BOTANICAL ALKYL HYDROQUINONE HQ17(3) INDUCES ENDOPLASMIC-RETICULUM STRESS AND AUTOPHAGY BEFORE THE ONSET OF CYTOTOXICITY ON THE T(9;22) PHILADELPHIA CHROMOSOME POSITIVE SUP-B15 ALL CELLS
(Abstract release date: 05/19/16)
EHA Library. Hu C. 06/09/16; 134503; PB1603
Dr. Chung-Yi Hu
Contributions
Contributions
Abstract
Abstract: PB1603
Type: Publication Only
Background
Acute lymphoblastic leukemia with Philadelphia chromosome (Ph+-ALL)(t(9;22)BCR-ABL) is a very high risk (VHR) hematological neoplasm. Constitutively active BCR-ABL oncoprotein together with multiple genetic lesions contribute to a very aggressive clinical cause that tyrosine kinase inhibitors (TKIs) combined with multi-agent chemotherapy fail to convey long-term disease control. Searching for agents selective to leukemias and investigating the molecule mechanisms involved in the inhibitory effects on leukemic cells will help to develop new anti-leukemic therapeutics for the Ph+-ALL. HQ17(3) [10’(Z),13’(E),15’(E)-heptadecatrienyl-hydroquinone], a natural product isolated from the sap of Rhus succedanea, exhibited very effective cytotoxic effect on the TKI-refractory Ph+-ALL SUP-B15 (IC50:1.9 µM) and other ALL cell lines, but spared normal peripheral blood mononuclear cells. Combination of HQ17(3) and Imatinib has synergistic cytotoxic effect on SUP-B15 cells.
Aims
To investigate the characters of, and the molecular pathways involved in the HQ17(3)-induced cytotoxic effects in Ph+-ALL SUP-B15 cells.
Methods
HQ17(3)-treated and control SUP-B15 cells were stained and analyzed by flow cytometry: membrane lipid disturbance was analyzed by Annexin V/PI stain, DNA fragmentation was defined as sub-G1 fraction of cellular DNA content after the PI staining, mitochondrial membrane potential (MMP) loss were stained by DiOC6(3). Pan-caspase inhibitor (zVAD-fmk), receptor interacting protein 1 (RIP1) inhibitor (necrostatin-1, Nec-1), iron-chelator (deferroxamine, DFO), 3-methyladenine (3MA) or chloroquine (CQ) (autophagy inhibitors), and lysosomal protease inhibitors were used in combination with HQ17(3) in some experiments. Acridine orange stain and confocal microscopy are used to visualize the changes of lysosomes in the presence of HQ17(3). Autophagic flow in response to HQ17(3) was revealed by accumulation of LC3B-II visualized by western blot analysis and aggregation of ectopically expressed GFP-LC3 indicator.
Results
Introduction of HQ17(3) induced extensive cell death within 24 hours characterized by losing plasma membrane integrity (PI+) concomitant with PS exposure (Annexin V+), which was not prevented by zVAD-fmk and/or Nec-1. Cell death displayed MMP loss and profound nuclear DNA fragmentation that could be attenuated by ROS scavengers. Acidic vesicles were significantly increased 4 hours after treatment of HQ17(3) then diminished when cell death was evident. Lysosomotropic DFO abolished the HQ17(3)-induced acidic vesicles and subsequent cell death. Application of AEBSF (serine protease inh.) and/or pepstatin/CA074-Me (cathepsin D/B inh.) did not rescue cells from death. Autophagy markers were enhanced in HQ17(3)-treated cells. Autophagy inhibitors (3MA and CQ) showed a modest protective effect. Further, HQ17(3) treatment gave rise to early and sustained AKT and mTOR activation and induced ER stress as shown unfolded protein response markers eIF2a phosphorylation and upregulated ER chaperon Grp78.
Conclusion
Naturally-derived HQ17(3) displayed significant cytotoxicity on Ph+-ALL SUP-B15 cells. HQ17(3) leads to ER stress and induces iron-dependent autophagy followed by necrotic-like, caspase-independent cell demise that is different from RIP1-mediated necroptosis or lysosomal protease-mediated cell death. These results suggest that agents selectively induce or sustain ROS in leukemic cells may induce ER stress and autophagy-associated cell death, and would potentially augment the treatment for VHR-ALL with t(9;22) translocation.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Cell line, Cytotoxicity
Type: Publication Only
Background
Acute lymphoblastic leukemia with Philadelphia chromosome (Ph+-ALL)(t(9;22)BCR-ABL) is a very high risk (VHR) hematological neoplasm. Constitutively active BCR-ABL oncoprotein together with multiple genetic lesions contribute to a very aggressive clinical cause that tyrosine kinase inhibitors (TKIs) combined with multi-agent chemotherapy fail to convey long-term disease control. Searching for agents selective to leukemias and investigating the molecule mechanisms involved in the inhibitory effects on leukemic cells will help to develop new anti-leukemic therapeutics for the Ph+-ALL. HQ17(3) [10’(Z),13’(E),15’(E)-heptadecatrienyl-hydroquinone], a natural product isolated from the sap of Rhus succedanea, exhibited very effective cytotoxic effect on the TKI-refractory Ph+-ALL SUP-B15 (IC50:1.9 µM) and other ALL cell lines, but spared normal peripheral blood mononuclear cells. Combination of HQ17(3) and Imatinib has synergistic cytotoxic effect on SUP-B15 cells.
Aims
To investigate the characters of, and the molecular pathways involved in the HQ17(3)-induced cytotoxic effects in Ph+-ALL SUP-B15 cells.
Methods
HQ17(3)-treated and control SUP-B15 cells were stained and analyzed by flow cytometry: membrane lipid disturbance was analyzed by Annexin V/PI stain, DNA fragmentation was defined as sub-G1 fraction of cellular DNA content after the PI staining, mitochondrial membrane potential (MMP) loss were stained by DiOC6(3). Pan-caspase inhibitor (zVAD-fmk), receptor interacting protein 1 (RIP1) inhibitor (necrostatin-1, Nec-1), iron-chelator (deferroxamine, DFO), 3-methyladenine (3MA) or chloroquine (CQ) (autophagy inhibitors), and lysosomal protease inhibitors were used in combination with HQ17(3) in some experiments. Acridine orange stain and confocal microscopy are used to visualize the changes of lysosomes in the presence of HQ17(3). Autophagic flow in response to HQ17(3) was revealed by accumulation of LC3B-II visualized by western blot analysis and aggregation of ectopically expressed GFP-LC3 indicator.
Results
Introduction of HQ17(3) induced extensive cell death within 24 hours characterized by losing plasma membrane integrity (PI+) concomitant with PS exposure (Annexin V+), which was not prevented by zVAD-fmk and/or Nec-1. Cell death displayed MMP loss and profound nuclear DNA fragmentation that could be attenuated by ROS scavengers. Acidic vesicles were significantly increased 4 hours after treatment of HQ17(3) then diminished when cell death was evident. Lysosomotropic DFO abolished the HQ17(3)-induced acidic vesicles and subsequent cell death. Application of AEBSF (serine protease inh.) and/or pepstatin/CA074-Me (cathepsin D/B inh.) did not rescue cells from death. Autophagy markers were enhanced in HQ17(3)-treated cells. Autophagy inhibitors (3MA and CQ) showed a modest protective effect. Further, HQ17(3) treatment gave rise to early and sustained AKT and mTOR activation and induced ER stress as shown unfolded protein response markers eIF2a phosphorylation and upregulated ER chaperon Grp78.
Conclusion
Naturally-derived HQ17(3) displayed significant cytotoxicity on Ph+-ALL SUP-B15 cells. HQ17(3) leads to ER stress and induces iron-dependent autophagy followed by necrotic-like, caspase-independent cell demise that is different from RIP1-mediated necroptosis or lysosomal protease-mediated cell death. These results suggest that agents selectively induce or sustain ROS in leukemic cells may induce ER stress and autophagy-associated cell death, and would potentially augment the treatment for VHR-ALL with t(9;22) translocation.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Cell line, Cytotoxicity
Abstract: PB1603
Type: Publication Only
Background
Acute lymphoblastic leukemia with Philadelphia chromosome (Ph+-ALL)(t(9;22)BCR-ABL) is a very high risk (VHR) hematological neoplasm. Constitutively active BCR-ABL oncoprotein together with multiple genetic lesions contribute to a very aggressive clinical cause that tyrosine kinase inhibitors (TKIs) combined with multi-agent chemotherapy fail to convey long-term disease control. Searching for agents selective to leukemias and investigating the molecule mechanisms involved in the inhibitory effects on leukemic cells will help to develop new anti-leukemic therapeutics for the Ph+-ALL. HQ17(3) [10’(Z),13’(E),15’(E)-heptadecatrienyl-hydroquinone], a natural product isolated from the sap of Rhus succedanea, exhibited very effective cytotoxic effect on the TKI-refractory Ph+-ALL SUP-B15 (IC50:1.9 µM) and other ALL cell lines, but spared normal peripheral blood mononuclear cells. Combination of HQ17(3) and Imatinib has synergistic cytotoxic effect on SUP-B15 cells.
Aims
To investigate the characters of, and the molecular pathways involved in the HQ17(3)-induced cytotoxic effects in Ph+-ALL SUP-B15 cells.
Methods
HQ17(3)-treated and control SUP-B15 cells were stained and analyzed by flow cytometry: membrane lipid disturbance was analyzed by Annexin V/PI stain, DNA fragmentation was defined as sub-G1 fraction of cellular DNA content after the PI staining, mitochondrial membrane potential (MMP) loss were stained by DiOC6(3). Pan-caspase inhibitor (zVAD-fmk), receptor interacting protein 1 (RIP1) inhibitor (necrostatin-1, Nec-1), iron-chelator (deferroxamine, DFO), 3-methyladenine (3MA) or chloroquine (CQ) (autophagy inhibitors), and lysosomal protease inhibitors were used in combination with HQ17(3) in some experiments. Acridine orange stain and confocal microscopy are used to visualize the changes of lysosomes in the presence of HQ17(3). Autophagic flow in response to HQ17(3) was revealed by accumulation of LC3B-II visualized by western blot analysis and aggregation of ectopically expressed GFP-LC3 indicator.
Results
Introduction of HQ17(3) induced extensive cell death within 24 hours characterized by losing plasma membrane integrity (PI+) concomitant with PS exposure (Annexin V+), which was not prevented by zVAD-fmk and/or Nec-1. Cell death displayed MMP loss and profound nuclear DNA fragmentation that could be attenuated by ROS scavengers. Acidic vesicles were significantly increased 4 hours after treatment of HQ17(3) then diminished when cell death was evident. Lysosomotropic DFO abolished the HQ17(3)-induced acidic vesicles and subsequent cell death. Application of AEBSF (serine protease inh.) and/or pepstatin/CA074-Me (cathepsin D/B inh.) did not rescue cells from death. Autophagy markers were enhanced in HQ17(3)-treated cells. Autophagy inhibitors (3MA and CQ) showed a modest protective effect. Further, HQ17(3) treatment gave rise to early and sustained AKT and mTOR activation and induced ER stress as shown unfolded protein response markers eIF2a phosphorylation and upregulated ER chaperon Grp78.
Conclusion
Naturally-derived HQ17(3) displayed significant cytotoxicity on Ph+-ALL SUP-B15 cells. HQ17(3) leads to ER stress and induces iron-dependent autophagy followed by necrotic-like, caspase-independent cell demise that is different from RIP1-mediated necroptosis or lysosomal protease-mediated cell death. These results suggest that agents selectively induce or sustain ROS in leukemic cells may induce ER stress and autophagy-associated cell death, and would potentially augment the treatment for VHR-ALL with t(9;22) translocation.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Cell line, Cytotoxicity
Type: Publication Only
Background
Acute lymphoblastic leukemia with Philadelphia chromosome (Ph+-ALL)(t(9;22)BCR-ABL) is a very high risk (VHR) hematological neoplasm. Constitutively active BCR-ABL oncoprotein together with multiple genetic lesions contribute to a very aggressive clinical cause that tyrosine kinase inhibitors (TKIs) combined with multi-agent chemotherapy fail to convey long-term disease control. Searching for agents selective to leukemias and investigating the molecule mechanisms involved in the inhibitory effects on leukemic cells will help to develop new anti-leukemic therapeutics for the Ph+-ALL. HQ17(3) [10’(Z),13’(E),15’(E)-heptadecatrienyl-hydroquinone], a natural product isolated from the sap of Rhus succedanea, exhibited very effective cytotoxic effect on the TKI-refractory Ph+-ALL SUP-B15 (IC50:1.9 µM) and other ALL cell lines, but spared normal peripheral blood mononuclear cells. Combination of HQ17(3) and Imatinib has synergistic cytotoxic effect on SUP-B15 cells.
Aims
To investigate the characters of, and the molecular pathways involved in the HQ17(3)-induced cytotoxic effects in Ph+-ALL SUP-B15 cells.
Methods
HQ17(3)-treated and control SUP-B15 cells were stained and analyzed by flow cytometry: membrane lipid disturbance was analyzed by Annexin V/PI stain, DNA fragmentation was defined as sub-G1 fraction of cellular DNA content after the PI staining, mitochondrial membrane potential (MMP) loss were stained by DiOC6(3). Pan-caspase inhibitor (zVAD-fmk), receptor interacting protein 1 (RIP1) inhibitor (necrostatin-1, Nec-1), iron-chelator (deferroxamine, DFO), 3-methyladenine (3MA) or chloroquine (CQ) (autophagy inhibitors), and lysosomal protease inhibitors were used in combination with HQ17(3) in some experiments. Acridine orange stain and confocal microscopy are used to visualize the changes of lysosomes in the presence of HQ17(3). Autophagic flow in response to HQ17(3) was revealed by accumulation of LC3B-II visualized by western blot analysis and aggregation of ectopically expressed GFP-LC3 indicator.
Results
Introduction of HQ17(3) induced extensive cell death within 24 hours characterized by losing plasma membrane integrity (PI+) concomitant with PS exposure (Annexin V+), which was not prevented by zVAD-fmk and/or Nec-1. Cell death displayed MMP loss and profound nuclear DNA fragmentation that could be attenuated by ROS scavengers. Acidic vesicles were significantly increased 4 hours after treatment of HQ17(3) then diminished when cell death was evident. Lysosomotropic DFO abolished the HQ17(3)-induced acidic vesicles and subsequent cell death. Application of AEBSF (serine protease inh.) and/or pepstatin/CA074-Me (cathepsin D/B inh.) did not rescue cells from death. Autophagy markers were enhanced in HQ17(3)-treated cells. Autophagy inhibitors (3MA and CQ) showed a modest protective effect. Further, HQ17(3) treatment gave rise to early and sustained AKT and mTOR activation and induced ER stress as shown unfolded protein response markers eIF2a phosphorylation and upregulated ER chaperon Grp78.
Conclusion
Naturally-derived HQ17(3) displayed significant cytotoxicity on Ph+-ALL SUP-B15 cells. HQ17(3) leads to ER stress and induces iron-dependent autophagy followed by necrotic-like, caspase-independent cell demise that is different from RIP1-mediated necroptosis or lysosomal protease-mediated cell death. These results suggest that agents selectively induce or sustain ROS in leukemic cells may induce ER stress and autophagy-associated cell death, and would potentially augment the treatment for VHR-ALL with t(9;22) translocation.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Cell line, Cytotoxicity
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