RESUMO
Nucleotide excision repair (NER) and cell cycle checkpoints impact the ability of the anti-cancer drug cisplatin to inhibit cell proliferation and induce cell death. Genetic studies have shown that both NER and cell cycle progression are impacted by the circadian clock, which has emerged as a novel pharmacological target for the treatment of various disease states. In this study, cultured human cell lines were treated with combinations of cisplatin and the circadian clock modulating compounds KS15 and SR8278, which enhance circadian clock transcriptional output by inhibiting the activities of the cryptochrome and REV-ERB proteins, respectively. Treatment of cells with KS15 and SR8278 protected cells against the anti-proliferative effects of cisplatin and increased the expression of NER factor XPA and cell cycle regulators Wee1 and p21 at the mRNA and protein level. Correlated with these molecular changes, KS15 and SR8278 treatment resulted in fewer unrepaired cisplatin-DNA adducts in genomic DNA and a higher fraction of cells in the G1 phase of the cell cycle. Thus, the use of pharmacological agents targeting the circadian clock could be a novel approach to modulate the responses of normal and cancer cells to cisplatin chemotherapy regimens.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Criptocromos/antagonistas & inibidores , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/farmacologia , Reparo do DNA/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Células A549 , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HaCaT , Humanos , Isoquinolinas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes.
Assuntos
Arabidopsis/metabolismo , Criptocromos/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/análise , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Sistema Livre de Células , Cotilédone/anatomia & histologia , Cotilédone/efeitos dos fármacos , Cotilédone/efeitos da radiação , Criptocromos/metabolismo , Criptocromos/efeitos da radiação , Meios de Cultura , Hipocótilo/anatomia & histologia , Hipocótilo/efeitos dos fármacos , Hipocótilo/efeitos da radiação , Processamento de Imagem Assistida por Computador , Luz , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Proteínas Recombinantes/biossíntese , Plântula/efeitos dos fármacos , Plântula/efeitos da radiação , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Mice lacking the core-clock components, cryptochrome-1 (CRY1) and cryptochrome-2 (CRY2) display a phenotype of hyperaldosteronism, due to the upregulation of type VI 3ß-hydroxyl-steroid dehydrogenase (Hsd3b6), the murine counterpart to the human type I 3ß-hydroxyl-steroid dehydrogenase (HSD3B1) gene. In the present study, we evaluated the role of CRY1 and CRY2 genes, and their potential interplay with HSD3B isoforms in adrenal pathophysiology in man. Forty-six sporadic aldosterone-producing adenomas (APAs) and 20 paired adrenal samples were included, with the human adrenocortical cells HAC15 used as the in vitro model. In our cohort of sporadic APAs, CRY1 expression was 1.7-fold [0.75â»2.26] higher (p = 0.016), while CRY2 showed a 20% lower expression [0.80, 0.52â»1.08] (p = 0.04) in APAs when compared with the corresponding adjacent adrenal cortex. Type II 3ß-hydroxyl-steroid dehydrogenase (HSD3B2) was 317-fold [200â»573] more expressed than HSD3B1, and is the main HSD3B isoform in APAs. Both dehydrogenases were more expressed in APAs when compared with the adjacent cortex (5.7-fold and 3.5-fold, respectively, p < 0.001 and p = 0.001) and HSD3B1 was significantly more expressed in APAs composed mainly of zona glomerulosa-like cells. Treatment with angiotensin II (AngII) resulted in a significant upregulation of CRY1 (1.7 ± 0.25-fold, p < 0.001) at 6 h, and downregulation of CRY2 at 12 h (0.6 ± 0.1-fold, p < 0.001), through activation of the AngII type 1 receptor. Independent silencing of CRY1 and CRY2 genes in HAC15 cells resulted in a mild upregulation of HSD3B2 without affecting HSD3B1 expression. In conclusion, our results support the hypothesis that CRY1 and CRY2, being AngII-regulated genes, and showing a differential expression in APAs when compared with the adjacent adrenal cortex, might be involved in adrenal cell function, and in the regulation of aldosterone production.
Assuntos
Adenoma/genética , Criptocromos/genética , Hipertensão/genética , Complexos Multienzimáticos/genética , Progesterona Redutase/genética , Esteroide Isomerases/genética , Adenoma/metabolismo , Adenoma/patologia , Aldosterona/biossíntese , Angiotensina II/genética , Animais , Linhagem Celular Tumoral , Criptocromos/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Hipertensão/patologia , CamundongosRESUMO
AIMS: We have previously identified a chemical scaffold possessing 2-ethoxypropanoic acid (designated as KS15) that directly binds to the C-terminal region of cryptochromes (CRYs: CRY1 and CRY2) and enhances E-box-mediated transcription. However, it is still unclear how KS15 impairs the feedback actions of the CRYs and which chemical moieties are functionally important for its actions. MAIN METHODS: The E-box-mediated transcriptional activities were mainly used to examine the effects of KS15 and its derivatives. Co-immunoprecipitation assays accompanied by immunoblotting were employed to monitor protein-protein associations. We also examined the effects of KS15 and selected derivatives on circadian molecular rhythms in cultured cells. KEY FINDINGS: The present study shows that KS15 inhibits the interaction between CRYs and Brain-Muscle-Arnt-Like protein 1 (BMAL1), thereby impairing the feedback actions of CRYs on E-box-dependent transcription by CLOCK:BMAL1 heterodimer, an indispensable transcriptional regulator of the mammalian circadian clock. Subsequent structure-activity relationship analyses using a well-designed panel of derivatives identified the structural requirements for the effects of KS15 on CRY-evoked regulation of E-box-mediated transcription. We found that KS15 and several derivatives significantly reduce the amplitude and delayed the phase of molecular circadian rhythms in fibroblast cultures. SIGNIFICANCE: Taken together, our results provide valuable information on the molecular mode-of-action as well as the chemical components of the CRYs inhibitor that pharmacologically impact on the transcriptional activity of the CLOCK:BMAL1 heterodimer.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Criptocromos/antagonistas & inibidores , Elementos E-Box , Compostos de Epóxi/farmacologia , Complexos Multiproteicos/metabolismo , Propionatos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Compostos de Epóxi/química , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Propionatos/química , Domínios ProteicosRESUMO
Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FADox) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Criptocromos/metabolismo , Modelos Moleculares , Mutação , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Ligação Competitiva , Biocatálise , Criptocromos/antagonistas & inibidores , Criptocromos/química , Criptocromos/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Temperatura Alta/efeitos adversos , Indazóis/química , Indazóis/metabolismo , Indazóis/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Desnaturação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia Estrutural de ProteínaRESUMO
Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer.
Assuntos
Antineoplásicos/farmacologia , Criptocromos/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Bibliotecas de Moléculas Pequenas/farmacologia , Apoptose/efeitos dos fármacos , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ritmo Circadiano/genética , Criptocromos/genética , Criptocromos/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Células MCF-7 , Especificidade de Órgãos , Transdução de Sinais , Tamoxifeno/farmacologiaRESUMO
In the current model of the mammalian circadian clock, PERIOD (PER) represses the activity of the circadian transcription factors BMAL1 and CLOCK, either independently or together with CRYPTOCHROME (CRY). Here, we provide evidence that PER has an entirely different function from that reported previously, namely, that PER inhibits CRY-mediated transcriptional repression through interference with CRY recruitment into the BMAL1-CLOCK complex. This indirect positive function of PER is consistent with previous data from genetic analyses using Per-deficient or mutant mice. Overall, our results support the hypothesis that PER plays different roles in different circadian phases: an early phase in which it suppresses CRY activity, and a later phase in which it acts as a transcriptional repressor with CRY. This buffering effect of PER on CRY might help to prolong the period of rhythmic gene expression. Additional studies are required to carefully examine the promoter- and phase-specific roles of PER.
Assuntos
Ritmo Circadiano/genética , Regulação da Expressão Gênica , Proteínas Circadianas Period/genética , Fatores de Transcrição ARNTL/antagonistas & inibidores , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/antagonistas & inibidores , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Células COS , Chlorocebus aethiops , Criptocromos/antagonistas & inibidores , Criptocromos/genética , Criptocromos/metabolismo , Células HEK293 , Humanos , Mamíferos , Camundongos , Células NIH 3T3 , Proteínas Circadianas Period/antagonistas & inibidores , Proteínas Circadianas Period/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , TransfecçãoRESUMO
Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Proteínas Circadianas Period/metabolismo , Preparações Farmacêuticas/administração & dosagem , Poluentes Químicos da Água/administração & dosagem , Fatores de Transcrição ARNTL/agonistas , Fatores de Transcrição ARNTL/antagonistas & inibidores , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Criptocromos/agonistas , Criptocromos/antagonistas & inibidores , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/induzido quimicamente , Obesidade/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Circadianas Period/agonistas , Proteínas Circadianas Period/antagonistas & inibidores , Proteínas Circadianas Period/genética , Testes de Toxicidade Crônica , Fatores de Transcrição/agonistas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
Circadian rhythms, biological oscillations with a period of about 24 h, are maintained by a genetically determined innate time-keeping system called the molecular circadian clockwork. Despite the physiological and clinical importance of the circadian clock, the development of small molecule modulators that directly target the core clock machinery has only been recently initiated. In the present study, we aimed to identify novel small molecule modulators influencing the molecular feedback loop of the circadian clock by applying our two-step cell-based screening strategy based on E-box-mediated transcriptional activity to test more than 1000 drug-like compounds. A derivative of 2-ethoxypropanoic acid designated as compound 15 was selected as the most promising candidate in terms of both efficacy and potency. We then performed pull-down assays with the biotinylated compound and find out that both cryptochrome (CRY)1 and 2 (CRY1/2), key negative components of the mammalian circadian clock, as molecular targets of compound 15. In accordance with the binding property, compound 15 enhanced E-box-mediated transcription in a CRY1/2-dependent manner, and more importantly, it attenuated the circadian oscillation of Per2-Luc and Bmal1-dLuc activities in cultured fibroblasts, indicating that compound 15 can functionally inhibit the effects of CRY1/2 in the molecular circadian clockwork. In conclusion, the present study describes the first novel chemical inhibitor of CRY1/2 that inhibits the repressive function of CRY1/2, thereby activating CLOCK-BMAL1-evoked E-box-mediated transcription. Further optimizations and subsequent functional studies of this compound may lead to development of efficient therapeutic strategies for a variety of physiological and metabolic disorders with circadian natures.
Assuntos
Relógios Circadianos/efeitos dos fármacos , Criptocromos/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Criptocromos/genética , Relação Dose-Resposta a Droga , Elementos E-Box/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Luciferases/genética , Medições Luminescentes , Camundongos , Estrutura Molecular , Células NIH 3T3 , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , TransfecçãoRESUMO
Very recently, the circadian rhythm was proved to play an important role in the pathogenesis of arthritis. The role of melatonin in the development and progress of rheumatoid arthritis has been implicated for decades. This study was aimed to investigate the effect of melatonin on the expression of circadian clock genes in mouse anti-type II collagen antibody-induced arthritis (CIA). Mice were divided into 3 groups: control, CIA, and CIA + melatonin treatment (MLT). Both mRNA and protein levels of circadian clock gene Cryptochrome1 (Cry1) were markedly decreased in CIA + MEL group compared with those in control and CIA groups. MLT increased paw thickness. Histologic and X-ray assessment also revealed increased infiltration of inflammatory cells, synovial hyperplasia, and the destruction of articular cartilage and bone by MLT. The concentrations of anti-type II collagen antibody in CIA + MEL group mice were significantly higher than those in control and CIA groups (P < 0.05). Serum concentrations of TNF-α (P < 0.005) and IL-6 (P < 0.05) in CIA + MLT group were also increased. Taken together, these results implicate that clock gene Cry1 may be involved in the aggravation of MLT-mediated arthritis in mice anti-type II collagen antibody-induced arthritis.