REVERSINE TRIGGERS MITOTIC CATASTROPHE AND APOPTOSIS IN BCR-ABL POSITIVE CELLS
(Abstract release date: 05/19/16)
EHA Library. Alves A. 06/09/16; 134714; PB1814
Mrs. Ana Alves
Contributions
Contributions
Abstract
Abstract: PB1814
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by the presence of the oncoprotein BCR-ABL1, which have a constitutive tyrosine kinase activity. The tyrosine kinase inhibitors for BCR-ABL1 (e.g. imatinib, dasatinib and nilotinib) can lead towards the elimination of the BCR-ABL1 clone and increase survival of CML patients. However, acquisition of resistance to tyrosine kinase inhibitors has emerged as a challenge for CML patients and the identification of novel targets is necessary. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Aurora kinase inhibition has proven to be an attractive anti-cancer approach. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1 positive K562 cell line.
Aims
Based on the evidence of participation of aurora kinases in the BCR-ABL1 signaling pathway and CML malignant phenotype, we explored the cellular effects of reversine in BCR-ABL1 positive cells.
Methods
K562 cell line was treated or not with the AURKA and AURKB pharmacological inhibitor, reversine, at 1, 10, 20 and 50µM for 24, 48 and 72 hours. After drug exposure, cells were evaluated for cell viability (MTT assay) and apoptosis (Annexin V/PI and cleavage caspase 3). For DNA content distribution analysis (Cycletest™ Plus DNA Reagent Kit) and Confocal immunofluorescence microscopy, K562 cells were treated or not with reversine at 1 and 10µM for 48 hours. Comparisons between the control and treated groups were performed by the ANOVA test and Bonferroni post-test. A p value <0.05 was considered statistically significant. At least three independent experiments for each method were tested.
Results
In K562 cells, reversine treatment significantly reduced cell viability in a dose- and time-dependent manner (all p<0.05). Using a nonlinear regression analysis, IC50 citotoxicity for K562 was 44.6 µM for 24 hours, 12.9 µM for 48 hours and 9.4 µM for 72 hours. We evaluate the DNA content distribution of K562 cells, since aurora kinases are known to be an important protein family for cell cycle progression and success of mitosis. We observed an increased percentage of K562 cells with tetraploidization (4N) or endopolyploidization (>4N) upon reversine treatment. These findings were confirmed by confocal microscopy analysis, which reveals large cells with several nucleus. Taken together, these data indicate that reversine is a potent inductor of mitotic catastrophe. Reversine significantly induced apoptosis in dose- and time-dependent manner (all p<0.05). In order to confirm our results regarding reversine-induced apoptosis, we also evaluated the caspase 3 cleavage in K562 cells and increased levels of cleaved-caspase 3 were observed in a dose-dependent manner.
Conclusion
Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anti-cancer opportunities for CML.
Session topic: E-poster
Keyword(s): Apoptosis, Cell cycle progression, Chronic myeloid leukemia
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by the presence of the oncoprotein BCR-ABL1, which have a constitutive tyrosine kinase activity. The tyrosine kinase inhibitors for BCR-ABL1 (e.g. imatinib, dasatinib and nilotinib) can lead towards the elimination of the BCR-ABL1 clone and increase survival of CML patients. However, acquisition of resistance to tyrosine kinase inhibitors has emerged as a challenge for CML patients and the identification of novel targets is necessary. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Aurora kinase inhibition has proven to be an attractive anti-cancer approach. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1 positive K562 cell line.
Aims
Based on the evidence of participation of aurora kinases in the BCR-ABL1 signaling pathway and CML malignant phenotype, we explored the cellular effects of reversine in BCR-ABL1 positive cells.
Methods
K562 cell line was treated or not with the AURKA and AURKB pharmacological inhibitor, reversine, at 1, 10, 20 and 50µM for 24, 48 and 72 hours. After drug exposure, cells were evaluated for cell viability (MTT assay) and apoptosis (Annexin V/PI and cleavage caspase 3). For DNA content distribution analysis (Cycletest™ Plus DNA Reagent Kit) and Confocal immunofluorescence microscopy, K562 cells were treated or not with reversine at 1 and 10µM for 48 hours. Comparisons between the control and treated groups were performed by the ANOVA test and Bonferroni post-test. A p value <0.05 was considered statistically significant. At least three independent experiments for each method were tested.
Results
In K562 cells, reversine treatment significantly reduced cell viability in a dose- and time-dependent manner (all p<0.05). Using a nonlinear regression analysis, IC50 citotoxicity for K562 was 44.6 µM for 24 hours, 12.9 µM for 48 hours and 9.4 µM for 72 hours. We evaluate the DNA content distribution of K562 cells, since aurora kinases are known to be an important protein family for cell cycle progression and success of mitosis. We observed an increased percentage of K562 cells with tetraploidization (4N) or endopolyploidization (>4N) upon reversine treatment. These findings were confirmed by confocal microscopy analysis, which reveals large cells with several nucleus. Taken together, these data indicate that reversine is a potent inductor of mitotic catastrophe. Reversine significantly induced apoptosis in dose- and time-dependent manner (all p<0.05). In order to confirm our results regarding reversine-induced apoptosis, we also evaluated the caspase 3 cleavage in K562 cells and increased levels of cleaved-caspase 3 were observed in a dose-dependent manner.
Conclusion
Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anti-cancer opportunities for CML.
Session topic: E-poster
Keyword(s): Apoptosis, Cell cycle progression, Chronic myeloid leukemia
Abstract: PB1814
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by the presence of the oncoprotein BCR-ABL1, which have a constitutive tyrosine kinase activity. The tyrosine kinase inhibitors for BCR-ABL1 (e.g. imatinib, dasatinib and nilotinib) can lead towards the elimination of the BCR-ABL1 clone and increase survival of CML patients. However, acquisition of resistance to tyrosine kinase inhibitors has emerged as a challenge for CML patients and the identification of novel targets is necessary. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Aurora kinase inhibition has proven to be an attractive anti-cancer approach. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1 positive K562 cell line.
Aims
Based on the evidence of participation of aurora kinases in the BCR-ABL1 signaling pathway and CML malignant phenotype, we explored the cellular effects of reversine in BCR-ABL1 positive cells.
Methods
K562 cell line was treated or not with the AURKA and AURKB pharmacological inhibitor, reversine, at 1, 10, 20 and 50µM for 24, 48 and 72 hours. After drug exposure, cells were evaluated for cell viability (MTT assay) and apoptosis (Annexin V/PI and cleavage caspase 3). For DNA content distribution analysis (Cycletest™ Plus DNA Reagent Kit) and Confocal immunofluorescence microscopy, K562 cells were treated or not with reversine at 1 and 10µM for 48 hours. Comparisons between the control and treated groups were performed by the ANOVA test and Bonferroni post-test. A p value <0.05 was considered statistically significant. At least three independent experiments for each method were tested.
Results
In K562 cells, reversine treatment significantly reduced cell viability in a dose- and time-dependent manner (all p<0.05). Using a nonlinear regression analysis, IC50 citotoxicity for K562 was 44.6 µM for 24 hours, 12.9 µM for 48 hours and 9.4 µM for 72 hours. We evaluate the DNA content distribution of K562 cells, since aurora kinases are known to be an important protein family for cell cycle progression and success of mitosis. We observed an increased percentage of K562 cells with tetraploidization (4N) or endopolyploidization (>4N) upon reversine treatment. These findings were confirmed by confocal microscopy analysis, which reveals large cells with several nucleus. Taken together, these data indicate that reversine is a potent inductor of mitotic catastrophe. Reversine significantly induced apoptosis in dose- and time-dependent manner (all p<0.05). In order to confirm our results regarding reversine-induced apoptosis, we also evaluated the caspase 3 cleavage in K562 cells and increased levels of cleaved-caspase 3 were observed in a dose-dependent manner.
Conclusion
Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anti-cancer opportunities for CML.
Session topic: E-poster
Keyword(s): Apoptosis, Cell cycle progression, Chronic myeloid leukemia
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by the presence of the oncoprotein BCR-ABL1, which have a constitutive tyrosine kinase activity. The tyrosine kinase inhibitors for BCR-ABL1 (e.g. imatinib, dasatinib and nilotinib) can lead towards the elimination of the BCR-ABL1 clone and increase survival of CML patients. However, acquisition of resistance to tyrosine kinase inhibitors has emerged as a challenge for CML patients and the identification of novel targets is necessary. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Aurora kinase inhibition has proven to be an attractive anti-cancer approach. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1 positive K562 cell line.
Aims
Based on the evidence of participation of aurora kinases in the BCR-ABL1 signaling pathway and CML malignant phenotype, we explored the cellular effects of reversine in BCR-ABL1 positive cells.
Methods
K562 cell line was treated or not with the AURKA and AURKB pharmacological inhibitor, reversine, at 1, 10, 20 and 50µM for 24, 48 and 72 hours. After drug exposure, cells were evaluated for cell viability (MTT assay) and apoptosis (Annexin V/PI and cleavage caspase 3). For DNA content distribution analysis (Cycletest™ Plus DNA Reagent Kit) and Confocal immunofluorescence microscopy, K562 cells were treated or not with reversine at 1 and 10µM for 48 hours. Comparisons between the control and treated groups were performed by the ANOVA test and Bonferroni post-test. A p value <0.05 was considered statistically significant. At least three independent experiments for each method were tested.
Results
In K562 cells, reversine treatment significantly reduced cell viability in a dose- and time-dependent manner (all p<0.05). Using a nonlinear regression analysis, IC50 citotoxicity for K562 was 44.6 µM for 24 hours, 12.9 µM for 48 hours and 9.4 µM for 72 hours. We evaluate the DNA content distribution of K562 cells, since aurora kinases are known to be an important protein family for cell cycle progression and success of mitosis. We observed an increased percentage of K562 cells with tetraploidization (4N) or endopolyploidization (>4N) upon reversine treatment. These findings were confirmed by confocal microscopy analysis, which reveals large cells with several nucleus. Taken together, these data indicate that reversine is a potent inductor of mitotic catastrophe. Reversine significantly induced apoptosis in dose- and time-dependent manner (all p<0.05). In order to confirm our results regarding reversine-induced apoptosis, we also evaluated the caspase 3 cleavage in K562 cells and increased levels of cleaved-caspase 3 were observed in a dose-dependent manner.
Conclusion
Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anti-cancer opportunities for CML.
Session topic: E-poster
Keyword(s): Apoptosis, Cell cycle progression, Chronic myeloid leukemia
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