RESUMO
OBJECTIVE: Light transmission aggregometry (LTA) is the gold standard method for assessing platelet function. Recently, a new parameter called adenosine diphosphate (ADP)-induced platelet aggregation level (APAL) was developed to aid interpretation of LTA results. APAL is a score calculated based on platelet aggregation patterns upon exposure to 1 µM and 10 µM ADP and is determined using an automated coagulation analyzer. We compared APAL and VerifyNow P2Y12 assay for neuroendovascular patients. METHODS: 42 patients who have received antiplatelet therapy were studied. Platelet function tests were performed on CS-2400 for APAL and VerifyNow P2Y12 assay was used for P2Y12 reaction unit (PRU) and % inhibition. RESULTS: Moderate correlations were observed between APAL and PRU (r=0.64, p<0.001) and between APAL and % inhibition (r=-0.74, p<0.001). The optimal threshold for APAL was 8.2 for PRU (threshold=240) and 8.1 for % inhibition (threshold=26%). The percentage of agreement between the above thresholds was 90.9% between PRU and APAL and 77.3% between % inhibition and APAL. CONCLUSIONS: The APAL system exhibits moderate correlation with PRU and % inhibition. APAL testing is a good choice for a clinical laboratory already in possession of Sysmex CS series analyzers. In this setting, APAL testing can significantly decrease the cost of platelet function testing for patients on antiplatelet therapy.
Assuntos
Testes de Coagulação Sanguínea/métodos , Agregação Plaquetária/fisiologia , Testes de Função Plaquetária/métodos , Difosfato de Adenosina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Testes de Coagulação Sanguínea/instrumentação , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Clopidogrel/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Testes de Função Plaquetária/instrumentaçãoRESUMO
BACKGROUND: Light transmission aggregometry (LTA) is the gold standard for platelet function assessment. The automated coagulation analyzer from Sysmex that performs LTA offers the advantage of being a walk-away technology. Recently, a new parameter "ADP-induced platelet aggregation level (APAL)" was developed to support the interpretation of results. APAL is calculated as a score from 0.0 to 10.0 based on platelet aggregation patterns with 1 and 10 µM adenosine diphosphate (ADP). Here, the basic performance of the newly developed APAL system and comparison with the maximum aggregation rate of ADP (ADP-MA) was evaluated. METHODS: The within-run precision was calculated by conducting five replicate analyses of the platelet-rich plasma (PRP) from healthy volunteers and 0.05 µM of cangrelor-spiked PRP. Cangrelor is a P2Y12 inhibitor that does not require liver CYP activation. The reference interval was calculated from the results of 67 healthy volunteers. The effect of the antiplatelet P2Y12 agent was evaluated using several concentrations of cangrelor. A comparative study was performed using 103 PRP samples with different levels of aggregation. Each test was analyzed with both APAL and ADP-MA. RESULTS: The percentage coefficient of variation in within-run precision was within 7% for APAL and 10 µM ADP-MA. Reference interval of APAL and 10 µM ADP-MA was 7.1 - 10.0 and 80.0 - 99.2%, respectively. APAL signifi-cantly decreased with the addition of 0.02 µM cangrelor, while 10 µM ADP-MA was barely affected. A significant correlation was observed between APAL and 10 µM ADP-MA (r = 0.94; p < 0.0001). CONCLUSIONS: The newly developed APAL system exhibited an acceptable performance. APAL score showed a good correlation with ADP-MA and was adequate to detect the weak effect of P2Y12 inhibitors. APAL is a new platelet aggregation scoring system with the potential to monitor the effects of P2Y12 inhibitor over a wide range.
Assuntos
Difosfato de Adenosina/farmacologia , Testes de Coagulação Sanguínea/instrumentação , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária/instrumentação , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Testes de Coagulação Sanguínea/métodos , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Testes de Função Plaquetária/métodos , Plasma Rico em Plaquetas/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
Inflammasomes are involved in innate immune responses. Several NOD-Like receptors (NLRs) participate in the formation of inflammasomes. NACHT, LRR and PYD domains-containing protein 3 (NALP3) belongs to the NLR family and recognizes adenosine triphosphate (ATP), crystals, and Reactive Oxygen Species (ROS). This study examined the effect of inflammasomes on alpha-galactosylceramide (GalCer)-induced liver injury using NALP3-knockout (KO) mice. GalCer administration induced inflammasome activation and IL-1ß-maturation. In NALP3-KO mice treated with GalCer, serum ALT levels were significantly reduced compared with those in GalCer-treated WT mice. Histological examination revealed decreased necrosis in NALP3-KO mice compared with WT mice, consistent with ALT levels. Expression of proinflammatory cytokines (such as IL-6, and TNF-α) and chemokines was also significantly suppressed in NALP3-KO mice. Moreover, flow cytometry analysis revealed fewer infiltrating immune cells in the livers of GalCer-treated NALP3-KO mice. Inportantly, the frequency of MDSCs (CD11b+Gr-1int cells), which suppress the immune response, was significantly increased in GalCer-treated NALP3-KO mice. In conclusion, NALP3 inhibition attenuated liver injury in GalCer-induced hepatitis. The inhibition of NALP3 signaling coused be a therapeutic target in immune-mediated liver injury.
Assuntos
Galactosilceramidas/imunologia , Hepatite/patologia , Inflamassomos/imunologia , Fígado/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Transdução de Sinais , Animais , Citocinas/análise , Citocinas/imunologia , Hepatite/genética , Hepatite/imunologia , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment.
Assuntos
Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Estilbenos/administração & dosagem , Ativação Transcricional/efeitos dos fármacos , Anticarcinógenos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Neoplasias Experimentais/patologia , Resveratrol , Esfingomielina Fosfodiesterase/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.
Assuntos
Ceramidase Ácida/metabolismo , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/enzimologia , Ceramidase Ácida/antagonistas & inibidores , Ceramidase Ácida/genética , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Endopeptidases/química , Endopeptidases/genética , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitina TiolesteraseRESUMO
Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.
Assuntos
Proliferação de Células/genética , Neoplasias do Colo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Diferenciação Celular , Neoplasias do Colo/patologia , Meios de Cultura Livres de Soro , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , RNA Mensageiro/biossínteseRESUMO
Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.