THE TERAPEUTIC POTENTIAL OF EMBRIONARY SIGNALING PATHWAYS INHIBITION IN ACUTE LYMPHOBLASTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Bela Sarmento-Ribeiro A. 06/09/16; 134489; PB1589
Prof. Ana Bela Sarmento-Ribeiro
Contributions
Contributions
Abstract
Abstract: PB1589
Type: Publication Only
Background
Embrionary signaling pathways, such as Wingless (WNT), Hedgehog (Hh), and Notch pathways, are essential for differentiation and self-regulation of stem cells, and their deregulation have been associated with several hematological neoplasias. Acute lymphoblastic leukemia (ALL) is characterized by the abnormal proliferation and accumulation of immature lymphoid cells within the bone marrow and lymphoid tissues, which can arise from the aberrant activation of embryonic signaling pathways. Therefore, these pathways may constitute new therapeutic targets for ALL treatment.
Aims
Evaluate the therapeutic potential of IWR-1, GDC-0449, and Gamma Secretase XXI inhibitor (GSXXI), inhibitors of WNT, Hh and Notch signaling pathways, respectively, in ALL in vitro models.
Methods
For this purpose, we used two ALL cell lines, CEM as a T cell ALL model, and KOPN-8 as a B cell ALL model. Both cell lines were cultured in absence and presence of different concentrations of IWR-1, GDC-0449 and GSXXI. Trypan blue assay was used to evaluate the effect of these inhibitors on cell viability and density. Cell death was determined by optical microscopy (May-Grunwald Giemsa staining) and by flow cytometry (FC) using the Annexin V and Propidium Iodide double staining. FC was also used to measure levels of apoptosis modulators, BAX and BCL-2, cell cycle (PI/RNase assay) and mitochondrial membrane potential (through the fluorescent probe JC1).
Results
Our results showed that IWR-1 reduces only the viability and proliferation of KOPN-8 cells in a time and dose dependent manner (being the maximal inhibitory concentration, IC50, approximately 50 μM at 48 hours of exposure), having no effect in CEM cells. On the other hand, GDC-0449 and GSXXI reduces cell viability and proliferation in a time, dose and cell line dependent manner, being the KOPN-8 cells the most sensitive. We found that the IC50 at 48 hours to GDC-0449 was 75 μM and 150 μM and to GSXXI was 25 μM and 30 μM, for KOPN-8 and CEM cells, respectively. All inhibitors induced cell death by apoptosis, confirmed by the BAX/BCL-2 ratio, morphological analysis, and mitochondrial membrane depolarization. The analysis of cell cycle progression revealed that all inhibitors induce cell cycle arrest in G0/G1 phase in both cell lines, except in CEM when incubated with IWR-1
Conclusion
In conclusion, our results suggest that IWR-1, GDC-0449 and GSXXI could be a potential new targeted therapy in acute lymphoblastic leukemia; however, the therapeutic efficacy is cell type-dependent. This work was supported by CIMAGO, GAI, FMUC, and Banco Santander Totta; Ana Pires by LPCC/Pfizer 2015, Joana Jorge by LPCC-NRC/CIMAGO, and Raquel Alves by FCT (SFRH/BD/51994/2012) grants.
Session topic: E-poster
Keyword(s): B cell acute lymphoblastic leukemia, Signaling, Signaling molecules, T cell acute lymphoblastic leukemia
Type: Publication Only
Background
Embrionary signaling pathways, such as Wingless (WNT), Hedgehog (Hh), and Notch pathways, are essential for differentiation and self-regulation of stem cells, and their deregulation have been associated with several hematological neoplasias. Acute lymphoblastic leukemia (ALL) is characterized by the abnormal proliferation and accumulation of immature lymphoid cells within the bone marrow and lymphoid tissues, which can arise from the aberrant activation of embryonic signaling pathways. Therefore, these pathways may constitute new therapeutic targets for ALL treatment.
Aims
Evaluate the therapeutic potential of IWR-1, GDC-0449, and Gamma Secretase XXI inhibitor (GSXXI), inhibitors of WNT, Hh and Notch signaling pathways, respectively, in ALL in vitro models.
Methods
For this purpose, we used two ALL cell lines, CEM as a T cell ALL model, and KOPN-8 as a B cell ALL model. Both cell lines were cultured in absence and presence of different concentrations of IWR-1, GDC-0449 and GSXXI. Trypan blue assay was used to evaluate the effect of these inhibitors on cell viability and density. Cell death was determined by optical microscopy (May-Grunwald Giemsa staining) and by flow cytometry (FC) using the Annexin V and Propidium Iodide double staining. FC was also used to measure levels of apoptosis modulators, BAX and BCL-2, cell cycle (PI/RNase assay) and mitochondrial membrane potential (through the fluorescent probe JC1).
Results
Our results showed that IWR-1 reduces only the viability and proliferation of KOPN-8 cells in a time and dose dependent manner (being the maximal inhibitory concentration, IC50, approximately 50 μM at 48 hours of exposure), having no effect in CEM cells. On the other hand, GDC-0449 and GSXXI reduces cell viability and proliferation in a time, dose and cell line dependent manner, being the KOPN-8 cells the most sensitive. We found that the IC50 at 48 hours to GDC-0449 was 75 μM and 150 μM and to GSXXI was 25 μM and 30 μM, for KOPN-8 and CEM cells, respectively. All inhibitors induced cell death by apoptosis, confirmed by the BAX/BCL-2 ratio, morphological analysis, and mitochondrial membrane depolarization. The analysis of cell cycle progression revealed that all inhibitors induce cell cycle arrest in G0/G1 phase in both cell lines, except in CEM when incubated with IWR-1
Conclusion
In conclusion, our results suggest that IWR-1, GDC-0449 and GSXXI could be a potential new targeted therapy in acute lymphoblastic leukemia; however, the therapeutic efficacy is cell type-dependent. This work was supported by CIMAGO, GAI, FMUC, and Banco Santander Totta; Ana Pires by LPCC/Pfizer 2015, Joana Jorge by LPCC-NRC/CIMAGO, and Raquel Alves by FCT (SFRH/BD/51994/2012) grants.
Session topic: E-poster
Keyword(s): B cell acute lymphoblastic leukemia, Signaling, Signaling molecules, T cell acute lymphoblastic leukemia
Abstract: PB1589
Type: Publication Only
Background
Embrionary signaling pathways, such as Wingless (WNT), Hedgehog (Hh), and Notch pathways, are essential for differentiation and self-regulation of stem cells, and their deregulation have been associated with several hematological neoplasias. Acute lymphoblastic leukemia (ALL) is characterized by the abnormal proliferation and accumulation of immature lymphoid cells within the bone marrow and lymphoid tissues, which can arise from the aberrant activation of embryonic signaling pathways. Therefore, these pathways may constitute new therapeutic targets for ALL treatment.
Aims
Evaluate the therapeutic potential of IWR-1, GDC-0449, and Gamma Secretase XXI inhibitor (GSXXI), inhibitors of WNT, Hh and Notch signaling pathways, respectively, in ALL in vitro models.
Methods
For this purpose, we used two ALL cell lines, CEM as a T cell ALL model, and KOPN-8 as a B cell ALL model. Both cell lines were cultured in absence and presence of different concentrations of IWR-1, GDC-0449 and GSXXI. Trypan blue assay was used to evaluate the effect of these inhibitors on cell viability and density. Cell death was determined by optical microscopy (May-Grunwald Giemsa staining) and by flow cytometry (FC) using the Annexin V and Propidium Iodide double staining. FC was also used to measure levels of apoptosis modulators, BAX and BCL-2, cell cycle (PI/RNase assay) and mitochondrial membrane potential (through the fluorescent probe JC1).
Results
Our results showed that IWR-1 reduces only the viability and proliferation of KOPN-8 cells in a time and dose dependent manner (being the maximal inhibitory concentration, IC50, approximately 50 μM at 48 hours of exposure), having no effect in CEM cells. On the other hand, GDC-0449 and GSXXI reduces cell viability and proliferation in a time, dose and cell line dependent manner, being the KOPN-8 cells the most sensitive. We found that the IC50 at 48 hours to GDC-0449 was 75 μM and 150 μM and to GSXXI was 25 μM and 30 μM, for KOPN-8 and CEM cells, respectively. All inhibitors induced cell death by apoptosis, confirmed by the BAX/BCL-2 ratio, morphological analysis, and mitochondrial membrane depolarization. The analysis of cell cycle progression revealed that all inhibitors induce cell cycle arrest in G0/G1 phase in both cell lines, except in CEM when incubated with IWR-1
Conclusion
In conclusion, our results suggest that IWR-1, GDC-0449 and GSXXI could be a potential new targeted therapy in acute lymphoblastic leukemia; however, the therapeutic efficacy is cell type-dependent. This work was supported by CIMAGO, GAI, FMUC, and Banco Santander Totta; Ana Pires by LPCC/Pfizer 2015, Joana Jorge by LPCC-NRC/CIMAGO, and Raquel Alves by FCT (SFRH/BD/51994/2012) grants.
Session topic: E-poster
Keyword(s): B cell acute lymphoblastic leukemia, Signaling, Signaling molecules, T cell acute lymphoblastic leukemia
Type: Publication Only
Background
Embrionary signaling pathways, such as Wingless (WNT), Hedgehog (Hh), and Notch pathways, are essential for differentiation and self-regulation of stem cells, and their deregulation have been associated with several hematological neoplasias. Acute lymphoblastic leukemia (ALL) is characterized by the abnormal proliferation and accumulation of immature lymphoid cells within the bone marrow and lymphoid tissues, which can arise from the aberrant activation of embryonic signaling pathways. Therefore, these pathways may constitute new therapeutic targets for ALL treatment.
Aims
Evaluate the therapeutic potential of IWR-1, GDC-0449, and Gamma Secretase XXI inhibitor (GSXXI), inhibitors of WNT, Hh and Notch signaling pathways, respectively, in ALL in vitro models.
Methods
For this purpose, we used two ALL cell lines, CEM as a T cell ALL model, and KOPN-8 as a B cell ALL model. Both cell lines were cultured in absence and presence of different concentrations of IWR-1, GDC-0449 and GSXXI. Trypan blue assay was used to evaluate the effect of these inhibitors on cell viability and density. Cell death was determined by optical microscopy (May-Grunwald Giemsa staining) and by flow cytometry (FC) using the Annexin V and Propidium Iodide double staining. FC was also used to measure levels of apoptosis modulators, BAX and BCL-2, cell cycle (PI/RNase assay) and mitochondrial membrane potential (through the fluorescent probe JC1).
Results
Our results showed that IWR-1 reduces only the viability and proliferation of KOPN-8 cells in a time and dose dependent manner (being the maximal inhibitory concentration, IC50, approximately 50 μM at 48 hours of exposure), having no effect in CEM cells. On the other hand, GDC-0449 and GSXXI reduces cell viability and proliferation in a time, dose and cell line dependent manner, being the KOPN-8 cells the most sensitive. We found that the IC50 at 48 hours to GDC-0449 was 75 μM and 150 μM and to GSXXI was 25 μM and 30 μM, for KOPN-8 and CEM cells, respectively. All inhibitors induced cell death by apoptosis, confirmed by the BAX/BCL-2 ratio, morphological analysis, and mitochondrial membrane depolarization. The analysis of cell cycle progression revealed that all inhibitors induce cell cycle arrest in G0/G1 phase in both cell lines, except in CEM when incubated with IWR-1
Conclusion
In conclusion, our results suggest that IWR-1, GDC-0449 and GSXXI could be a potential new targeted therapy in acute lymphoblastic leukemia; however, the therapeutic efficacy is cell type-dependent. This work was supported by CIMAGO, GAI, FMUC, and Banco Santander Totta; Ana Pires by LPCC/Pfizer 2015, Joana Jorge by LPCC-NRC/CIMAGO, and Raquel Alves by FCT (SFRH/BD/51994/2012) grants.
Session topic: E-poster
Keyword(s): B cell acute lymphoblastic leukemia, Signaling, Signaling molecules, T cell acute lymphoblastic leukemia
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