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1.
J Thromb Thrombolysis ; 51(3): 625-632, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32803738

RESUMO

The impact of inhibition of multidrug resistance protein 4 (MRP4) on nitric oxide (NO) resistance and on ADP-induced platelet aggregation is unknown. The aim of this investigation was to verify whether platelet NO resistance correlates with MRP4 expression and evaluate whether this can be reduced by in vitro MRP4 inhibition mediated by cilostazol. Moreover, we assessed if inhibition of MRP4-mediated transport reduces ADP-induced platelet reactivity. The inhibitory effect of sodium nitroprusside (SNP), a NO-donor that enhances cyclic guanosine monophosphate (cGMP) cytosolic concentration, was assessed in platelets obtained from aspirin treated patients and in a control population. The inhibitory effect of SNP was evaluated by ADP-induced aggregation in SNP-treated platelets. The impact of MRP4 on ADP-induced platelet aggregation was performed in high on aspirin residual platelet reactivity (HARPR) patients and compared to healthy volunteers (HV), and a control cohort (CTR). In aspirin-treated patients with high levels of MRP4, reduced SNP inhibition was found compared to those with low levels of MRP4. MRP4 inhibition by cilostazol significantly reduced ADP-induced platelet aggregation in HARPR population, and to a lesser extent in HV and CTR populations. In conclusion, cilostazol can mitigate the hyper-reactive platelet phenotype of HARPR patients by reducing residual ADP-induced platelet aggregation and increasing NO-dependent endothelial antiplatelet effects.


Assuntos
Aspirina/farmacologia , Plaquetas , Cilostazol/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Óxido Nítrico/metabolismo , Ativação Plaquetária , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Perfilação da Expressão Gênica , Humanos , Nitroprussiato/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Inibidores da Agregação Plaquetária/farmacologia
4.
Thromb Haemost ; 119(5): 726-734, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30759486

RESUMO

Chronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y1 receptor exerts its effects via a Gq-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y1 expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y1 mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y1 mRNA expression was higher than in mock culture. P2Y1 expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y1 expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y1 up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y1 is coupled to the same Gq-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway.


Assuntos
Aspirina/uso terapêutico , Plaquetas/fisiologia , Células Progenitoras de Megacariócitos/fisiologia , Inibidores da Agregação Plaquetária/uso terapêutico , Receptores Purinérgicos P2Y1/metabolismo , Difosfato de Adenosina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , PPAR alfa/agonistas , Ativação Plaquetária , Agregação Plaquetária , Pirimidinas/farmacologia , Receptores Purinérgicos P2Y1/genética
5.
J Cardiovasc Med (Hagerstown) ; 19(10): 611-613, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30015780

RESUMO

BACKGROUND: Multidrug resistance protein-4 (MRP4) is an ATP binding cassette membrane transporter, actively involved in the efflux of important pharmacological and physiological molecules. Recently, its over-expression has been associated with reduced aspirin (ASA) efficacy after by-pass surgery. MicroRNAs (miRNAs) are small molecules of non-coding RNA involved in the regulation of many physiological and pathophysiological pathways, are abundant in platelets, and can be modulated by several drugs. In the present study, we assessed the role of platelet miRNAs in modulating MRP4 function in response to ASA. METHODS: MRP4 mRNA expression has been analyzed by RealTime PCR in platelets from patients on chronic ASA treatment versus a control group. A panel of miRNAs was run on the pool of each cohort. MiRNAs validation was performed by RealTime PCR. To verify whether MRP4 is the target of miR-26b also in platelets, miR-26b was transfected in platelet and DAMI cells with miRNA mimic technology. MRP4 expression was evaluated by flow cytometry and western blotting. RESULTS: We observed a higher MRP4 mRNA expression in platelets of patients under ASA treatment compared to the control group (p<0.005). MiR-26b was found significantly down-regulated in patients on ASA treatment as compared to control group (P < 0.005) and this was validated by RealTime PCR. MiR-26b transfection in platelets was associated to a significant down-regulation of MRP4 expression (p<0.005). MiR-26b transfection in DAMI cells was associated to a significant reduction of MRP4 mRNA and protein level (P < 0.05). CONCLUSION: We found that miR-26b is down-regulated in platelets in patients on chronic ASA treatment. Importantly, miR-26b can specifically downregulate MRP4. Thus, miR-26b seems to be involved in MRP4 modulation and may contribute to ASA resistance.


Assuntos
Aspirina/administração & dosagem , Plaquetas/efeitos dos fármacos , Resistência a Medicamentos , MicroRNAs/sangue , Proteínas Associadas à Resistência a Múltiplos Medicamentos/sangue , Inibidores da Agregação Plaquetária/administração & dosagem , Aspirina/efeitos adversos , Plaquetas/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Regulação para Baixo , Esquema de Medicação , Resistência a Medicamentos/genética , Humanos , MicroRNAs/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Inibidores da Agregação Plaquetária/efeitos adversos , Fatores de Tempo
6.
Res Pract Thromb Haemost ; 2(3): 596-606, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30046765

RESUMO

BACKGROUND: A mechanism involved in high on-aspirin treatment residual platelet reactivity is platelet multidrug resistance protein 4 (MRP4) overexpression. Aspirin enhances platelet MRP4 expression with a PPARα-dependent mechanism and reduces miR-21 expression that, in turn, downregulates PPARα expression. OBJECTIVE: The aim of our study was to verify the relationship between miR-21 and MRP4-PPARα levels induced by aspirin treatment. METHODS: We evaluated the changes in MRP4-PPARα, mRNA, MRP4 protein, and miR-21 expression induced by aspirin in: (i) in vitro-treated megakaryoblastic cell line (DAMI), (ii) primary megakaryocytes cultures and derived platelets, (iii) healthy volunteers' platelets treated with aspirin, and (iv) aspirinated patients (aspirin-treated patients) and in a control population (control). RESULTS: We observed an aspirin-induced reverse relationship between the expression of miR-21 and PPARα-MRP4. In DAMI cells the miR-21 mimic transfection reduces PPARα and MRP4 expression, even if cells were treated with aspirin after transfection. MiR-21 inhibitor transfection induces PPARα and MRP4 expression that are not enhanced by aspirin treatment. In human megakaryocytes, aspirin treatment lead to a miR-21 downregulation and a MRP4 upregulation and this trend is confirmed in derived platelets. In aspirin-treated volunteers, an inverse relationship between miR-21 and MRP4 platelet expression was found after aspirin treatment. A similar negative relationship was found in aspirin-treated patients vs the control population. CONCLUSION: The results reported in this study provide information that aspirin induces the modulation of platelet miR-21 expression levels and this modulation can be responsible for MRP4 enhancement in circulating platelets.

7.
Thromb Haemost ; 118(3): 490-501, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29448294

RESUMO

Platelet multidrug resistance protein 4 (MRP4) plays a modulating role on platelet activation. Platelet function and thrombus formation are impaired in MRP4 knockout mice models, and, among aspirin-treated patients, high on-aspirin residual platelet reactivity (HARPR) positively correlates with MRP4 levels. To better understand the effects of MRP4 on platelet function, the aim of this investigation was to assess the impact of cilostazol-induced inhibition of MRP4-mediated transport and assess aspirin-induced antiplatelet effects and rates of HARPR in human subjects.Cilostazol-dependent inhibition of MRP4-mediated transport was assessed with the release of the fluorescent adduct bimane-glutathione and aspirin entrapment. Effect of Cilostazol on cAMP inhibition was evaluated by vasodilator-stimulated phosphoprotein (VASP). Platelet function was studied by collagen and TRAP-6-induced platelet aggregation and secretion.Cilostazol reduced the release of bimane-glutathione and enhanced aspirin entrapment demonstrating an inhibitory effect on MRP4 in platelets. VASP phosphorylation was absent until 10 seconds after addition of cilostazol, and becomes evident after 30 seconds. An inhibitory effect on platelet aggregation and secretion was found in activated platelets, with threshold concentration of agonists, 10 seconds after addition of cilostazol, supporting a role of MRP4 on platelet function that is cAMP independent. Cilostazol effects were also shown in aspirin-treated platelets. A reduction of platelet aggregation and secretion were observed in aspirin-treated patients with HARPR.This study supports the role of MRP4 on modulating platelet function which occurs through cAMP-independent mechanisms. Moreover, inhibition of MRP4 induced by cilostazol enhances aspirin-induced antiplatelet effects and reduces HARPR.


Assuntos
Aspirina/administração & dosagem , Plaquetas/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Testes de Função Plaquetária , Idoso , Plaquetas/metabolismo , Cilostazol/farmacologia , Estudos de Coortes , AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Ativação Plaquetária , Agregação Plaquetária , Inibidores da Agregação Plaquetária/administração & dosagem , Prostaglandinas H/metabolismo , Ácido Salicílico/administração & dosagem , Trombose/tratamento farmacológico
8.
Front Immunol ; 8: 1946, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29375570

RESUMO

Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of in vitro differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin "in vivo" and "in vitro" not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1.

9.
Thromb Haemost ; 116(6): 1100-1110, 2016 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-27683757

RESUMO

Platelet multidrug resistance protein4 (MRP4)-overexpression has a role in reducing aspirin action. Aspirin in vivo treatment enhances platelet MRP4 expression and MRP4 mediated transport inhibition reduces platelet function and delays thrombus formation. The aim of our work was to verify whether MRP4 expression is enhanced in platelets obtained from patients under chronic aspirin treatment and whether it correlates with residual platelet reactivity. We evaluated changes on mRNA and protein-MRP4 expression and platelet aggregation in four populations: healthy volunteers (HV), aspirin-free control population (CTR), patients who started the treatment less than one month ago (ASA<1 month patients) and aspirinated patients who started the treatment more than two months ago (ASA>2 months patients). In platelets obtained from ASA>2 months patients, it was found a statistically significant MRP4 enhancement of both mRNA and protein expression compared to HV, CTR and ASA<1 month patients. Platelets obtained from ASA>2 months patients that present high levels of platelet MRP4, have higher serum TxB2 levels and collagen-induced platelet aggregation compared to patient with low levels of MRP4 in platelets. In addition collagen induced platelet aggregation is higher in in vitro aspirinated platelets obtained from patients with high levels of MRP4 patients compared to those obtained from patients with low MRP4 levels. We can assert that, in patients under chronic aspirin treatment, platelets that present high MRP4 levels have an increase of residual platelet reactivity, which is due in part to incomplete COX-1 inhibition, and in part to COX-1-independent mechanism.


Assuntos
Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Plaquetas/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária , Inibidores da Agregação Plaquetária , Testes de Função Plaquetária
10.
Oncol Lett ; 12(3): 2133-2138, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27602152

RESUMO

microRNA (miR/miRNA) are small non-coding RNAs that control gene expression at the post-transcriptional level by targeting mRNAs. Aberrant expression of miRNAs is often observed in different types of cancer. Specific miRNAs function as tumor suppressors or oncogenes and interfere with various aspects of carcinogenesis, including differentiation, proliferation and invasion. Upregulation of miRNAs 221 and 222 has been shown to induce a malignant phenotype in numerous human cancers via inhibition of phosphatase and tensin homolog (PTEN) expression. Neuroblastoma is the most common extracranial solid malignancy in children, which is characterized by cellular heterogeneity that corresponds to different clinical outcomes. The different cellular phenotypes are associated with different gene mutations and miRs that control genetic and epigenetic factors. For this reason miRs are considered a potential therapeutic target in neuroblastoma. The aim of the present study was to investigate the mechanisms by which extracellular high mobility group box 1 (HMGB1) promotes cell growth in neuroblastoma. SK-N-BE(2) and SH-SY5Y neuroblastoma derived cell lines were transfected with the antisense oligonucleotides, anti-miR-221 and -222, followed by treatment with HMGB1 to investigate the expression of the oncosuppressor PTEN. In this study, it was demonstrated that HMGB1, which is released by damaged cells and tumor cells, upregulates miR-221/222 oncogenic clusters in the two human neuroblastoma derived cell lines. The results revealed that the oncogenic cluster miRs 221/222 were more highly expressed by the most undifferentiated cell line [SK-N-BE(2)] compared with the the less tumorigenic cell line (SH-SY5Y) and that exogenous HMGB1 increases this expression. In addition, HMGB1 modulates PTEN expression via miR-221/222, as demonstrated by transiently blocking miR-221/222 with anti-sense oligonucleotides. These results may lead to the development of novel therapeutic strategies for neuroblastoma.

11.
Vascul Pharmacol ; 76: 11-7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26141932

RESUMO

Platelet Multidrug Resistance Protein 4 (MRP4)-overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin induces platelet MRP4 over-expression, through megakaryocytes genomic modulation. Aim of our work was to verify whether other non-steroidal antiinflammatory drugs (NSAIDs) enhance platelet MRP4 expression and evaluate platelet function in patients who overexpressed MRP4. We evaluated MRP4-mRNA in a human megakacaryoblastic cell line (DAMI), treated with both COX-2 inhibitor (celecoxib) and traditional NSAIDs (diclofenac and naproxen). Osteoarthritis patients, who reported to take NSAIDs twice a week for at least four continuous weeks and a control population, who didn't take any drugs during the previous month, were enrolled. We evaluated platelet MRP4 amount, by both mRNA levels and protein expression (Western-Blot) and ADP induced platelet aggregation. DAMI cells treated with celecoxib, diclofenac, and naproxen showed a significant increase in MRP4-mRNA expression compared to the mock culture. Osteoarthritis patient platelets presented a higher expression of MRP4 (both at mRNA and protein levels) and an increase in ADP-induced platelet aggregation compared to the control population. NSAID treatment induced platelet MRP4 overexpression. Osteoarthritis patients, who overexpress MRP4, showed platelet hyper-reactivity. These evidences could explain in part the increased cardiovascular risk present during NSAID treatment.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Adulto , Idoso , Aspirina/farmacologia , Estudos de Casos e Controles , Celecoxib/farmacologia , Células Cultivadas , Inibidores de Ciclo-Oxigenase 2/farmacologia , Diclofenaco/farmacologia , Feminino , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Pessoa de Meia-Idade , Naproxeno/farmacologia , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , RNA Mensageiro/metabolismo
12.
Chin J Cancer Res ; 27(5): 491-6, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26543336

RESUMO

BACKGROUND: Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. METHODS: The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®). RESULTS: Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively. CONCLUSIONS: We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms.

13.
Mediators Inflamm ; 2015: 607957, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26491233

RESUMO

Overexpression of efflux transporters, in human cells, is a mechanism of resistance to drug and also to chemotherapy. We found that multidrug resistance protein-4 (MRP4) overexpression has a role in reducing aspirin action in patients after bypass surgery and, very recently, we found that aspirin enhances platelet MRP4 levels through peroxisome proliferator activated receptor-α (PPARα). In the present paper, we verified whether exposure of human embryonic kidney-293 cells (Hek-293) to aspirin modifies MRP4 gene expression and its correlation with drug elimination and cell toxicity. We first investigated the effect of high-dose aspirin in Hek-293 and we showed that aspirin is able to increase cell toxicity dose-dependently. Furthermore, aspirin effects, induced at low dose, already enhance MRP4 gene expression. Based on these findings, we compared cell viability in Hek-293, after high-dose aspirin treatment, in MRP4 overexpressing cells, either after aspirin pretreatment or in MRP4 transfected cells; in both cases, a decrease of selective aspirin cell growth inhibition was observed, in comparison with the control cultures. Altogether, these data suggest that exposing cells to low nontoxic aspirin dosages can induce gene expression alterations that may lead to the efflux transporter protein overexpression, thus increasing cellular detoxification of aspirin.


Assuntos
Aspirina/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transporte Biológico/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Morte Celular , Linhagem Celular , Separação Celular , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Resistência a Medicamentos , Citometria de Fluxo , Regulação da Expressão Gênica , Células HEK293 , Humanos , PPAR alfa/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
15.
Platelets ; 26(8): 783-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25734355

RESUMO

Serum thromboxane-B2 (TxB2), together with arachidonic acid (AA)-induced platelet aggregation, are, at the moment, the most used tests to identify patients displaying high on-aspirin treatment platelet reactivity (HAPR). Both tests are specific for aspirin action on cyclooxygenase-1. While the correlation between serum TxB2 assay and clinical outcome is established, data are conflicting with regard to aspirin treatment and a possible association with AA-stimulated platelet markers and clinical outcome. To understand such discrepancy, we performed a retrospective study to compare both assays. We collected data from 132 patients receiving a daily dose of aspirin (100 mg/day) and data from 48 patients receiving aspirin on alternate days. All Patients who received a daily dose of aspirin were studied for AA-induced platelet aggregation together with serum TxB2 levels and AA-induced TxB2 formation was also studied in 71 patients out of entire population. Consistent with recommendations in the literature, we defined HAPR by setting a cut-off point at 3.1 ng/ml for serum levels of thromboxane B2 and 20% for AA-induced platelet aggregation. According to this cut-off point, we divided our overall population into two groups: (1) TxB2 < 3.1 ng/ml and (2) TxB2 > 3.1 ng/ml. We found low agreement between such tests to identify patients displaying HAPR. Our results show that AA-induced platelet aggregation >20% identify a smaller number of HAPR patients in comparison with TxB2. A good correlation between serum TxB2 and arachidonic acid-induced TxB2 production was found (r = 0.76619).


Assuntos
Ácido Araquidônico/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Testes de Função Plaquetária , Idoso , Idoso de 80 Anos ou mais , Aspirina/uso terapêutico , Resistência a Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Estudos Retrospectivos , Tromboxano B2/sangue , Tromboxano B2/metabolismo
16.
Br J Clin Pharmacol ; 78(6): 1343-53, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24902864

RESUMO

AIM: The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα). METHODS: The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV). RESULTS: In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment. CONCLUSIONS: The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.


Assuntos
Aspirina/farmacologia , Megacariócitos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Megacariócitos/metabolismo , Pessoa de Meia-Idade , PPAR alfa/genética , RNA Mensageiro/análise , Regulação para Cima
17.
Mol Carcinog ; 52(7): 526-34, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22389255

RESUMO

Reactivation of the HMGA1 protoncogene is very frequent in human cancer, but still very little is known on the molecular mechanisms leading to this event. Prompted by the finding of putative E2F binding sites in the human HMGA1 promoter and by the frequent deregulation of the RB/E2F1 pathway in human carcinogenesis, we investigated whether E2F1 might contribute to the regulation of HMGA1 gene expression. Here we report that E2F1 induces HMGA1 by interacting with a 193 bp region of the HMGA1 promoter containing an E2F binding site surrounded by three putative Sp1 binding sites. Both gain and loss of function experiments indicate that Sp1 functionally interacts with E2F1 to promote HMGA1 expression. However, while Sp1 constitutively binds HMGA1 promoter, it is the balance between different E2F family members that tunes the levels of HMGA1 expression between quiescence and proliferation. Finally, we found increased HMGA1 expression in pituitary and thyroid tumors developed in Rb(+/-) mice, supporting the hypothesis that E2F1 is a novel important regulator of HMGA1 expression and that deregulation of the RB/E2F1 path might significantly contribute to HMGA1 deregulation in cancer.


Assuntos
Fator de Transcrição E2F1/metabolismo , Proteína HMGA1a/genética , Neoplasias Hipofisárias/metabolismo , Fator de Transcrição Sp1/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Imunoprecipitação da Cromatina , Fator de Transcrição E2F1/genética , Proteína HMGA1a/metabolismo , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Neoplasias Hipofisárias/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteína do Retinoblastoma/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/genética , Neoplasias da Glândula Tireoide/genética , Ativação Transcricional
18.
Platelets ; 24(7): 554-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23249278

RESUMO

Ca(2+)influx might occur through K(+)-dependent Na(+)/Ca(2+) exchanger operating in reverse mode (rNCKX). In a cellular model different from platelets, an interaction between canonical transient receptor potential cation (TRPC) channels and NCX has been found. The aim of this study was to verify whether the TRPC/NCKX interaction operates in human platelets. Our results showed that the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced rNCKX-mediated Ca(2+) influx through TRPC-mediated Na(+) influx. DAG-induced activation of TRPC/NCKX occurs independently of protein kinase C (PKC) activation, as PKC inhibitor did not modify OAG-mediated Ca(2+) influx. Moreover, as both rNCKX and TRPC inhibitors reduced OAG-induced platelet aggregation which, conversely, was increased by flufenamic acid, known to develop TRPC activity, it could be suggested that the TRPC/NCKX interaction has a role in OAG-dependent platelet aggregation.


Assuntos
Plaquetas/efeitos dos fármacos , Canais de Cálcio/sangue , Cálcio/sangue , Diglicerídeos/farmacologia , Proteína Quinase C/sangue , Canais de Cátion TRPC/sangue , Plaquetas/citologia , Plaquetas/metabolismo , Ativação Enzimática , Humanos , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Transdução de Sinais
19.
Mol Cancer Res ; 9(1): 67-77, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21173028

RESUMO

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma.


Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia , Bleomicina/farmacologia , Western Blotting , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Mutação , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Serina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
FASEB J ; 23(12): 4276-87, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19713529

RESUMO

MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/tratamento farmacológico , Neuropeptídeos/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Bases , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Proteínas da Matriz Extracelular/genética , Humanos , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neuropeptídeos/genética , Proteína Reelina , Serina Endopeptidases/genética , Tretinoína/farmacologia
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