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1.
Br J Cancer ; 122(2): 194-208, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31819176

RESUMO

BACKGROUND: Although produced by several types of tumours, the role of serotonin on cancer biology is yet to be understood. METHODS: The effects of serotonin (5-HT) on human breast cancer cells proliferation, signalling pathways and metabolic profile were evaluated by cytometry, western blotting, qPCR, enzymology and confocal microscopy. RESULTS: Our results revealed that incubation of MCF-7 cells with 10 µM 5-HT increased cell growth rate by 28%, an effect that was prevented by the 5-HTR2A/C antagonist, ketanserin. Conversely, increasing concentrations of 5-HT promoted glucose consumption and lactate production by MCF-7 cells. We also showed that increased glucose metabolism is provoked by the upregulation of pyruvate kinase M2 (PKM2) isoform through 5-HTR2A/C-triggered activation of Jak1/STAT3 and ERK1/2 subcellular pathways. However, we noticed a decrease in the rate of produced lactate per consumed glucose as a function of the hormone concentration, suggesting a disruption of the Warburg effect. The latter effect is due to 5-HTR2A/C-dependent mitochondrial biogenesis and metabolism, which is triggered by adenylyl cyclase/PKA, enhancing the oxidation of lactate within these cells. CONCLUSIONS: We showed that serotonin, through 5-HTR2A/C, interferes with breast cancer cells proliferation and metabolism by triggering two distinct signalling pathways: Jak1/STAT3 that boosts glycolysis through upregulation of PKM2, and adenylyl cyclase/PKA that enhances mitochondrial biogenesis.

2.
J Bioenerg Biomembr ; 50(2): 93-105, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29589262

RESUMO

Breast cancer is the major cause of death by cancer in women worldwide and in spite of the many drugs for its treatment, there is still the need for novel therapies for its control. Ocimum species have been used by traditional medicine to control several diseases, including cancer. We have previously characterized the antidiabetic properties of the unfractionated aqueous leaf extracts of Ocimum basilicum (OB) and Ocimum gratissimum (OG), modulating glucose metabolism in diabetic mice. Since glucose metabolism is primordial for cancer cells survival, we hypothesized that these extracts are effective against cancer cells. The unfractionated aqueous leaf extracts of OB and OG were chemically characterized and tested for their cytotoxic, cytostatic and anti-proliferative properties against the human breast cancer cell line MCF-7. Both extracts presented cytostatic effects with an 80% decrease in MCF-7 cell growth at 1 mg/mL. However, only OB promoted cytotoxic effects, interfering with the cell viability even after interruption of the treatment. Moreover, OB but not OG affected the cell proliferation and metabolism, evaluated in terms of lactate production and intracellular ATP content. After 24 h of treatment, OB treated cells presented an apoptotic profile, while OG treated cells were more necrotic. The treatment with both extracts also activated AMPK, but OB was much more efficient than OG in promoting this. The activation of mTOR signaling, another survival pathway was promoted by OB, whereas OG failed to activate it. In the end, we conclude that OB extract is efficient against the human breast cancer cell line.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Redes e Vias Metabólicas/efeitos dos fármacos , Ocimum basilicum/toxicidade , Ocimum/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo
3.
PLoS One ; 10(6): e0130555, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26098874

RESUMO

BACKGROUND: Although demonstrated as a selective anticancer drug, the clinical use of clotrimazole (CTZ) is limited due to its low solubility in hydrophilic fluids. Thus, we prepared a water-soluble nanomicellar formulation of CTZ (nCTZ) and tested on the human breast cancer cell line MCF-7 biology. METHODOLOGY/PRINCIPAL FINDINGS: CTZ was nanoencapsulated in tween 80 micelles, which generated nanomicelles of, approximately, 17 nm of diameter. MCF-7 cells were treated with nCTZ and unencapsulated DMSO-solubilized drug (sCTZ) was used for comparison. After treatment, the cells were evaluated in terms of metabolism, proliferation, survival and structure. We found that nCTZ was more efficient than sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes. Moreover, this increased activity was also observed for lactate production, intracellular ATP content, ROS production and antioxidant potential. As a consequence, nCTZ-treated MCF-7 cells displayed alterations to the plasma membrane, mitochondria and the nucleus. Finally, nCTZ induced both apoptosis and necrosis in MCF-7 cells. CONCLUSIONS/SIGNIFICANCE: MCF-7 cells are more sensible to nCTZ than to sCTZ. This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism. Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Clotrimazol/farmacologia , Nanopartículas/administração & dosagem , Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Antioxidantes/química , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica/métodos , Clotrimazol/química , Feminino , Glicólise/efeitos dos fármacos , Humanos , Ácido Láctico/metabolismo , Células MCF-7 , Micelas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanopartículas/química , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Solubilidade
4.
Int J Biochem Cell Biol ; 62: 132-41, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25794423

RESUMO

Clotrimazole (CTZ) has been proposed as an antitumoral agent because of its properties that inhibit glycolytic enzymes and detach them from the cytoskeleton. However, the broad effects of the drug, e.g., acting on different enzymes and pathways, indicate that CTZ might also affect several signaling pathways. In this study, we show that CTZ interferes with the human breast cancer cell line MCF-7 after a short incubation period (4 h), thereby diminishing cell viability, promoting apoptosis, depolarizing mitochondria, inhibiting key glycolytic regulatory enzymes, decreasing the intracellular ATP content, and permeating plasma membranes. CTZ treatment also interferes with autophagy. Moreover, when the incubation is performed under hypoxic conditions, certain effects of CTZ are enhanced, such as phosphatidylinositol-3-phosphate kinase (PI3K), which is inhibited upon CTZ treatment; this inhibition is potentiated under hypoxia. CTZ-induced PI3K inhibition is not caused by upstream effects of CTZ because the drug does not affect the interaction of the PI3K regulatory subunit and the insulin receptor substrate (IRS)-1. Additionally, CTZ directly inhibits human purified PI3K in a dose-dependent and reversible manner. Pharmacologic and in silico results suggest that CTZ may bind to the PI3K catalytic site. Therefore, we conclude that PI3K is a novel, putative target for the antitumoral effects of CTZ, interfering with autophagy, apoptosis, cell division and viability.


Assuntos
Antineoplásicos/farmacologia , Clotrimazol/farmacologia , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Células MCF-7 , Neoplasias/metabolismo
5.
IUBMB Life ; 66(5): 361-70, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24817132

RESUMO

The aim of this study was to evaluate the antidiabetic potential of a leaf extract and flavonoids from Sedum dendroideum (SD). Additionally, our goals were to establish a possible structure/activity relationship between these flavonoids and to assess the most active flavonoid on the glycolytic enzyme 6-phosphofructo-1-kinase (PFK). SD juice (LJ), a flavonoid-rich fraction (BF), and separately five flavonoids were evaluated intraperitoneally for their acute hypoglycemic activity in normal and streptozotocin-induced diabetic mice. First, the major flavonoids kaempferol 3,7-dirhamnoside or kaempferitrin (1), kaempferol 3-glucoside-7-rhamnoside (2), and kaempferol 3-neohesperidoside-7-rhamnoside (3) were tested. Then, the monoglycosides kaempferol 7-rhamnoside (5) and kaempferol 3-rhamnoside (6) were assayed to establish their structure/activity relationship. The effect of 1 on PFK was evaluated in skeletal muscle, liver, and adipose tissue from treated mice. LJ (400 mg/kg), BF (40 mg/kg), and flavonoid 1 (4 mg/kg) reduced glycemia in diabetic mice (120 min) by 52, 53, and 61%, respectively. Flavonoids 2, 3, 5, and 6 were inactive or showed little activity, suggesting that the two rhamnosyl moieties in kaempferitrin are important requirements. Kaempferitrin enhanced the PFK activity chiefly in hepatic tissue, suggesting that it is able to stimulate tissue glucose utilization. This result is confirmed testing kaempferitrin on C2C12 cell line, where it enhanced glucose consumption, lactate production, and the key regulatory glycolytic enzymes. The hypoglycemic activity of kaempferitrin depends on the presence of both rhamnosyl residues in the flavonoid structure when intraperitoneally administered. Our findings show for the first time that a flavonoid is capable of stimulating PFK in a model of diabetes and that kaempferitrin stimulates glucose-metabolizing enzymes. This study contributes to the knowledge of the mechanisms by which this flavonoid exerts its hypoglycemic activity.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Quempferóis/farmacologia , Fosfofrutoquinases/metabolismo , Extratos Vegetais/farmacologia , Sedum/química , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Diabetes Mellitus Experimental/enzimologia , Avaliação Pré-Clínica de Medicamentos , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/uso terapêutico , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/enzimologia , Quempferóis/isolamento & purificação , Quempferóis/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Mioblastos/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico
6.
Biochimie ; 95(6): 1336-43, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23454376

RESUMO

Cancer cells are highly dependent on glycolysis to supply the energy and intermediates required for cell growth and proliferation. The enzyme 6-phosphofructo-1-kinase (PFK) is critical for glycolysis, and its activity is directly correlated with cellular glucose consumption. Resveratrol is a potential anti-tumoral drug that decreases glucose metabolism and viability in cancer cells. However, the mechanism involved in resveratrol-mediated anti-tumor activity is not entirely clear. In this work, it is demonstrated that resveratrol decreases viability, glucose consumption and ATP content in the human breast cancer cell line MCF-7. These effects are directly correlated with PFK inhibition by resveratrol in these cells. Moreover, resveratrol directly inhibits purified PFK, promoting the dissociation of the enzyme from fully active tetramers into less active dimers. This effect is exacerbated by known negative regulators of the enzyme, such as ATP and citrate. On the other hand, positive modulators that stabilize the tetrameric form of the enzyme, such as fructose-2,6-bisphosphate and ADP, prevent the inhibition of PFK activity by resveratrol, an effect not observed with increased pH. In summary, our results provide evidence that resveratrol directly inhibits PFK activity, therefore disrupting glucose metabolism and reducing viability in cancer cells.


Assuntos
Antioxidantes/farmacologia , Neoplasias da Mama/metabolismo , Glucose/metabolismo , Fosfofrutoquinase-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estilbenos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Resveratrol
7.
PLoS One ; 7(2): e30462, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22347377

RESUMO

BACKGROUND: Clotrimazole is an azole derivative with promising anti-cancer effects. This drug interferes with the activity of glycolytic enzymes altering their cellular distribution and inhibiting their activities. The aim of the present study was to analyze the effects of clotrimazole on the growth pattern of breast cancer cells correlating with their metabolic profiles. METHODOLOGY/PRINCIPAL FINDINGS: Three cell lines derived from human breast tissue (MCF10A, MCF-7 and MDA-MB-231) that present increasingly aggressive profiles were used. Clotrimazole induces a dose-dependent decrease in glucose uptake in all three cell lines, with K(i) values of 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231, respectively. Furthermore, the drug also decreases intracellular ATP content and inhibits the major glycolytic enzymes, hexokinase, phosphofructokinase-1 and pyruvate kinase, especially in the highly metastatic cell line, MDA-MB-231. In this last cell lineage, clotrimazole attenuates the robust migratory response, an effect that is progressively attenuated in MCF-7 and MCF10A, respectively. Moreover, clotrimazole reduces the viability of breast cancer cells, which is more pronounced on MDA-MB-231. CONCLUSIONS/SIGNIFICANCE: Clotrimazole presents deleterious effects on two human breast cancer cell lines metabolism, growth and migration, where the most aggressive cell line is more affected by the drug. Moreover, clotrimazole presents little or no effect on a non-tumor human breast cell line. These results suggest, at least for these three cell lines studied, that the more aggressive the cell is the more effective clotrimazole is.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Clotrimazol/farmacologia , Glicólise/efeitos dos fármacos , Antineoplásicos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clotrimazol/uso terapêutico , Feminino , Humanos , Metaboloma
8.
Int. j. high dilution res ; 10(36): 170-171, september 30, 2011.
Artigo em Inglês | LILACS-Express | ID: hom-10710

RESUMO

Biotherapics are homeopathic remedies prepared from organic products that are chemically undefined and can be used for treatment of diseases like influenza. There are several classes of biotherapics and, among these, there are some called "living biotherapics" or "Roberto Costa?s Biotherapics". This study aimed to compare the cellular and biochemical effects of biotherapics prepared from intact influenza virus diluted in water and the one obtained from the same viral sample inactivated by ethanol 70% (v / v), both in the potencies of 12x and 30x. Transmission electron microscopy (TEM) analyses were performed on both preparations to assess the integrity of viral particles, which showed that ethanol 70% (v/v) induced a complete denaturation of viral particles. In contrast, the integrity of virus particles was preserved when water was used as the biotherapic solvent. Cellular and biochemical alterations induced by the preparations on MDCK cells were analyzed and compared with those induced by respective controls (water 30x-treated and untreated cells). Cellular viability analyzed by MTT method showed statistically significant differences (p <0.05) in MDCK cells treated with intact biotherapic for 5 (3 stimuli) and 30 (18 stimuli) days in comparison with untreated control. TEM analysis did not show significant cellular changes when the different experimental groups were compared. The enzymatic activity of phosphofructokinase 1 (PFK), an important enzyme in the glycolytic pathway, presented a statistically significant increase (p <0.05) after 30 days of treatment when compared to control groups. The results obtained suggest that inactivation of viral sample with ethanol 70% induces lysis and disruption of viral particles. In addition, preliminary results indicated that treatment with intact biotherapic seems to induce higher variations on MDCK cells responses when compared to inactivated-biotherapic-treated cells. Further analyses are ongoing, including scanning electron microscopy and quantification of the number of mitosis, in order to elucidate the mechanisms involved with biochemical and cellular responses induced by theses biotherapics.(AU)


Assuntos
Influenzavirus A , Bioterápicos
9.
IUBMB Life ; 63(6): 435-45, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21698747

RESUMO

6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism.


Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Glicólise , Fosfofrutoquinase-1/metabolismo , Regulação Alostérica , Animais , Antifúngicos/metabolismo , Catálise , Clotrimazol/metabolismo , Dimerização , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Frutose-Bifosfato Aldolase/química , Músculo Esquelético/enzimologia , Fosfofrutoquinase-1/antagonistas & inibidores , Fosfofrutoquinase-1/química , Conformação Proteica , Coelhos , Espectrometria de Fluorescência
10.
Arch Biochem Biophys ; 497(1-2): 62-7, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20346906

RESUMO

Clotrimazole (CTZ) has been proposed as a potential anti-neoplastic agent, which inhibits glucose metabolism. The present work aimed to evaluate the effects of CTZ on the kinetic mechanism of 6-phosphofructo-1-kinase (PFK). We show that CTZ promotes a dose-dependent inhibition of PFK, presenting a K(i) of 28 +/- 2 microM. Inhibition occurs through the dissociation of the enzyme tetramers, as demonstrated through fluorescence spectroscopy and gel filtration chromatography. Moreover, the affinities of the enzyme for ATP and fructose-6-phosphate are reduced 50% and 30%, respectively. Furthermore, the affinity of PFK for ATP at the inhibitory site becomes 2-fold higher. Altogether, the results presented here suggest that PFK inhibition by CTZ involves a decrease in the affinity of PFK for its substrates at the catalytic site with the concomitant potentiation of the inhibitory properties of ATP.


Assuntos
Trifosfato de Adenosina/metabolismo , Antifúngicos/metabolismo , Clotrimazol/metabolismo , Fosfofrutoquinase-1/antagonistas & inibidores , Regulação Alostérica , Dimerização , Frutosefosfatos/metabolismo , Glicólise , Cinética , Ligação Proteica , Especificidade por Substrato
11.
Biochem Pharmacol ; 73(10): 1520-7, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17291460

RESUMO

Clotrimazole is an antifungal azole derivative recently recognized as a calmodulin antagonist with promising anticancer effects. This property has been correlated with the ability of the drug to decrease the viability of tumor cells by inhibiting their glycolytic flux and consequently decreasing the intracellular concentration of ATP. The effects of clotrimazole on cell glycolysis and ATP production are considered to be due to the detachment of the glycolytic enzymes from the cytoskeleton. Here, we show that clotrimazole directly inhibits the key glycolytic enzyme 6-phosphofructo-1-kinase (PFK). This property is independent of the anti-calmodulin activity of the drug, since it is not mimicked by the classical calmodulin antagonist compound 48/80. However, the clotrimazole-inhibited enzyme can be activated by calmodulin, even though calmodulin has no effect on PFK activity in the absence of the drug. Clotrimazole alone induces the dimerization of PFK reducing the population of tetramers, which is not observed when calmodulin is also present. Since PFK dimers are less active than PFK tetramers, this can explain the inhibitory effect of clotrimazole on the enzyme. Additionally, clotrimazole positively modulates the association of PFK with erythrocyte membranes. Altogether, our data support a hitherto unrecognized action of clotrimazole as a negative modulator of glycolytic flux through direct inhibition of the key enzyme PFK.


Assuntos
Clotrimazol/farmacologia , Membrana Eritrocítica/metabolismo , Glicólise/efeitos dos fármacos , Fosfofrutoquinases/metabolismo , Antifúngicos/farmacologia , Calmodulina/metabolismo , Citoesqueleto/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Glucose-6-Fosfato/metabolismo , Humanos , Fosfofrutoquinases/química , Fosfofrutoquinases/efeitos dos fármacos , Conformação Proteica , p-Metoxi-N-metilfenetilamina/farmacologia
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