A proteomic study of myeloproliferative neoplasms using reverse-phase protein arrays.

Myeloproliferative neoplasms polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis constitute a group of haematological diseases. The comprehensive assessment of signaling pathway activation in blood cells may aid the understanding of MPN pathophysiology. Thus, levels of post-translational protein modifications and total protein expression were determined in MPN patients and control leukocytes by using reverse-phase protein arrays (RPPA). Compared to control samples, p-SRC, p-CTNNB1, c-MYC, MCL-1, p-MDM2, BAX and CCNB1 showed higher expression in PV samples than controls. P-JAK2/JAK2 and pro-apoptotic BIM showed differential expression between JAK2V617F-positive and -negative ET patients. Apoptosis, cancer and PI3K/AKT pathways proteins showed differential expression among the studied groups. For most of the proteins analyzed using Western-Blot and RPPA, RPPA showed higher sensitivity to detect subtle differences. Taken together, our data indicate deregulated protein expression in MPN patients compared to controls. Thus, RPPA may be a useful method for broad proteome analysis in MPN patients´ leukocytes.

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 venom and BmooLAAO-I downmodulate CXCL8/IL-8 and CCL2/MCP-1 production and oxidative burst response, and upregulate CD11b expression in human neutrophils.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation.

BACKGROUND: Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. METHODS: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. RESULTS: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. CONCLUSIONS: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.

l-Amino acid oxidase isolated from Calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines.

BACKGROUND: Myeloproliferative neoplasms are Philadelphia chromosome-negative diseases characterized by hyperproliferation of mature myeloid cells, associated or not with the Janus kinase 2 tyrosine kinase mutation, JAK2V617F. As there is no curative therapy, researchers have been investigating new drugs to treat myeloproliferative neoplasms, including l-amino acid oxidase from Calloselasma rhodostoma snake venom (CR-LAAO), which is a toxin capable of eliciting apoptosis in several tumor cell lines. OBJECTIVE: To evaluate the effects of l-amino acid oxidase from C. rhodostoma snake venom in the apoptotic machinery of JAK2-mutated cell lines. METHODS: The HEL 92.1.7 and SET-2 cell lines were cultured with l-amino acid oxidase and catalase for 12h at 37°C in 5% carbon dioxide. The cell viability was assessed by the multi-table tournament method, the level of apoptosis was measured by flow cytometry, and the expression of cysteine-dependent aspartate-specific proteases and cleaved Poly(ADP-ribose) polymerase were analyzed by Western blotting. RESULTS: l-Amino acid oxidase from C. rhodostoma snake venom was cytotoxic to HEL 92.1.7 and SET-2 cells (50% inhibitory concentration=0.15µg/mL and 1.5µg/mL, respectively) and induced apoptosis in a concentration-dependent manner. Cell treatment with catalase mitigated the l-amino acid oxidase toxicity, indicating that hydrogen peroxide is a key component of its cytotoxic effect.The activated caspases 3 and 8 expression and cleaved PARP in HEL 92.1.7 and SET-2 cells confirmed the apoptosis activation by CR-LAAO. CONCLUSIONS: l-Amino acid oxidase from C. rhodostoma snake venom is a potential antineoplastic agent against HEL 92.1.7 and SET-2 JAK2V617F-positive cells as it activates the extrinsic apoptosis pathway.

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.

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)

BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. Methods: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. Results: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. Conclusions: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.(AU)

L-amino acid oxidase isolated from Bothrops pirajai induces apoptosis in BCR-ABL-positive cells and potentiates imatinib mesylate effect.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the presence of Philadelphia chromosome and by BCR-ABL1, which encodes the BCR-ABL oncoprotein. Although imatinib mesylate (IM) is effective for CML treatment, patients in accelerated and blastic phases of the disease are often refractory to this therapy, and there are also cases of IM resistance in patients in the chronic phase. Therefore, potential new drugs are being investigated to improve the efficiency of the therapy of CML such as snake venoms and their compounds. In this investigation, Bothrops pirajai L-amino acid oxidase (BpirLAAO-I) effect on normal peripheral blood mononuclear cells (PBMC) and on BCR-ABL(+) cell line was assessed to explore its potential against leukaemic cells. MTT viability assay, lymphocyte subsets quantification and cell activation markers expression were performed to evaluate BpirLAAO-I effect on normal PBMC. The effect of BpirLAAO-I on HL-60 and HL-60.BCR-ABL cell lines was assessed by apoptosis detection. BpirLAAO-I was able to induce apoptosis in HL-60 and HL-60.BCR-ABL cell lines in a dose-dependent manner, promoted caspases 3, 8 and 9 activation and enhanced IM effect while not affecting the viability of normal cells. In addition, BpirLAAO-I promoted immune cells activation and lymphocytes subsets changes on normal PBMC. The results indicate that BpirLAAO-I induces apoptosis and potentiates IM effect on BCR-ABL(+) cells.

l-Amino acid oxidase isolated from calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines

BACKGROUND: Myeloproliferative neoplasms are Philadelphia chromosome-negative diseases characterized by hyperproliferation of mature myeloid cells, associated or not with the Janus kinase 2 tyrosine kinase mutation, JAK2V617F. As there is no curative therapy, researchers have been investigating new drugs to treat myeloproliferative neoplasms, including l-amino acid oxidase from Calloselasma rhodostoma snake venom (CR-LAAO), which is a toxin capable of eliciting apoptosis in several tumor cell lines. OBJECTIVE: To evaluate the effects of l-amino acid oxidase from C. rhodostoma snake venom in the apoptotic machinery of JAK2-mutated cell lines. METHODS: The HEL 92.1.7 and SET-2 cell lines were cultured with l-amino acid oxidase and catalase for 12 h at 37 °C in 5% carbon dioxide. The cell viability was assessed by the multi-table tournament method, the level of apoptosis was measured by flow cytometry, and the expression of cysteine-dependent aspartate-specific proteases and cleaved Poly(ADP-ribose) polymerase were analyzed by Western blotting. RESULTS: l-Amino acid oxidase from C. rhodostoma snake venom was cytotoxic to HEL 92.1.7 and SET-2 cells (50% inhibitory concentration = 0.15 µg/mL and 1.5 µg/mL, respectively) and induced apoptosis in a concentration-dependent manner. Cell treatment with catalase mitigated the l-amino acid oxidase toxicity, indicating that hydrogen peroxide is a key component of its cytotoxic effect.The activated caspases 3 and 8 expression and cleaved PARP in HEL 92.1.7 and SET-2 cells confirmed the apoptosis activation by CR-LAAO. CONCLUSIONS: l-Amino acid oxidase from C. rhodostoma snake venom is a potential antineoplastic agent against HEL 92.1.7 and SET-2 JAK2V617F-positive cells as it activates the extrinsic apoptosis pathway.