-piperazin-1-ylmethyl)- (IVa) 3H-pyrrolo[3,2-f]quinoline derivatives were studied in rat-tail arteries. In tissues precontracted with 0.5 microM 5-hydroxytryptamine (5-HT), 3 microM phenylephrine or 80 mM KCl, Ia and IVa showed endothelium-independent relaxing action. In a preliminary study on the cellular mechanisms of Ia, the influence of propranolol, a beta-receptor antagonist, and ketanserin, a 5-HT(2A)-receptor antagonist, was checked. In the presence of phenylephrine, the vasorelaxing effect of Ia was not affected by these inhibitors.
Assuntos
Piperazinas/farmacologia , Pirróis/farmacologia , Quinolinas/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Artérias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Bases de Mannich , Músculo Liso Vascular/efeitos dos fármacos , Piperazinas/síntese química , Piperazinas/química , Pirróis/síntese química , Pirróis/química , Quinolinas/síntese química , Quinolinas/química , Ratos , Ratos Wistar , Cauda/irrigação sanguínea , Vasodilatadores/síntese química , Vasodilatadores/químicaRESUMO
We studied the effects of 5-HT in resistance vessels. Compounds acting on 5-HT2A and 5-HT1B receptors were tested on rat-tail arterial rings in varying experimental conditions. We tested 5-HT from 50 nM to 100 microM: the pD2 was 6.4 +/- 0.1. We evaluated vasoconstriction by 5-HT in tissues slightly depolarized with 30 mM KCl. In this condition, the 5-HT concentration-related contraction started at lower concentration in comparison to control tissues. Preincubation with 50 nM ketanserin, a 5-HT2A-antagonist, and 1 microM prazosin, an alpha1-antagonist, strongly inhibited concentration-related contraction by 5-HT: the pD2 was 3.2 +/- 0.2. Moreover, we experimented alpha-methyl-5-HT (alpha-me-5-HT) and 5-carboxamidotryptamine (5-CT), selective agonists at 5-HT2A and 5-HT1B receptors, respectively. Both agonists induced concentration-related contraction; the potency order observed was 5-HT > alpha-me-5-HT > 5-CT. Finally, we studied SB 224289, a selective 5-HT1B-antagonist, on contraction by 5-HT in control and in depolarized conditions. 0.2 microM SB 224289 significantly inhibited vasoconstriction induced by 5-HT in depolarized vascular tissues. The data indicate that vasoconstriction induced by 5-HT is mainly dependent on 5-HT2A receptors; however, 5-HT1B receptors are also present in rat-tail artery.
Assuntos
Receptor 5-HT1B de Serotonina/fisiologia , Receptor 5-HT2A de Serotonina/fisiologia , Serotonina/análogos & derivados , Vasoconstrição/fisiologia , Animais , Artérias/efeitos dos fármacos , Artérias/fisiologia , Ketanserina/farmacologia , Masculino , Piperidonas/farmacologia , Prazosina/farmacologia , Ratos , Ratos Wistar , Receptor 5-HT1B de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Compostos de Espiro/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
Tryptophan metabolism along the kynurenine pathway in normolipidemic and diet-induced hyperlipidemic New Zealand rabbits was studied. The activities of liver tryptophan 2,3-dioxygenase small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate semialdehyde decarboxylase (picolinic carboxylase) were determined. Liver tryptophan 2,3-dioxygenase (TDO) was present only as a holoenzyme. In fact, similar results were found in the absence or presence of the cofactor haematin. In addition, TDO activity was higher in normolipidemic than in hyperlipidemic rabbits. Small intestine indole 2,3-dioxygenase did not change significantly among the two groups of animals, but was higher than liver TDO. Liver and kidney kynurenine 3-monooxygenase activities did not change significantly whereas kynurenine-oxoglutarate transaminase activity decreased only per g of fresh kidney in hyperlipidemic rabbits. Kynureninase activity decreased significantly per g of fresh tissue only in liver. 3-Hydroxyanthranilate 3,4-dioxygenase, both as specific activity and per g of fresh tissue and aminocarboxymuconate-semialdehyde decarboxylase activities showed significantly lower values per g of fresh kidney in hyperlipidemic rabbits compared with controls. Therefore, hyperlipidemia can influence enzyme activities along the kynurenine pathway, leading to a reduction in the biosynthesis of NAD coenzymes.
Assuntos
Hiperlipidemias/metabolismo , Cinurenina/metabolismo , Animais , Hiperlipidemias/enzimologia , Intestino Delgado/enzimologia , Rim/enzimologia , Fígado/enzimologia , Masculino , Coelhos , Triptofano/metabolismo , Triptofano Oxigenase/metabolismoRESUMO
Fifteen plant polyphenols, including flavonoids, cinnamic acids, coumarins and capsaicin, were investigated for their capacity to suppress cell growth and regulate the cell cycle of in vitro human ovarian carcinoma (2008 cell line) and cervix squamous carcinoma cells (A431), and their cisplatin (CDDP)-resistant subclones (C13 and A431Pt, respectively). Evaluation of the cytotoxic effects of the polyphenols (0.01-100 µM) indicated that especially rhein and quercetin were almost equiactive in wild type and CDDP-resistant cells, indicating lack of cross-resistance with cisplatin. Capsaicin was more potent in CDDP-resistant subclones than in wild type cells. The order of their potencies is flavonoids > anthraquinones > vanilloids > coumarins > phenols, cinnamic acids. The natural phenols which were most cytotoxic (rhein, quercetin and capsaicin) were able to cause the arrest of the cancer cell cycle, suggesting that specific cell cycle regulatory proteins are possibly involved in their intracellular mechanism of action. In particular, the natural compounds were revealed to be more active in CDDP-resistant cells than in wild types, especially inducing apoptotic death.
Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cisplatino/farmacologia , Fenóis/farmacologia , Capsaicina/farmacologia , Linhagem Celular Tumoral , Cinamatos/farmacologia , Cumarínicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , HumanosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Croton lechleri Mull. Arg. (Euphorbiaceae) is a traditional medicinal plant which produces a red sap, traditionally known as "Sangre de Drago"; it is used in folk medicine externally for wounds, fractures, and haemorrhoids, internally for intestinal and stomach ulcers and also for the empirical cure of cancers. MATERIALS AND METHODS: We investigated the effects of Croton lechleri sap and taspine in comparison with taxol and vinblastine on the growth of human cancer cell lines of SK23 (melanoma), LoVo and HT29 (colorectal cancer) using MTT and Trypan blue assays. Further, we studied cell cycle by flow cytometry and detected acetylated-α-tubulin by confocal microscope. RESULTS: Croton lechleri inhibited cell proliferation starting from 1 µg/mL in SK23 cells, whereas 10 times higher concentrations were required for growth inhibition of HT-29 and LoVo cell lines. Also taspine (0.1 µg/mL) inhibited the SK23 and HT29 cell proliferation. Further, assay was assessed on SK23 and HT29 cell lines with 24-48 h treatment with sap and taspine. Both sap and taspine inhibited cancer cell proliferation; taspine showed higher activity on SK23 cells, which was significantly increased after 48 h of SK23 treatment. Using confocal microscopy we observed that Croton lechleri (1 µg/mL) caused a loss of microtubule structure, whereas taspine (0.5 µg/mL) caused an increase in acetylated α-tubulin and a modification of cellular morphology, mainly in SK23 cells. Croton lechleri sap 10 and 50 µg/mL influence cell cycle; 50 µg/mL sap caused a dramatic reduction of cells in G(1)/G(0) and S phases with a great increase of subG(0) cells. CONCLUSIONS: The data showed that Croton lechleri and taspine could inhibit cell proliferation with higher potency against melanoma SK23 cells, supporting the empirical use of the sap as anticancer in ethnomedicine and taspine as a possible anticancer agent.
Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Croton , Extratos Vegetais/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Células HT29 , Humanos , Melanoma/tratamento farmacológico , Paclitaxel/farmacologia , Vimblastina/farmacologiaRESUMO
Zinc is one of the most important trace elements in the body and it is essential as a cofactor for the structure and function of a number of cellular molecules including enzymes, transcription factors, cellular signalling proteins and DNA repair enzymes. On the other hand, recent studies have shown that zinc could play a role both in the development of various cancers and in the induction of apoptosis in some cell types, however, no established common relationships of zinc with cancer development and progression have been identified. To date, in our research group different metal-dithiocarbamato complexes have been designed that were expected to resemble the main features of cisplatin together with higher activity, improved selectivity and bioavailability, and lower side-effects. On the basis of the obtained encouraging achievements with other metals (such as gold and copper) we have decided to enlarge the studies to the complexes of zinc(II) using the same ligands. Hereby, we report the results on the synthesis and characterisation of ZnL(2) complexes with five different dithiocarbamato derivatives, such as dimethyl-(DMDT), pyrrolidine-(PyDT), methyl-(MSDT), ethyl-(ESDT) and tert-butyl-(TSDT) sarcosinedithiocarbamate. All the obtained compounds have fully been characterised by means of several spectroscopic techniques. In addition, the crystal structure of [Zn(MSDT)(2)](2) dinuclear complex is also reported. In order to evaluate the in vitro cytotoxic properties, some biological assays have been carried out on a panel of human tumour cell lines sensible and resistant to cisplatin. Some of the tested compounds show cytotoxicity levels comparable or even greater than the reference drug (cisplatin).
Assuntos
Antineoplásicos/química , Complexos de Coordenação/química , Tiocarbamatos/química , Zinco/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Metoxicloro/química , Tiocarbamatos/farmacologiaRESUMO
OBJECTIVES: Thienopyridines are prodrugs currently used as anti-aggregating agents. The aim of this study was to determine if these compounds might have vascular activity independent of hepatic bioactivation. METHODS: The direct activity of thienopyridines was studied in rat caudal arterial rings and aortic smooth muscle cells in culture. KEY FINDINGS: Both compounds (0.01 µm-100 µm) showed a concentration-dependent vasorelaxation in arterial tissues precontracted with phenylephrine, 5-hydroxytryptamine and KCl. The relaxation induced by 100 µm ticlopidine and clopidogrel was greater than 80%. The relaxation by ticlopidine was compared with the activity of acetylcholine. These two agents showed similar potency, although ticlopidine was slightly more active. Pretreatment with the nitric oxide synthase inhibitor L-NAME inhibited the relaxation by acetylcholine but not that by ticlopidine. To further study vasorelaxation by ticlopidine, other pharmacological inhibitors including propranolol, nifedipine and suramin were used. These compounds lacked inhibitory effects on the vasorelaxation by ticlopidine. In vascular smooth muscle cells, 1 µm ticlopidine induced a decrease in cell proliferation, while incubation with both ticlopidine and ADP or 2-methioADP led to an additive effect. CONCLUSIONS: The data suggest that ticlopidine and clopidogrel cause relaxation of arterial tissues and influence vascular smooth muscle cell proliferation directly without hepatic biotransformation. Furthermore, the arterial relaxation induced in vitro by thienopyridines is endothelium independent, and ß-adrenergic and P2 receptors are not involved.