RESUMO
BACKGROUND: Loss or mutations of the BRCA1 gene are associated with increased risk of breast and ovarian cancers and with prostate cancer (PCa) aggressiveness. Previously, we identified GADD153 as a target of BRCA1 protein, which increases doxorubicin sensitivity in human p53 -/- PCa cells (PC3). Considering that p53 is a crucial target in cancer therapy, in this work we investigated p53 role in the regulation of transcription of GADD153. METHODS: We performed reverse transcription quantitative PCR (RT-qPCR), western blot and luciferase assays to analyze GADD153 and/or BRCA1 expression in response to ultraviolet or doxorubicin exposure in PC3 p53 stable-transfected cells and LNCaP (p53+/+) cells. BRCA1 protein recruitment to GADD153 promoter was studied by chromatin immunoprecipitation-qPCR. To assess expression of BRCA1 and/or p53 target genes, we used a panel of stable-transfected PCa cell lines. We finally analyzed these genes in vivo using BRCA1-depleted PCa xenograft models. RESULTS: We found that GADD153 was highly induced by doxorubicin in PC3 cells; however, this response was totally abolished in LNCaP (p53wt) and in p53-restituted PC3 cells. Furthermore, BRCA1 protein associates to GADD153 promoter after DNA damage in the presence of p53. Additionally, we demonstrated that BRCA1 and/or p53 modulate genes involved in DNA damage and cell cycle regulation (cyclin D1, BLM, BRCA2, DDB2, p21(WAF1/CIP1), H3F3B, GADD153, GADD45A, FEN1, CCNB2), EMT (E-cadherin, ß-catenin, vimentin, fibronectin, slug, snail) and Hedgehog pathways (SHH, IHH, DHH, Gli1, PATCH1). Furthermore, xenograft studies demonstrated that BRCA1 knockdown in PC3 cells increased tumor growth and modulated these genes in vivo. CONCLUSIONS: Although BRCA1 induces GADD153 in a p53 independent manner, p53 abolished GADD153 induction in response to DNA damage. In addition, several important PCa targets are modulated by BRCA1 and p53. Altogether, these data might be important to understand the therapy response of PCa patients.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Próstata/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína BRCA1/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA , Ouriços/genética , Ouriços/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
1. The effect of in vitro glycation on delta-aminolevulinic dehydratase (ALA-D) under several experimental conditions was investigated. When preincubated with 500 mM glucose at 37 degrees C for 20 hr, ALA-D was 80% inactivated and glycated hemoglobin levels were increased more than fourfold. 2. Thiobarbituric acid species were not modified during glycation; therefore ALA-D inactivation cannot be attributed to glucose autoxidation. 3. Acetyl salicylic acid was effective in preventing both hemoglobin glycation and ALA-D inactivation by glucose. 4. A method has been developed for measuring protein glycation in vitro, in a crude preparation of red blood cells, which can also be applied to sugars other than glucose.
Assuntos
Aspirina/farmacologia , Glucose/metabolismo , Sintase do Porfobilinogênio/metabolismo , Metabolismo dos Carboidratos , Carboidratos/farmacologia , Glucose/farmacologia , Hemoglobinas Glicadas/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Sintase do Porfobilinogênio/antagonistas & inibidores , Temperatura , Fatores de TempoRESUMO
1. The effect of long-term griseofulvin (GRIS) topical administration on some indicators of liver damage was examined. 2. Liver porphyrin accumulation was significant; however, no porhyrin crystals were observed under light microscopy. 3. An earlier onset of hepatopathy was established (3-fold) increase of direct bilirubin values after 7 days of treatment; hepatic injury was confirmed by measuring a 6-fold increase of free bilirubin. 4. Enhanced values of alkaline phosphatase and glutamic oxalacetic transaminase (GOT) confirmed the onset of cholestasis. 5. Topical application of GRIS induced measurable hepatopathy. Nevertheless, we cannot discard the possibility that this hepatopathy could also be attributed in part to a direct reaction to xenobiotics.
Assuntos
Antifúngicos/farmacologia , Griseofulvina/farmacologia , Heme/metabolismo , Fígado/efeitos dos fármacos , Administração Tópica , Animais , Antifúngicos/administração & dosagem , Biomarcadores , Griseofulvina/administração & dosagem , Fígado/enzimologia , Fígado/patologia , Testes de Função Hepática , Masculino , CamundongosRESUMO
1. The effect of chronic enflurane or isoflurane anesthesia on hepatic heme regulation and the drug-metabolizing system in mice treated or not with phenobarbital (PB) was investigated. 2. delta-Aminolevulinic acid synthetase was induced 50-170% in all cases. Urinary porphyrin precursor excretion was also enhanced, but these values were lower when animals also received PB. 3. Cytochrome (CYT) P-450 levels were enhanced in animals treated with enflurane whether or not they were given PB. 4. Gluthatione-S-transferase activity was induced by enflurane (138%) or isoflurane (174%), and even more in animals receiving PB also. Sulfatase activity was increased more than 60% with anesthetics. Isoflurane produced a 50% increase of beta-glucuronidase activity and a 35% diminution of tryptophan pyrrolase. 5. The association between anesthetics and PB produced diverse effects on the metabolizing enzyme system. 6. Data suggest that both anesthetics, chemically related, could act through two different mechanisms, however, with the same final effect: heme pathway deregulation.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Anestésicos Inalatórios/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Enflurano/farmacologia , Heme/metabolismo , Hipnóticos e Sedativos/farmacologia , Isoflurano/farmacologia , Fígado/efeitos dos fármacos , Fenobarbital/farmacologia , Ácido Aminolevulínico/urina , Animais , Fígado/enzimologia , Masculino , Camundongos , Porfobilinogênio/urina , Porfirinas/metabolismoRESUMO
1. The effect of enflurane or isoflurane anesthesia (1 ml/kg, i.p.) in animals chronically treated with ethanol (30%, v/v, in drinking water during a week) on heme metabolism and its regulation was investigated. 2. In those animals previously intoxicated with ethanol that received isoflurane, ALA-S activity was increased (control values: 0.071 +/- 0.022 nmol/mg, n = 10; treated animals: 0.110 +/- 0.034 nmol/mg, n = 8) and blood PBGase and deaminase were strikingly diminished (control values, n = 10: PBGase: 0.101 +/- 0.015 nmol/mg, deaminase: 0.242 +/- 0.075 nmol/mg; treated animals, n = 6: PBGase: 0.063 +/- 0.013 nmol/mg; deaminase: 0.145 +/- 0.045 nmol/mg). 3. The time-response study showed that liver ALA-S is enhanced at shorter times of anesthesia with isoflurane and that blood PBGase and deaminase appeared inhibited later in animals previously treated with ethanol. 4. Results reproduce some biochemical alterations known to occur in acute intermittent porphyria.
Assuntos
Etanol/farmacologia , Éteres/farmacologia , Heme/metabolismo , Animais , Enflurano/farmacologia , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
A frequent coexistence of diabetes and porphyria disease has been reported. Under normal conditions, porphyrin biosynthesis is well regulated to only form the amount of heme required for the synthesis of the various hemoproteins. The activity of some heme enzymes and rhodanese in streptozotocin (STZ) induced diabetic mice and in allylisopropylacetamide (AIA) induced experimental acute porphyria mice has been examined. The role of alpha-tocopherol (alpha-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic delta-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. alpha-Tocopherol did not modify ALA-S activity in either group. delta-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. alpha-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but alpha-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of alpha-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.
Assuntos
Alilisopropilacetamida/toxicidade , Diabetes Mellitus Experimental/enzimologia , Porfiria Aguda Intermitente/enzimologia , Vitamina E/farmacologia , 5-Aminolevulinato Sintetase/sangue , 5-Aminolevulinato Sintetase/metabolismo , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Heme/metabolismo , Masculino , Camundongos , Nucleosídeo Desaminases/sangue , Nucleosídeo Desaminases/metabolismo , Sintase do Porfobilinogênio/sangue , Sintase do Porfobilinogênio/metabolismo , Porfiria Aguda Intermitente/induzido quimicamente , Estreptozocina/toxicidade , Tiossulfato Sulfurtransferase/metabolismoRESUMO
1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-amino-levulinate synthetase (ALA-S), cytoplasmic and mitochondrial rhodanese were determined in tumor (T) and liver of both normal mice (NM) and T-bearing mice (TBM). 2. Rhodanese tumoral mitochondrial levels were higher than the hepatic normal mitochondrial fraction, while the cytoplasmic activity was nearly equal in all sources. 3. In neither case was the activity of tumoral ALA-S and rhodanese altered by any of the porphyrinogenic drugs. 4. Mitochondrial and cytoplasmic rhodanese activity was also measured in tumor and liver of TBM at different intervals after transplantation. We concluded that the behaviour of rhodanese is a property inherent to the tissue and not one attained with time.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Tiossulfato Sulfurtransferase/metabolismo , 5-Aminolevulinato Sintetase/biossíntese , Alilisopropilacetamida/farmacologia , Animais , Barbital/farmacologia , Citoplasma/enzimologia , Indução Enzimática/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Tiossulfato Sulfurtransferase/biossínteseRESUMO
1. A clear biphasic response of the enzyme activities as a function of intoxication time due to the topical cutaneous griseofulvin treatment was observed. 2. The initial acute induction of ALA-S activity would be due to depletion of free heme in the regulatory pool caused by cytochrome P 450 destruction. 3. The second induction peak, would be due to less heme formation, secondary to the ferrochelatase inhibition, as expected for the erythropoietic protoporphyria model. 4. The biphasic response of hepatic ALA-D and PBGase activities would be related to ALA-S activity changes and the subsequent augmented available substrates. 5. Endogenous liver porphyrin distribution in cytosolic, mitochondrial and nuclear fractions was investigated. 6. The in vitro biosynthesis of porphyrins confirmed both the biphasic model and the hepatic porphyrins subcellular distribution. 7. Two mechanisms to explain the action of griseofulvin at shorter and longer times of intoxication are proposed.
Assuntos
Griseofulvina/farmacologia , Heme/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Amônia-Liases/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Sintase do Porfobilinogênio/metabolismo , Porfirinas/metabolismo , Proteínas/metabolismoRESUMO
1. Some in vitro studies were performed to elucidate the action of S-adenosyl-L-methionine (SAM) and thiosulphate on liver rhodanese, delta-amino-levulinic acid dehydratase (Al A-D) and cytochrome oxidase affected by cyanide in the experimental conditions. 2. SAM was unable to interact with the sulfur substituted rhodanese complex suggesting that SAM would blockade the thiosulphate binding sites on rhodanese. 3. Cyanide and thiosulphate inhibited ALA-D activity when both compounds were present in the incubation or the preincubation mixture. Cyanide binding on the enzyme was irreversible. 4. Cyanide inhibited cytochrome oxidase activity and the reversible nature of the binding was demonstrated by gel filtration. 5. SAM had no effect on either ALA-D or cytochrome oxidase activities.
Assuntos
Cianetos/farmacologia , S-Adenosilmetionina/farmacologia , Tiossulfato Sulfurtransferase/antagonistas & inibidores , Tiossulfatos/farmacologia , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Sintase do Porfobilinogênio/metabolismoRESUMO
1. Several sub-lethal doses of cyanide were assayed with the aim of obtaining significant differences in the parameters studied. A dose of 4 mg/kg s.c. was selected. 2. Present studies were carried out to determine the time dependence of the effects produced by s.c. administration of a single dose of potassium cyanide as well as the recovery time of some of the toxicity indicative parameters. 3. It was found that cyanide effects continued for at least 3 days; after the parameters had recovered normal values.