Influence of glutathione s-transferase on the ototoxicity caused by aminoglycosides

The process of hair cell damage and death as a result of exposure to noise and ototoxins seems to be mediated by reactive oxygen species. AIM: To investigate the relationship between genetic polymorphisms in the Glutathione S-transferase and the susceptibility to hearing loss induced by aminoglycosides. MATERIALS AND METHODS: Null genotypes were analyzed by multiplex-PCR in the DNA samples from 50 patients and 72 controls. The patients were divided into 3 groups, 10 with hearing loss using aminoglycosides (group A), 20 with hearing loss without exposure to the drug (group B) and 20 hearing individuals who used the antibiotic (group C). STUDY DESIGN: Experimental. RESULTS: Polymorphisms in the GSTM1 and GSTT1 genes were found in 16% and 42% of patients and in 18% and 53% of the control group, respectively. After statistical analysis no significant difference was observed between the control groups and A (p=0.86) and (p=0.41), controls and B (p=0.27) and (p=0.24), controls and C (p=0.07) and (p=0.47), controls and A + C (p=0.09) and (p=0.47), C and A (p=0.32) and (p=0.75), GSTT1 and GSTM1, respectively. CONCLUSION: Our data show that polymorphisms in GSTM1 and GSTT1 genes have no influence on the ototoxicity of aminoglycosides.O processo de morte e danos em células ciliadas devido à exposição ao ruído e ototoxinas parece ser mediado por espécies reativas de oxigênio. OBJETIVO: Investigar a relação entre polimorfismos gênicos na Glutationa S-transferase e a susceptibilidade à deficiência auditiva induzida pelos aminoglicosídeos. CASUÍSTICA E MÉTODO: Genótipos nulos foram analisados por PCR-multiplex em amostras de DNA de 50 pacientes e 72 controles. Os pacientes foram divididos em três grupos, sendo 10 com deficiência auditiva e uso de aminoglicosídeos (grupo A), 20 com deficiência auditiva sem exposição à droga (grupo B), e 20 ouvintes que utilizaram o antibiótico (grupo C). FORMA DE ESTUDO: Experimental. RESULTADOS: Polimorfismos nos genes GSTT1 e GSTM1 foram encontrados em 16% e 42% dos pacientes e em 18% e 53% do grupo controle, respectivamente. Após a análise estatística nenhuma diferença significativa foi observada entre os grupos controle e A (p=0,86) e (p=0,41), controle e B (p=0,27) e (p=0,24), controle e C (p=0,07) e (p=0,47), controle e A+C (p=0,09) e (p=0,47), C e A (p=0,32) e (p=0,75), GSTT1 e GSTM1, respectivamente. CONCLUSÃO: Nossos dados demonstram que polimorfismos na GSTT1 e GSTM1 não exercem influência sobre a ototoxicidade dos aminoglicosídeos.30630

Influence of polymorphisms CYP2B6 G15631T, GSTM1, GSTT1, NQO1 C609T and MDR-1 C3435T in treatment response of acute leukemia and myelodysplastic syndrome

Orientador: Sara Teresinha Olalla SaadDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: As síndromes mielodisplásicas (SMD) são um grupo heterogêneo de doenças hematopoiéticas caracterizadas pela hematopoiese ineficaz resultando em citopenia no sangue periférico; cerca de 30% das SMDs evolui para leucemia mielóide aguda secundária. Leucemias agudas (LA) são doenças malignas do sangue caracterizadas por acúmulo de blastos podendo ser classificadas em mielóide agudas (LMA), quando há envolvimento de mieloblastos, ou linfóides agudas (LLA), quando há envolvimento de linfoblastos. A sobrevida média dos pacientes com leucemia aguda ainda é muito baixa e muitos deles são resistentes ao tratamento ou apresentam recaída. O melhor entendimento sobre os mecanismos de progressão da mielodisplasia e da resposta ao tratamento em leucemias agudas poderia melhorar a taxa de resposta ao tratamento e aumentar a sobrevida dos pacientes. O metabolismo e o efluxo de drogas são mecanismos de defesa responsáveis pela proteção contra agentes tóxicos e estão envolvidos na biotransformação de diversos xenobióticos. O metabolismo de drogas pode ser divido em duas fases (Fase I: Oxidação; Fase II: Conjugação), sendo ambas mediadas por enzimas metabolizadoras de drogas. O efluxo de drogas é outro mecanismo de proteção contra tóxicos, similar ao metabolismo de drogas, porém mediado por proteínas de membrana. Essas proteínas são polimórficas e esses polimorfismos alteram a atividade enzimática, podendo modificar a resposta ao tratamento e a sua resistência. O gene CYP2B6 codifica uma enzima da fase I do metabolismo responsável pela ativação dos fármacos. Esse gene possui o polimorfismo G15631T onde há troca do aminoácido (Gln172His) resultando em perda da atividade enzimática. Os genes GSTM1, GSTT1 e NQO1 codificam enzimas da fase II do metabolismo, responsáveis pela conjugação com outras substâncias para facilitar a excreção. Os genes GSTM1 e GSTT1 possuem um polimorfismo que causa deleção homozigota do gene; e o gene NQO1 possui o polimorfismo C609T que resulta em troca do aminoácido codificado (Pro187Ser). Esses polimorfismos levam a perda da atividade enzimática. O gene MDR-1 codifica a P-glicoproteína que é uma proteína de membrana responsável pelo efluxo de drogas. Esse gene possui o polimorfismo C3435T que apesar de ser silencioso (Ile1142Ile) diminui a expressão de P-glicoproteína. Assim, o objetivo deste estudo foi identificar a influência dos polimorfismos CYP2B6 G15631T, GSTT1, GSTM1, NQO1 C609T e MDR-1 C3435T no risco de leucemias agudas e SMD, na progressão de SMD e resposta ao tratamento de leucemia aguda. Foram analisados 90 pacientes com leucemia aguda (66 LMA e 24 LLA), 68 pacientes com SMD e 100 controles normais utilizando os métodos de PCR-RFLP e Multiplex. Não houve diferença estatística na freqüência dos polimorfismos entre pacientes e grupo controle. Em SMD encontramos maior frequência de deleções de GST em pacientes que progrediram comparados aos pacientes que não progrediram: 50% e 21% (P=0,019). Também encontramos menor frequência do alelo polimórfico T do polimorfismo MDR-1 C3435T em pacientes que progrediram comparada a dos pacientes que não progrediram: 50% e 81% (P=0,012). Na resposta ao tratamento de leucemias agudas, encontramos uma tendência à maior frequência do polimorfismo NQO1 C609T em pacientes com falha de indução quando comparados a pacientes com remissão em leucemias agudas, em geral, (P=0,093) e pacientes somente com LMA (P=0,125); e quando comparamos falha de indução com o grupo controle em leucemias agudas, em geral, (P=0,101) e somente em LMA (P=0,08). Observamos a mesma tendência quando comparamos a frequência do polimorfismo NQO1 C609T em pacientes com óbito precoce versus a população normal (P=0,058). Em conclusão, estes resultados sugerem que os polimorfismos não estão relacionados ao risco de leucemia aguda e SMD, embora a amostra aqui analisada possa ter sido insuficiente; as deleções GST e o polimorfismo MDR-1 C3435T estão envolvidos na progressão de SMD e o polimorfismo NQO1 C609T tem uma tendência a estar relacionado à falha de indução e ao óbito precoce em pacientes com leucemias agudas, em geral, e somente LMAAbstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by ineffective hematopoiesis resulting in peripheral blood cytopenia, about 30% of MDS patients progresses to acute myeloid leukemia. Acute leukemia (AL) are malignant blood diseases characterized by accumulation of blasts and they can be classified into acute myeloid (AML), when there is myeloblasts involvement or acute lymphoid (ALL), when there is lymphoblasts involvement. The median survival of acute leukemia patients is very low and many of them are resistant to treatment or relapsed. The better understanding of the myelodysplasia progression mechanisms and the acute leukemia response to treatment could improve the treatment response rate and patients survival. The metabolism and drug efflux are defense mechanisms responsible for protection against toxic agents and are involved in the biotransformation of various xenobiotics. The drug metabolism can be divided into two phases (Phase I: Oxidation; Phase II: Conjugation), both being mediated by drug metabolizing enzymes. The drug efflux is a similar mechanism of protection but is mediated by membrane proteins. These enzymes are polymorphic and these polymorphisms alter the enzyme activity and may modify treatment response and resistance. The CYP2B6 gene encodes a phase I enzyme responsible for drug activation. This gene has the G15631T polymorphism where there is exchange of the amino acid (Gln172His) resulting in loss of enzyme activity. The GSTM1, GSTT1 and NQO1 genes encoding phase II metabolizing enzymes that are responsible for combining with other substances to facilitate drug excretion. GSTM1 and GSTT1 genes have a polymorphism that causes homozygous deletion of the gene; and the NQO1 gene has the C609T polymorphism that results in amino acid changes (Pro187Ser). These polymorphisms lead to loss of enzyme activity. The MDR-1 gene encodes P-glycoprotein (P-gp) which is a membrane protein responsible for drug efflux. This gene has the C3435T polymorphism that despite being silent (Ile1142Ile) leads to lower P-gp expression. The aim of this study was to identify the influence of CYP2B6 G15631T, GSTT1, GSTM1, NQO1 C609T and MDR-1 C3435T polymorphisms in acute leukemia and MDS risk, MDS progression and acute leukemia response to treatment. We analyzed 90 patients with acute leukemia (66 AML and 24 ALL), 68 MDS patients and 100 normal controls using the PCRRFLP and Multiplex methods. There was no statistical difference in the frequency of polymorphisms between patients and control group. In MDS we found higher frequency of GST deletions in patients who progressed compared to patients who did not progress: 50% and 21% (P = 0.019). We also found less frequently polymorphic allele T of MDR-1 C3435T polymorphism in patients who progressed compared to patients who did not progress: 50% and 81% (P = 0.012). In acute leukemia response to the treatment, we found a trend toward a higher frequency of NQO1 C609T polymorphism in patients with induction failure compared to patients in remission, with acute leukemia in general, (P = 0.093) and AML patients only (P = 0.125); and induction failure when compared with the control group in acute leukemia in general (P = 0.101) and only in AML patients (P = 0.08). We observed the same trend when comparing the frequency of NQO1 C609T polymorphism in patients with early death versus normal population (P = 0.058). In conclusion, these results suggest that theses polymorphisms are not related to acute leukemia and MDS risk, although the sample analyzed here may have been insufficient; GST deletions and MDR-1 C3435T polymorphism are involved in MDS progression and NQO1 C609T polymorphism has a tendency to be related to induction failure and early death in patients with acute leukemia, in general, and AML onlyMestradoBiologia Estrutural, Celular, Molecular e do DesenvolvimentoMestre em Fisiopatologia Médic

Influence of VEGFA and SEMA3A in the pathogenesis of myelodysplastic syndromes and acute leukemias

Orientador: Sara Teresinha Olalla SaadTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A interação entre as células-tronco hematopoéticas (HSCs) e o microambiente da medula óssea (MO) é essencial para a regulação da hematopoese; e a interação entre as células leucêmicas e o microambiente é essencial para a leucemogênese. O microambiente é composto por diversos tipos celulares como: células endoteliais, osteoblastos e células mesenquimais estromais (MSCs). As células leucêmicas são capazes de modificar a interação com as MSCs e promover a leucemogênese. Nosso grupo de pesquisa identificou, por ensaio de microarranjo, a hiperexpressão do fator de crescimento vascular endotelial A (VEGFA) em células CD34+ e a hiperexpressão de Semaforina 3A (SEMA3A) em MSCs da MO de pacientes com síndrome mielodisplásica (SMD) do tipo ARSA. VEGFA é o fator pró-angiogênico mais estudado, está aumentado em pacientes com leucemia mielóide aguda (LMA) e correlaciona-se com pior prognóstico. SEMA3A é uma proteína secretada que parece ter efeitos antiangiogênicos em tumores sólidos. A competição entre VEGFA e SEMA3A pelo receptor neuropilina 1 (NRP1) tem sido discutida. O objetivo desse estudo foi entender a interação entre VEGFA e SEMA3A na MO de pacientes com SMD e LMA e sua função na patogênese. Para investigar a expressão gênica de VEGFA em células CD34+, recrutamos 18 pacientes com SMD e 16 com LMA e descrevemos, por RT-PCR, aumento da expressão de VEGFA em pacientes com LMA de novo comparado ao grupo controle (5,06 [0,42¿57,52] vs 1,00 [0,8¿2,19], P=0,0073). Para investigar a expressão gênica de SEMA3A em MSCs, recrutamos 25 pacientes com SMD e 17 com LMA e encontramos aumento da expressão de SEMA3A em todas as amostras comparando ao grupo controle: SMD de baixo risco (2,97 [0,64¿24,39] vs 0,83 [0,43¿2,86], P=0,028); SMD de alto risco (9,50 [2,34¿22,60] vs 0,83 [0,43¿2,86], P=0,005); LMA secundária (3,07 [0,97¿6,97] vs 0,83 [0,43¿2,86], P=0,05) e LMA de novo (5,73 [1,10¿28,21] vs 0,83 [0,43¿2,86], P=0,007). Para investigar os efeitos de VEGFA em células CD34+ normais e em células leucêmicas, nós hiperexpressamos VEGFA em células leucêmicas KG1 e células CD34+ de sangue de cordão umbilical. A hiperexpressão de VEGFA aumentou a viabilidade de células KG1 (124,4± 23,03% vs 100± 0,62%; P=0,03) e de células CD34+ (159,6± 41,2% vs 102,5 ± 16,93%; P=0,045); e a proliferação de células KG1 (107,7± 2,82%; P=0,042) e de células CD34+ (134,0± 7,68%; P=0,047); comparado ao controle. Para estudar a interação entre VEGFA e SEMA3A, hiperexpressamos SEMA3A em células HS5 e realizamos ensaios de cocultura. A cocultura de células KG1 ou CD34+ hiperexpressando VEGFA com células HS5 aumentou a proliferação (176,9± 46,34%; P=0,045) de células KG1 de células CD34+ (131,8± 7,9%; P=0,02). A cocultura de células KG1 ou CD34+ com células HS5 hiperexpressando SEMA3A diminuiu a proliferação (93,15± 0,79%; P=0,004) de células KG1; e de células CD34+ (72,73± 4,72%; P=0,009). Quando combinamos a hiperexpressão de VEGFA e de SEMA3A, os efeitos de SEMA3A são dominantes. Para investigar se o efeito dominante de SEMA3A está relacionado a competição por NRP1, realizamos ensaio de imunoprecipitação utilizando células HS5 e KG1 tratadas com as proteínas recombinantes VEGFA e SEMA3A. No tratamento combinado das duas proteínas recombinantes, há maior formação de complexos entre os receptores Plexina A4 e NRP1, favorecendo a sinalização de SEMA3A. Nossos resultados sugerem que a hiperexpressão de VEGFA confere vantagens às células leucêmicas, aumentando a proliferação, e que SEMA3A parece anular os efeitos causados por VEGFA. Portanto, acreditamos que o uso de inibidores de VEGFA combinado com a proteína SEMA3A pode ser benéfico no tratamento dos pacientesAbstract: The cross-talk between hematopoietic stem cells (HSCs) and the bone marrow (BM) microenvironment is essential for hematopoiesis regulation; and the cross-talk between leukemic stem cells (LSCs) and the bone marrow (BM) microenvironment is essential for leukemogenesis. The BM microenvironment compromises several cellular types such as endothelial cells, osteoblasts and mesenchymal stromal cells (MSCs). LSCs are capable of modifying the interaction with MSCs and promoting leukemogenesis. Our group performed a microarray assay and identified VEGFA overexpression in CD34+ cells and SEMA3A overexpression in MSCs from bone marrow of myelodysplastic syndrome RARS patients. VEGFA is the most studied angiogenic factor; is increased in acute myeloid leukemia (AML) patients, and correlates with a worse prognosis. SEMA3A is a secreted protein that appears to have antiangiogenic effects in solid tumors. A competition between VEGFA and SEMA3A for the neuropilin 1 (NRP1) receptor binding has been reported. The aim of this study was to reach a better understanding of VEGFA and SEMA3A interaction in the bone marrow of MDS and AML patients and the role of this interaction in pathogenesis. To investigate VEGFA expression in CD34+ cells, we recruited 18 MDS and 15 AML patients and described, by RT-PCR, increased VEGFA expression in de novo AML patients compared to the control group (5.06 [0.42¿57.52] vs 1.00 [0.8¿2.19], P=0.0073). To investigate SEMA3A expression in MSCs, we collected 25 MDS and 17 AML bone marrow samples and we found increased SEMA3A expression in all samples compared to the control group: low-risk MDS (2.97 [0.64¿24.39] vs 0.83 [0.43¿2.86], P=0,028); High-risk MDS (9.50 [2.34¿22.60] vs 0.83 [0.43¿2.86], P=0.005); secondary AML (3.07 [0.97¿6.97] vs 0.83 [0.43¿2.86], P=0.05) and de novo AML (5.73 [1.10¿28.21] vs 0.83 [0.43¿2.86], P=0.007). In order to investigate VEGFA effects in AML cells and CD34+ normal cells, we overexpressed VEGFA in KG1 leukemic cells and in CD34+ from umbilical cord blood. The VEGFA overexpression increased KG1 (124.4± 23.03% vs 100± 0.62%; P=0.045) and CD34+ cells (159.6± 41.2% vs 102.5 ± 16.93%; P=0.042) viability; and KG1 (107.7± 2.82%; P=0.042) and CD34+ cells (134.0± 7.68%; P=0.047) proliferation compared to control cells. In order to investigate the interaction between SEMA3A and VEGFA, we overexpressed SEMA3A in HS5 stromal cell line and performed co-culture assays. The co-culture of KG1 or CD34+ cells overexpressing VEGFA with HS5 cells showed increased KG1 proliferation (176.9± 46.34%; P=0.045) and CD34+ proliferation (131.8± 7.9%; P=0.02). The co-culture of KG1 or CD34+ cells with HS5 cells overexpressing SEMA3A showed decreased KG1 proliferation (93.15± 0.79%; P=0.004) and CD34+ proliferation (72.73± 4.72%; P=0.009). When we combined VEGFA and SEMA3A overexpression, the effects of SEMA3A were dominant over VEGFA. To investigate whether the dominant effect of SEMA3A could be due to NRP1 competition, we performed immunoprecipitation assay using KG1 and HS5 treated with VEGFA and SEMA3A recombinant proteins. The combined treatment of VEGFA and SEMA3A proteins induced increased complex formation between NRP1 and Plexin A4 receptors, favoring SEMA3A signaling. Ours results suggest that VEGFA overexpression confer AML cells advantages by increasing proliferation. SEMA3A seems to reverse the effects of VEGFA. We suggested that the use of SEMA3A protein combined with VEGFA inhibitors could be beneficial to AML patient¿s treatmentDoutoradoFisiopatologia MédicaDoutora em Ciências2012/00529-9FAPES

Hematopoietic Cell Kinase (HCK) Is Highly Expressed in Myelodysplastic Syndrome (MDS) and Regulates Proliferation, Cell Cycle Progression and Hematopoietic Differentiation

54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH)1202

Mdr-1 And Gst Polymorphisms Are Involved In Myelodysplasia Progression.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by abnormal hematopoietic differentiation and maturation, which progress toward acute leukemia in approximately 30% of the cases. Drug metabolism polymorphisms in Cytochrome P450 2B6 (CYP2B6), Glutathione S-transferase (GST) and Dehydrogenase Quinone 1 (NQO1) enzymes and P-glycoprotein (MDR-1) could modify enzyme activity. Thus, the aim of this study was to identify the influence of CYP2B6 G15631T, GSTT1, GSTM1, NQO1 C609T and MDR-1 C3435T polymorphisms on MDS progression. We analyzed 78 MDS patients using the PCR-RFLP and multiplex method. The frequency of GST deletions and MDR-1 CC genotype was lower in progression-free patients compared to patients with progression; GST: 17% vs. 35% (P=0.018); MDR-1 gene: 19% vs. 48% (P=0.012). We also verified the influence of GST deletions and MDR-1 C3435T on patient overall survival and found no significant difference (RR=0.75; P=0.599 and RR=0.79; P=0.594 respectively). We concluded that GSTM1 deletion may contribute toward MDS progression probably due to toxic metabolite accumulation which generates cell toxicity and DNA damage. Moreover, MDR-1 C3435T may have a protective effect against MDS progression because the expected lower expression of P-glycoprotein would lead to a higher degree of cell death. To the best of our knowledge, this is the first study showing the relationship of these polymorphisms with MDS progression.37970-

SEMA3A partially reverses VEGF effects through binding to neuropilin-1

Cross-talk between hematopoietic stem cells (HSCs) and bone marrow stromal cells (BMSCs) is essential for HSCs regulation and leukemogenesis. Studying bone marrow of myelodysplasia patients, a pre-leukemic condition, we found mRNA overexpression of vascular endothelial growth factor A (VEGFA) in CD34+ HSCs and semaphorin 3A (SEMA3A) in BMSCs. To better understand the role of VEGFA and SEMA3A in leukemogenesis, we recruited 30 myelodysplastic syndrome (MDS) patients, 29 acute myeloid leukemia (6 secondary to MDS) patients and 12 controls. We found higher VEGFA expression in de novo AML patients (without prior MDS) group (p = 0.0073) and higher SEMA3A expression in all BMSCs patient's samples compared to control group. We then overexpressed VEGFA in an acute myelogenous leukemia cell line, KG1 cells, and in normal CD34+ cells. This overexpression increased KG1 (p = 0.045) and CD34+ cell (p = 0.042) viability and KG1 (p = 0.042) and CD34+ cell (p = 0.047) proliferation. Moreover, KG1 and CD34+ cells overexpressing VEGFA also had increased proliferation when co-cultured with human marrow stromal HS5 cells (p = 0.045 and p = 0.02, respectively). However, co-culture of these transformed cells with HS5 cells overexpressing SEMA3A reduced KG1 (p = 0.004) and CD34+ (p = 0.009) proliferation. Co-culture of KG1 transformed cells with HS27 cells overexpressing SEMA3A reduced KG1 proliferation as well (p = 0.01). To investigate whether the dominant SEMA3A effect over VEGFA could be due to competition for neuropilin1 receptor (NRP1), we performed immunoprecipitation with anti-NRP1 antibody of cell extracts of co-cultured KG1 and HS5 cells, induced or not by VEGFA and SEMA3A recombinant proteins. Results showed a preferential association of NRP1 with SEMA3A, suggesting that SEMA3A can partially reverse the effects caused by the VEGFA preventing its binding with the NRP1 receptor. Since both hematopoietic cells, leukemic and normal, showed similar behavior, we suppose that the attempt to reversion of VEGF effects by SEMA3A is a homeostatic phenomenon in the hematopoietic niche. Finally, we conclude that VEGFA overexpression confers AML cell advantages and SEMA3A may partially reverse this effect; thus, SEMA3A protein combined with VEGFA inhibitors could be beneficial for AML treatment