Contributions
Abstract: PB1350
Type: Publication Only
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Acute leukemias are a group of hematological neoplasias characterized by a dysregulation in cell proliferation and/or an arrest in the differentiation process of hematopoietic stem cells. Oxidative stress (OS) has been referred as one of the mechanisms involved in the pathophysiology of these diseases. The OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor (TF) that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy. Based on this, the modulation of this transcription factor may represent a therapeutic approach in acute leukemias.
Aims
The aim of this study was to investigate the therapeutical potential of the NRF2 modulator Oltipraz (Olt) in acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cell lines.
Methods
To this propose, 3 ALLs cell lines, two from T lineage (CEM and MOLT-4 cells) and one B lineage (KOPN8 cells), and two AML (HEL and NB4 cells) were incubated in the absence and presence of Oltipraz in several concentrations for 72h. Metabolic activity was evaluated by resazurin assay. Cell death was assessed by flow cytometry (FC), using the Annexin V and 7-AAD double staining, and by optic microscopy (May-Grünwald-Giemsa staining). Cell cycle analysis was evaluated by FC using a PI/RNAse solution. The intracellular levels of ROS and GSH were quantified by FC (DCFH2-DA, DHE, and MO probes). JC-1 probe was used to determine the mitochondrial membrane potential (Ymit) by FC. The statistical analysis was performed, considering a significance level of 95%.
Results
Oltipraz reduced metabolic activity in a time, dose, and cell line-dependent manner. After 72h of incubation, B-ALL cell line was the most sensitive to Olt (KOPN8 IC50= 4.5µM) comparing to T-ALL (MOLT-4 IC50= 87.4µM; CEM IC50= 40.4µM) and AML (HEL IC50= 63µM, NB4 IC50= 21µM) models. This drug induced a cytotoxic effect mediated by apoptosis in AML cells. It was also observed a cytostatic effect in B-ALL cells, with a cell cycle arrest in G0/G1 phase, and in AML cell lines with a cell cycle arrest in S phase. Moreover, Olt increased the intracellular levels of ROS and ROS/GSH ratio and decreased the mitochondrial membrane potential (Ymit) in all cell lines. These results support the role of OS and mitochondria dysfunction in the therapeutic effects mediated by Oltipraz in acute leukemias.
Conclusion
Our results demonstrate that lymphoid and myeloid acute leukemias were sensitive to NRF2 modulation using Oltipraz, suggesting that this drug may constitute a new therapeutic approach in these hematological neoplasms. However, the drug efficacy is cell type dependent and could be also related with molecular/genetic characteristics of the leukemia.
This work was supported by FMUC, CIMAGO and FCT (SFRH/BD/145531/2019).
Keyword(s): Acute leukemia, Targeted therapy, Transcription factor
Abstract: PB1350
Type: Publication Only
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Acute leukemias are a group of hematological neoplasias characterized by a dysregulation in cell proliferation and/or an arrest in the differentiation process of hematopoietic stem cells. Oxidative stress (OS) has been referred as one of the mechanisms involved in the pathophysiology of these diseases. The OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor (TF) that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy. Based on this, the modulation of this transcription factor may represent a therapeutic approach in acute leukemias.
Aims
The aim of this study was to investigate the therapeutical potential of the NRF2 modulator Oltipraz (Olt) in acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cell lines.
Methods
To this propose, 3 ALLs cell lines, two from T lineage (CEM and MOLT-4 cells) and one B lineage (KOPN8 cells), and two AML (HEL and NB4 cells) were incubated in the absence and presence of Oltipraz in several concentrations for 72h. Metabolic activity was evaluated by resazurin assay. Cell death was assessed by flow cytometry (FC), using the Annexin V and 7-AAD double staining, and by optic microscopy (May-Grünwald-Giemsa staining). Cell cycle analysis was evaluated by FC using a PI/RNAse solution. The intracellular levels of ROS and GSH were quantified by FC (DCFH2-DA, DHE, and MO probes). JC-1 probe was used to determine the mitochondrial membrane potential (Ymit) by FC. The statistical analysis was performed, considering a significance level of 95%.
Results
Oltipraz reduced metabolic activity in a time, dose, and cell line-dependent manner. After 72h of incubation, B-ALL cell line was the most sensitive to Olt (KOPN8 IC50= 4.5µM) comparing to T-ALL (MOLT-4 IC50= 87.4µM; CEM IC50= 40.4µM) and AML (HEL IC50= 63µM, NB4 IC50= 21µM) models. This drug induced a cytotoxic effect mediated by apoptosis in AML cells. It was also observed a cytostatic effect in B-ALL cells, with a cell cycle arrest in G0/G1 phase, and in AML cell lines with a cell cycle arrest in S phase. Moreover, Olt increased the intracellular levels of ROS and ROS/GSH ratio and decreased the mitochondrial membrane potential (Ymit) in all cell lines. These results support the role of OS and mitochondria dysfunction in the therapeutic effects mediated by Oltipraz in acute leukemias.
Conclusion
Our results demonstrate that lymphoid and myeloid acute leukemias were sensitive to NRF2 modulation using Oltipraz, suggesting that this drug may constitute a new therapeutic approach in these hematological neoplasms. However, the drug efficacy is cell type dependent and could be also related with molecular/genetic characteristics of the leukemia.
This work was supported by FMUC, CIMAGO and FCT (SFRH/BD/145531/2019).
Keyword(s): Acute leukemia, Targeted therapy, Transcription factor