Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells.

BACKGROUND: Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. METHODS: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. RESULTS: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. CONCLUSION: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)

MLL2/KMT2D and MLL3/KMT2C expression correlates with disease progression and response to imatinib mesylate in chronic myeloid leukemia.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm whose pathogenesis is linked to the Philadelphia chromosome presence that generates the BCR-ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR-ABL tyrosine kinase. The disease shows three distinct clinical-laboratory stages: chronic phase, accelerated phase and blast crisis. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the lysine methyltransferase MLL2/KMT2D and MLL3/KMT2C. METHODS: Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression. RESULTS: In patients, both methyltransferases were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. A rescue in the expression of both MLL genes was observed in KCL22S, a CML cell line sensitive to IM, after treatment with dasatinib or nilotinib which was associated with a higher rate of apoptosis, an enhanced expression of p21 (CDKN1A) and a concomitant decrease in the expression of CDK2, CDK4 and Cyclin B1 (CCNB1) in comparison to untreated KCL22S control or IM resistant KCL22R cell line, which suggests involvement of p53 regulated pathway. CONCLUSION: Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.

Expression of Hippo signaling pathway and Aurora kinase genes in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from clonal expansion of hematopoietic stem cells positive for the Philadelphia chromosome. The CML pathogenesis is associated with expression of the BCR-ABL1 oncogene, which encodes the Bcr-Abl protein with tyrosine kinase activity, promoting the leukemic cell exacerbated myeloproliferation and resistance to apoptosis. CML patients are usually treated with tyrosine kinase inhibitors (TKI), but some of them acquire resistance or are refractory to TKI. Thus, it is still relevant to elucidate the CML pathogenesis and seek new therapeutic targets, such as the Hippo signaling pathway and cell cycle regulatory genes from the Aurora kinase family. The present study quantified the expression level of genes encoding components of the Hippo signaling pathway (LATS1, LATS2, YAP, and TAZ), AURKA and AURKB in CML patients at different stages of the disease, who were resistant or sensitive to imatinib mesylate therapy, and in healthy individuals. The expression levels of the target genes were correlated with the CML Sokal's prognostic score. The most striking results were the LATS2 and AURKA overexpression in CML patients, the overexpression of TAZ and AURKB in CML patients at advanced phases and TAZ in CML IM-resistant. The development of drugs and/or identification of tumor markers for the Hippo signaling pathway and the Aurora kinase family, either alone or in combination, can optimize CML treatment by enhancing the susceptibility of leukemic cells to apoptosis and leading to a better disease prognosis.

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells.

BACKGROUND: Chronic myeloid leukemia (CML) is a BCR-ABL1 + myeloproliferative neoplasm marked by increased myeloproliferation and presence of leukemic cells resistant to apoptosis. The current first-line therapy for CML is administration of the tyrosine kinase inhibitors imatinib mesylate, dasatinib or nilotinib. Although effective to treat CML, some patients have become resistant to this therapy, leading to disease progression and death. Thus, the discovery of new compounds to improve CML therapy is still challenging. Here we addressed whether MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, affects the viability of imatinib mesylate-resistant Bcr-Abl+ cell lines. METHODS: We examined the cytotoxic and pro-apoptotic effect of MjTX-I in K562-S and K562-R Bcr-Abl+ cells and in the non-tumor HEK-293 cell line and peripheral blood mononuclear cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the hypotonic fluorescent solution methods, associated with detection of caspases 3, 8, and 9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. We also analyzed the MjTX-I potential to modulate the expression of apoptosis-related genes in K562-S and K562-R cells. RESULTS: MjTX-I decreased the viability of K562-S and K562-R cells by 60 to 65%, without affecting the viability of the non-tumor cells, i.e. it exerted selective cytotoxicity towards Bcr-Abl+ cell lines. In leukemic cell lines, the toxin induced apoptosis, activated caspases 3, 8, and 9, cleaved PARP, downregulated expression of the anti-apoptotic gene BCL-2, and upregulated expression of the pro-apoptotic gene BAD. CONCLUSION: The antitumor effect of MjTX-I is associated with its potential to induce apoptosis and cytotoxicity in Bcr-Abl positive cell lines sensitive and resistant to imatinib mesylate, indicating that MjTX-I is a promising candidate drug to upgrade the CML therapy.

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells

A leucemia mieloide crônica (LMC) é uma neoplasia mieloproliferativa BCR-ABL1 + marcada por aumento da mieloproliferação e presença de células leucêmicas resistentes à apoptose. A terapia de primeira linha atual para a LMC é a administração de inibidores da tirosina quinase, mesilato de imatinibe, dasatinibe ou nilotinibe. Embora eficaz no tratamento da LMC, alguns pacientes se tornaram resistentes a essa terapia, levando à progressão da doença e à morte. Assim, a descoberta de novos compostos para melhorar a terapia da LMC ainda é um desafio. Aqui, os destinatários se MjTX-I, uma fosfolipase A 2 isolado a partir de Bothrops moojeni de veneno de cobra, afecta a viabilidade de Bcr-Abl de mesilato de imatinib-resistente + linhas celulares. Métodos: Examinamos o efeito citotóxico e pró-apoptótico de MjTX-I em células K562-S e K562-R Bcr-Abl + e na linha de células HEK-293 não tumorais e células mononucleares de sangue periférico, usando o 3- (4, Brometo de 5-dimetiltiazol-2-il) -2,5-difeniltetrazólio e os métodos de solução fluorescente hipotônica, associados à detecção de ativação de caspases 3, 8 e 9 e clivagem de poli (ADP-ribose) polimerase (PARP). Também analisamos o potencial MjTX-I para modular a expressão de genes relacionados à apoptose em células K562-S e K562-R. Resultados: O MjTX-I diminuiu a viabilidade das células K562-S e K562-R em 60 a 65%, sem afetar a viabilidade das células não tumorais, ou seja, exerceu citotoxicidade seletiva para as linhagens celulares Bcr-Abl + . Em linhas de células leucêmicas, a toxina induziu apoptose, caspases 3, 8 e 9 ativadas, PARP clivada, expressão negativa do gene anti-apoptótico BCL-2 e expressão aumentada do gene pró-apoptótico BAD. Conclusão: O efeito antitumoral de MjTX-I está associado ao seu potencial para induzir apoptose e citotoxicidade em linhagens celulares positivas para Bcr-Abl sensíveis e resistentes ao mesilato de imatinibe, indicando que MjTX-I é um candidato promissor a fármaco para atualizar a terapia de LMC.(AU)

Hippo pathway deregulation: implications in the pathogenesis of haematological malignancies.

The Hippo pathway participates in the regulation of cell proliferation, differentiation and apoptosis. It is composed by a large array of proteins whose deregulation has been associated with pro-oncogenic and antioncogenic processes. The present review focuses on the Hippo pathway signalling network and discusses its dual role in oncogenesis, particularly in haematological malignancies.

The L-amino acid oxidase from Calloselasma rhodostoma snake venom modulates apoptomiRs expression in Bcr-Abl-positive cell lines.

Anti-apoptotic genes and apoptomiRs deregulated expression contribute to apoptosis resistance in chronic myeloid leukemia (CML) Bcr-Abl(+) cells. Here, the L-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) venom altered the apoptotic machinery regulation by modulating the expression of the miR-145, miR-26a, miR-142-3p, miR-21, miR-130a, and miR-146a, and of the apoptosis-related proteins Bid, Bim, Bcl-2, Ciap-2, c-Flip, and Mcl-1 in Bcr-Abl(+) cells. CR-LAAO is a potential tool to instigate apoptomiRs regulation that contributes to drive CML therapy.

CR-LAAO antileukemic effect against Bcr-Abl(+) cells is mediated by apoptosis and hydrogen peroxide.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Bcr-Abl tyrosine kinase protein, which confers resistance to apoptosis in leukemic cells. Tyrosine kinase inhibitors (TKIs) are effectively used to treat CML; however, CML patients in the advanced (CML-AP) and chronic (CML-CP) phases of the disease are usually resistant to TKI therapy. Thus, it is necessary to seek for novel agents to treat CML, such as the enzyme l-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) snake venom. We examined the antitumor effect of CR-LAAO in Bcr-Abl(+) cell lines and peripheral blood mononuclear cells (PBMC) from healthy subjects and CML patients. CR-LAAO was more cytotoxic towards Bcr-Abl(+) cell lines than towards healthy subjects' PBMC. The H2O2 produced during the enzymatic action of CR-LAAO mediated its cytotoxic effect. The CR-LAAO induced apoptosis in Bcr-Abl(+) cells, as detected by caspases 3, 8, and 9 activation, loss of mitochondrial membrane potential, and DNA damage. CR-LAAO elicited apoptosis in PBMC from CML-CP patients without TKI treatment more strongly than in PBMC from healthy subjects and TKI-treated CML-CP and CML-AP patients. The antitumor effect of CR-LAAO against Bcr-Abl(+) cells makes this toxin a promising candidate to CML therapy.

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways.

BACKGROUND: Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked by Bothrops snakes. The present study aimed to assess whether Bothrops jararacussu and Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways. METHODS: Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t(½) values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model. RESULTS: At the highest concentration tested (120 µg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 µg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 µg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum. CONCLUSION: Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system by B. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 g/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 g/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 g/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 μg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 μg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 μg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.(AU)