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1.
Neurosci Lett ; 638: 189-195, 2017 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-28007652

RESUMO

Glioblastoma is the most aggressive primary brain tumor. Surgical resection, radiotherapy and temozolomide (TMZ), an alkylating agent, is the standard of care. Glioma cells may synthetize the antioxidant glutathione by importing cystine through a cystine/glutamate antiporter, which is inhibited by sulfasalazine (SAS). C6 rat glioma cells are largely used in in vitro and in vivo models for developing new glioblastoma treatment strategies. We treated C6 cells with 25µM TMZ and/or 0.25mM or 0.5mM SAS for 1, 3 or 5days and evaluated viability, apoptosis, total glutathione levels and metalloproteinase MMP2 and MMP9 activities. TMZ treatment slightly reduced cell viability by 9.5% compared with vehicle treatment (0.1% dimethyl sulfoxide) only after 5days. In addition, TMZ did not modify apoptosis, glutathione content or MMP2/MMP9 activities. The 0.25mM SAS treatment reduced cell viability by 31.1% and 19.4% after the first and third days, respectively. This effect was not sustained after the fifth day of treatment. In contrast, 0.5mM SAS caused a reduction in cell viability by nearly 100%, total glutathione depletion and apoptosis induction. Moreover, the effect of 0.5mM SAS was greater than that of TMZ in terms of cell viability reduction, total glutathione depletion and apoptosis induction. MMP9 activity was reduced by 40% after 5days of 25µM TMZ and 0.5mM SAS co-administration. Considering previous data from our group, we verified that the cellular viability results differed between rat and human cells; C6 cells were more vulnerable to 0.5mM SAS than human A172 and T98G glioblastoma lineages. We propose that C6 cells may not be appropriate for studying human glioblastoma and that the results obtained using these cells should be interpreted with caution.


Assuntos
Antineoplásicos/farmacologia , Sulfassalazina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Glioma , Glutationa/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Temozolomida
2.
Molecules ; 19(4): 5360-78, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24879583

RESUMO

Neglected agricultural products (NAPs) are defined as discarded material in agricultural production. Corn cobs are a major waste of agriculture maize. Here, a methanolic extract from corn cobs (MEC) was obtained. MEC contains phenolic compounds, protein, carbohydrates (1.4:0.001:0.001). We evaluated the in vitro and in vivo antioxidant potential of MEC. Furthermore, its antiproliferative property against tumor cells was assessed through MTT assays and proteins related to apoptosis in tumor cells were examined by western blot. MEC showed no hydroxyl radical scavenger capacity, but it showed antioxidant activity in Total Antioxidant Capacity and DPPH scavenger ability assays. MEC showed higher Reducing Power than ascorbic acid and exhibited high Superoxide Scavenging activity. In tumor cell culture, MEC increased catalase, metallothionein and superoxide dismutase expression in accordance with the antioxidant tests. In vivo antioxidant test, MEC restored SOD and CAT, decreased malondialdehyde activities and showed high Trolox Equivalent Antioxidant Capacity in animals treated with CCl4. Furthermore, MEC decreased HeLa cells viability by apoptosis due an increase of Bax/Bcl-2 ratio, caspase 3 active. Protein kinase C expression increased was also detected in treated tumor cells. Thus, our findings pointed out the biotechnological potential of corn cobs as a source of molecules with pharmacological activity.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Extratos Vegetais/farmacologia , Zea mays/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/isolamento & purificação , Células HeLa , Humanos , Concentração Inibidora 50 , Metalotioneína/metabolismo , Metanol/química , Oxirredução , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ratos Wistar , Extração em Fase Sólida , Solventes/química , Superóxido Dismutase/metabolismo , Superóxidos/química
3.
Biometals ; 27(1): 183-94, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24442571

RESUMO

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG-DTA) analyses, and FT-IR and UV-Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC50 values below 5.7 µM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.


Assuntos
Antiprotozoários/farmacologia , Benzimidazóis/química , Leishmania/efeitos dos fármacos , Compostos Organoáuricos/síntese química , Compostos Organoáuricos/farmacologia , Células 3T3 , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antiprotozoários/síntese química , Antiprotozoários/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Compostos Organoáuricos/química , Teoria Quântica , Relação Estrutura-Atividade
4.
Neurochem Res ; 37(9): 1967-81, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22684283

RESUMO

Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5 h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45 min); however the phosphorylation level returned to baseline after 2 h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Junção Neuromuscular/metabolismo , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Western Blotting , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imunofluorescência , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Laminina/biossíntese , Masculino , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/efeitos dos fármacos , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Fosforilação , Ratos , Ratos Wistar
5.
J Pharm Pharmacol ; 64(6): 832-42, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22571261

RESUMO

OBJECTIVES: To investigate the molecular interaction between ß-cyclodextrin (ßCD) or hydroxypropyl-ß-cyclodextrin (HPßCD) and riboflavin (RF), and to test the anticancer potential of these formulations. METHODS: The physicochemical characterization of the association between RF and CDs was performed by UV-vis absorption, fluorescence, differential scanning calorimetry and NMR techniques. Molecular dynamics simulation was used to shed light on the mechanism of interaction of RF and CDs. Additionally, in-vitro cell culture tests were performed to evaluate the cytotoxicity of the RF-CD complexes against prostate cancer cells. KEY FINDINGS: Neither ßCD nor HPßCD led to substantial changes in the physicochemical properties of RF (with the exception of solubility). Additionally, rotating frame Overhauser effect spectroscopy experiments detected no spatial correlations between hydrogens from the internal cavity of CDs and RF, while molecular dynamics simulations revealed 'out-of-ring' RF-CD interactions. Notwithstanding, both RF-ßCD and RF-HPßCD complexes were cytotoxic to PC3 prostate cancer cells. CONCLUSIONS: The interaction between RF and either ßCD or HPßCD, at low concentrations, seems to be made through hydrogen bonding between the flavonoid and the external rim of both CDs. Regardless of the mechanism of complexation, our findings indicate that RF-CD complexes significantly increase RF solubility and potentiate its antitumour effect.


Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Neoplasias da Próstata/tratamento farmacológico , Riboflavina/química , beta-Ciclodextrinas/química , 2-Hidroxipropil-beta-Ciclodextrina , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Ligação de Hidrogênio , Masculino , Riboflavina/farmacologia , Riboflavina/uso terapêutico , Solubilidade
6.
Dev Growth Differ ; 53(1): 88-96, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21261614

RESUMO

MC3T3-E1 cells grown in the presence of ascorbic acid and ß-glycerophosphate (AA/ß-GP) express alkaline phosphatase and produce an extensive collagenous extracellular matrix. Differentiated MC3T3-E1 cells are more sensitive to hydrogen peroxide-induced oxidative stress than undifferentiated cells. In this study, we compared the profile of antioxidant enzymes and molecular markers of apoptosis in undifferentiated and differentiated MC3T3-E1 cells (cell differentiation was induced by treatment with AA/ß-GP). Differentiated osteoblasts showed lower expression and activity of catalase, glutathione S-transferase and glutathione peroxidase. The total superoxide dismutase activity and the expression of Cu/Zn superoxide dismutase were also lower, while the expression of Mn superoxide dismutase was higher in differentiated osteoblasts. The level of malondialdehyde, a widely used marker for oxidative stress, was lower in the AA/ß-GP group compared with control cells, but this difference was not significant. Western blotting showed that treatment with AA/ß-GP increased the Bax/Bcl-2 ratio used as an index of cellular vulnerability to apoptosis. In addition, the activities of caspases 3, 8 and 9 and cleaved poly (ADP) ribose polymerase were significantly higher in differentiated cells. These findings provide new insights into how changes in the activities of major antioxidant enzymes and in the signaling pathways associated with apoptosis may influence the susceptibility of bone cells to oxidative stress.


Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Glicerofosfatos/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Peróxido de Hidrogênio/farmacologia , Camundongos
7.
J Cell Biochem ; 112(1): 71-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20626033

RESUMO

Despite numerous reports on the ability of ascorbic acid and ß-glycerophosphate (AA/ß-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/ß-GP-induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/ß-GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR-1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood.


Assuntos
Ácido Ascórbico/metabolismo , Diferenciação Celular , Glicerofosfatos/metabolismo , Osteoblastos/citologia , Transdução de Sinais , Animais , Células Cultivadas , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fosforilação
8.
Toxicol In Vitro ; 24(7): 1911-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20688149

RESUMO

Photoderivatives of riboflavin can modulate the proliferation and survival of cancer cells. In this work, we examined the influence of riboflavin and photoderivatives on osteoblast differentiation induced by ascorbic acid and ß-glycerophosphate. These compounds decreased the osteoblast proliferation, increased the alkaline phosphatase activity, promoted a reduction in matrix metalloproteinase-2 activity and the decreased in the OPG/RANKL ratio. The effects of flavins on osteoblasts were unrelated to the antioxidant activity of these compounds. The biological activity of osteogenic medium containing riboflavin and its photoderivatives involved the activation of different signaling pathways (AKT, FAK, CaMKII), caspases-3, -8 and -9, and up-regulation of the expression and/or stabilization of osteoblastic transcription factors (Runx2 and ß-catenin). These findings suggest a potential use of flavins as adjuvants to improve bone metabolism.


Assuntos
Flavinas/farmacologia , Fotólise , Riboflavina/farmacologia , Complexo Vitamínico B/farmacologia , Células 3T3 , Animais , Caspases/efeitos dos fármacos , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos , Osteoblastos/metabolismo , Riboflavina/química , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Complexo Vitamínico B/química
10.
J Cell Biochem ; 108(1): 134-44, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19562668

RESUMO

Pre-osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre-osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell-cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho-PP2A (at Tyr307). Also, it is plausible to suggest during pre-osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho-Src (Y416). In addition, it was observed that MAPKp38, but not MAPK-erk, played a key role on pre-osteoblast adhesion by phosphorylating MAPKAPK-2 and ATF-2 (also called CRE-BP1). Also, the up-modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement.


Assuntos
Citoesqueleto/metabolismo , Osteoblastos/citologia , Transdução de Sinais , Animais , Adesão Celular , Ciclo Celular , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Modelos Biológicos , Osteoblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismo
11.
Biochimie ; 90(6): 843-54, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18294971

RESUMO

Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular basis for these actions remains only partly understood and hence we investigated the effects of ferruginol on androgen-independent human prostate cancer cells (PC3), a known model for solid tumor cells with an exceptional resistance to therapy. The results show that ferruginol induces PC3 cell death via activation of caspases as well as apoptosis-inducing factor (AIF) as confirmed by its translocation into the nucleus. In order to clarify the biochemical mechanism responsible for the anti-tumor activity of ferruginol, we analyzed a set of molecular mediators involved in tumor cell survival, progression and aggressiveness. Ferruginol was able to trigger inhibition/downregulation of Ras/PI3K, STAT 3/5, protein tyrosine phosphatase and protein kinases related to cell cycle regulation. Importantly, the toxic effect of ferruginol was dramatically impeded in a more reducing environment, which indicates that at least in part, the anti-tumoral activity of ferruginol might be related to redox status modulation. This study supports further examination of ferruginol as a potential agent for both the prevention and treatment of prostate cancer.


Assuntos
Abietanos/toxicidade , Antineoplásicos/toxicidade , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacos , Abietanos/química , Androgênios , Antineoplásicos/química , Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Masculino , Oxirredução , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição STAT/metabolismo
12.
Cancer Lett ; 258(1): 126-34, 2007 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-17933458

RESUMO

Riboflavin is a potent photosensitizer as well as part of the vitamin B complex. Recently we demonstrated that the products generated by irradiation of riboflavin have potential as anti-leukaemic therapy. The possible action, however, of the riboflavin photoproducts in solid cancers has not been addressed. Hence, we investigated the effects of irradiated riboflavin on androgen-independent human prostate cancer cells (PC3), a known model for solid tumour cells with an exceptional resistance to therapy. Our results show that riboflavin photoproducts are cytotoxic to these cells in a FasL-Fas-dependent manner. Furthermore, irradiated riboflavin inhibited matrix-degrading proteases, caused downregulation of VEGF and upregulation of TIMP1 suggesting anti-metastatic potential. Together, these results show that the anti-neoplastic action of riboflavin photoproducts is not limited to haematological malignancies, warranting clinical studies in solid tumours.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Fármacos Fotossensibilizantes/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Riboflavina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Células Cultivadas/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Neoplasias Hormônio-Dependentes/secundário , Próstata/citologia , Próstata/efeitos dos fármacos , Próstata/metabolismo , Ratos
13.
J Enzyme Inhib Med Chem ; 22(4): 439-44, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17847710

RESUMO

Flavonoids, polyphenolic phytochemicals, are ubiquitous in plants and are commonly present in the human diet. They may exert diverse beneficial effects, including antioxidant and anticarcinogenic activities. The present study was designed to evaluate three biomolecules that play important roles in the apoptotic process: mitogen-activated protein kinases, protein phosphatases and NFkappaB, using HL60 cells treated with fisetin as an experimental model. Our results demonstrated that cells treated with fisetin presented high expression of NFkappaB, activation of MAPK p38 and an increase of phosphoprotein levels; inhibition of enzymes involved in redox status maintenance were also observed. Our findings reinforce the hypothesis that fisetin is likely to exert beneficial and/or toxic actions on cells not through its potential as antioxidant but rather through its modulation of protein kinase and phosphatase signaling cascades. Additionally, our results also indicate that the cellular effects of fisetin will ultimately depend on the cell type and on the extent to which they associate with the cells, either by interactions at the membrane or by uptake into the cytosol.


Assuntos
Flavonoides/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/biossíntese , Fosfoproteínas/química , Antioxidantes/química , Antioxidantes/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavonóis , Células HL-60 , Humanos , Linfócitos/metabolismo , Modelos Químicos , Estresse Oxidativo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Apoptosis ; 11(10): 1761-71, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16927017

RESUMO

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at microM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.


Assuntos
Morte Celular/efeitos dos fármacos , Leucemia/patologia , Riboflavina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Luz , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Riboflavina/efeitos da radiação , Riboflavina/uso terapêutico
15.
Exp Hematol ; 34(2): 188-96, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16459187

RESUMO

OBJECTIVE: Tetrahydroxyquinone is a molecule best known as a primitive anticataract drug but is also a highly redox active molecule that can take part in a redox cycle with semiquinone radicals, leading to the formation of reactive oxygen species (ROS). Its potential as an anticancer drug has not been investigated. METHODS: The effects of tetrahydroxyquinone on HL60 leukemia cells are investigated using fluorescein-activated cell sorting-dependent detection of phosphatidylserine exposure combined with 7-amino-actinomycin D exclusion, via Western blotting using phosphospecific antibodies, and by transfection of constitutively active protein kinase B. RESULTS: We observe that in HL60 leukemia cells tetrahydroxyquinone causes ROS production followed by apoptosis through the mitochondrial pathway, whereas cellular physiology of normal human blood leukocytes was not affected by tetrahydroxyquinone. The antileukemic effect of tetrahydroxyquinone is accompanied by reduced activity of various antiapoptotic survival molecules including the protein kinase B pathway. Importantly, transfection of protein kinase B into HL60 cells and thus artificially increasing protein kinase B activity inhibits tetrahydroxyquinone-dependent cytotoxicity. CONCLUSION: Tetrahydroxyquinone provokes cytotoxic effects on leukemia cells by reduced protein kinase B-dependent survival signaling followed by apoptosis through the mitochondrial pathway. Thus, tetrahydroxyquinone may be representative of a novel class of chemotherapeutic drugs, inducing apoptosis in cancer cells through diminished survival signaling possibly as a consequence of ROS generation.


Assuntos
Apoptose/efeitos dos fármacos , Hidroquinonas/farmacologia , Leucemia/tratamento farmacológico , Quinonas/farmacologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células HL-60 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Quinonas/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
16.
Mol Cell Biochem ; 265(1-2): 133-40, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15543943

RESUMO

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site.


Assuntos
Brometos/farmacologia , Proteínas Tirosina Fosfatases/química , Sulfatos/farmacologia , Animais , Ânions , Bioquímica/métodos , Brometos/química , Cátions , Bovinos , Relação Dose-Resposta a Droga , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Cloreto de Sódio/química , Eletricidade Estática , Sulfatos/química , Ésteres do Ácido Sulfúrico , Tensoativos/química , Tensoativos/farmacologia , Fatores de Tempo
17.
Blood ; 104(5): 1459-64, 2004 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15130948

RESUMO

Violacein, a pigment isolated from Chromobacterium violaceum in the Amazon River, presents diverse biologic properties and attracts interest as a consequence of its antileukemic activity. Elucidation of the molecular mechanism mediating this activity will provide further relevant information for understanding its effects on the cellular physiology of untransformed cells and for considering its possible clinical application. Here, we show that violacein causes apoptosis in HL60 leukemic cells but is ineffective in this respect in other types of leukemia cells or in normal human lymphocytes and monocytes. Violacein cytotoxicity in HL60 cells was preceded by activation of caspase 8, transcription of nuclear factor kappaB (NF-kappaB) target genes, and p38 mitogen-activated protein (MAP) kinase activation. Thus, violacein effects resemble tumor necrosis factor alpha (TNF-alpha) signal transduction in these cells. Accordingly, infliximab, an antibody that antagonizes TNF-alpha-induced signaling abolished the biologic activity of violacein. Moreover, violacein directly activated TNF receptor 1 signaling, because a violacein-dependent association of TNF receptor-associated factor 2 (TRAF2) to this TNF receptor was observed in coimmunoprecipitation experiments. Hence, violacein represents the first member of a novel class of cytotoxic drugs mediating apoptosis of HL60 cells by way of the specific activation of TNF receptor 1.


Assuntos
Morte Celular/efeitos dos fármacos , Indóis/farmacologia , Leucemia Promielocítica Aguda , Antígenos CD/metabolismo , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HL-60 , Humanos , Células K562 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Células U937 , Regulação para Cima/efeitos dos fármacos
18.
Phytochemistry ; 65(7): 831-6, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15081282

RESUMO

In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaGu, was calculated at 25 degrees C as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole.


Assuntos
Fosfatase Ácida/química , Glycine max/enzimologia , Guanidina/química , Ureia/química , Fosfatase Ácida/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Sementes/enzimologia , Espectrometria de Fluorescência , Termodinâmica
19.
Plant Physiol Biochem ; 42(1): 15-20, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15061079

RESUMO

In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.


Assuntos
Fosfatase Ácida/metabolismo , Glycine max/enzimologia , Glycine max/crescimento & desenvolvimento , Escuridão , Germinação , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Cinética , Nitrofenóis/metabolismo , Organofosfatos/química , Organofosfatos/metabolismo , Compostos Organofosforados/metabolismo , Fosfatos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Especificidade por Substrato , Temperatura
20.
Mol Cell Biochem ; 266(1-2): 11-5, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15646022

RESUMO

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.


Assuntos
Fosfatase Ácida/química , Nitrofenóis/química , Compostos Organofosforados/química , Proteínas de Plantas/química , Ricinus communis/enzimologia , Sementes/enzimologia , Estabilidade Enzimática , Temperatura Alta , Fosfatos/química , Desnaturação Proteica , Termodinâmica
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