Contributions
Abstract: PB1611
Type: Publication Only
Background
Patients suffering from Acute lymphoblastic leukemias (ALLs) harboring t(9;22) genetic abnormality are classified very high risk (VHR) ALLs displaying poor clinical outcome irrespective of intensive chemotherapies and tyrosine kinase inhibitor treatment. Development of new adjuvant therapeutics will provide great value. HQ17(3)[10’(Z),13’(E),15’(E)-heptadecatrienyl hydroquinone] isolated from sap of the lacquer tree showed potent cytotoxic effect within 24 hours at micromolar concentration on several ALL cell lines, including TKI (Imatinib, IM)-refractory Ph+ B-ALL cell line SUP-B15 cells, but spare normal PB leukocytes, and were non-toxic in experimental rats after 28-day HQ17(3) injection. Thus HQ17(3) presents as a potential anti-leukemic agent and serves a model for design anti-leukemic regimen. We previously showed HQ17(3)-induced rapid cell demise, characterized by oxidative stress, loss of membrane integrity, mitochondrial membrane potential disturbance and nuclear DNA fragmentation. Neither pan-caspase inhibitor nor Nec-1 (RIP-1 inhihitor) protected SUP-B15 cells from HQ17(3)-induced cell death. The cell death program elicited by HQ17(3) is caspase-independent, and is different from the RIP1-mediated controlled necroptosis.
Aims
To investigate the characters of, and the molecular pathways involved in the HQ17(3)-induced non-classical death on VHR-ALL SUP-B15 cells and help developing effective therapeutic strategies for the VHR-ALLs.
Methods
Cell growth inhibition in response to HQ17(3) w/wo inhibitors was analyzed by ACP assay. Cells were stained by Annexin V/PI and analyzed by flow cytometry for cell death. Lysosomal protease inhibitors (AEBSF (serine protease inh.), pepstatin/CA074-Me (cathepsin D/B inh.)) or autophagy inhibitors (Bafilomycin A1) were used in combination with HQ17(3) in some experiments. Acridine orange stain and confocal microscopy are used to visualize the changes of acidic vesicles. Autophagic flow in response to HQ17(3) was revealed by aggregation of ectopically expressed EGFP-LC3. Western blot analysis were used to analyze p- eIF2a, ER chaperone Grp78, spliced XBP-1 (markers for ER stress). Lenti-viral delivery of shRNAs was used to repress the expression of Beclin-1. Nuclear accumulation of apoptosis inducing factor (AIF) was revealed by fluorescence microscopy.
Results
Enlarged acidic vesicles accumulated soon after HQ17(3) treatment, and deminished when cell death ensued. HQ17(3)-induced cell death could not be attributed to cathepsins released from lysosomal membrane permeabilization (LMP) as cathepsin inhibitors did not attenuate the cell death. HQ17(3) enhanced autophagy as revealed by aggregation of ectopically expressed EGFP-LC3. Inhibition of autophagy by Bafilomycin A1 or knockdown the essential autophagy-related Beclin1 by shRNA could partially attenuate HQ17(3)-induced cell death. Further, HQ17(3) treatment gave rise to early ER stress as revealed by enhancement of eIF2a phosphorylation and up-regulation of ER chaperone Grp78. HQ17(3) induced nuclear translocation of AIF, in compatible with mitochondria disturbance and caspase-independent cell death thereafter.
Conclusion
In Ph+-ALL SUP-B15 cells, HQ17(3) acts in multi-facet: a) lead to oxidative stress and perturb mitochondria membrane integrity, b) induce ER stress and calcium mobilization to mitochondria, cleave and release AIF to mediate nuclear chromatin cleavage, c) HQ17(3)-induced autophagy may be implicated cell death. This study shows agents that are capable of eliciting an intricate effector network in therapy-induced cytotoxicity will have potential as adjuvants controlling the VHR-Ph+-ALL cells refractory to conventional high dose chemotherapies and TKI regime.
Session topic: 1. Acute lymphoblastic leukemia - Biology
Keyword(s): Cytotoxicity, Cell line, Ph+ ALL
Abstract: PB1611
Type: Publication Only
Background
Patients suffering from Acute lymphoblastic leukemias (ALLs) harboring t(9;22) genetic abnormality are classified very high risk (VHR) ALLs displaying poor clinical outcome irrespective of intensive chemotherapies and tyrosine kinase inhibitor treatment. Development of new adjuvant therapeutics will provide great value. HQ17(3)[10’(Z),13’(E),15’(E)-heptadecatrienyl hydroquinone] isolated from sap of the lacquer tree showed potent cytotoxic effect within 24 hours at micromolar concentration on several ALL cell lines, including TKI (Imatinib, IM)-refractory Ph+ B-ALL cell line SUP-B15 cells, but spare normal PB leukocytes, and were non-toxic in experimental rats after 28-day HQ17(3) injection. Thus HQ17(3) presents as a potential anti-leukemic agent and serves a model for design anti-leukemic regimen. We previously showed HQ17(3)-induced rapid cell demise, characterized by oxidative stress, loss of membrane integrity, mitochondrial membrane potential disturbance and nuclear DNA fragmentation. Neither pan-caspase inhibitor nor Nec-1 (RIP-1 inhihitor) protected SUP-B15 cells from HQ17(3)-induced cell death. The cell death program elicited by HQ17(3) is caspase-independent, and is different from the RIP1-mediated controlled necroptosis.
Aims
To investigate the characters of, and the molecular pathways involved in the HQ17(3)-induced non-classical death on VHR-ALL SUP-B15 cells and help developing effective therapeutic strategies for the VHR-ALLs.
Methods
Cell growth inhibition in response to HQ17(3) w/wo inhibitors was analyzed by ACP assay. Cells were stained by Annexin V/PI and analyzed by flow cytometry for cell death. Lysosomal protease inhibitors (AEBSF (serine protease inh.), pepstatin/CA074-Me (cathepsin D/B inh.)) or autophagy inhibitors (Bafilomycin A1) were used in combination with HQ17(3) in some experiments. Acridine orange stain and confocal microscopy are used to visualize the changes of acidic vesicles. Autophagic flow in response to HQ17(3) was revealed by aggregation of ectopically expressed EGFP-LC3. Western blot analysis were used to analyze p- eIF2a, ER chaperone Grp78, spliced XBP-1 (markers for ER stress). Lenti-viral delivery of shRNAs was used to repress the expression of Beclin-1. Nuclear accumulation of apoptosis inducing factor (AIF) was revealed by fluorescence microscopy.
Results
Enlarged acidic vesicles accumulated soon after HQ17(3) treatment, and deminished when cell death ensued. HQ17(3)-induced cell death could not be attributed to cathepsins released from lysosomal membrane permeabilization (LMP) as cathepsin inhibitors did not attenuate the cell death. HQ17(3) enhanced autophagy as revealed by aggregation of ectopically expressed EGFP-LC3. Inhibition of autophagy by Bafilomycin A1 or knockdown the essential autophagy-related Beclin1 by shRNA could partially attenuate HQ17(3)-induced cell death. Further, HQ17(3) treatment gave rise to early ER stress as revealed by enhancement of eIF2a phosphorylation and up-regulation of ER chaperone Grp78. HQ17(3) induced nuclear translocation of AIF, in compatible with mitochondria disturbance and caspase-independent cell death thereafter.
Conclusion
In Ph+-ALL SUP-B15 cells, HQ17(3) acts in multi-facet: a) lead to oxidative stress and perturb mitochondria membrane integrity, b) induce ER stress and calcium mobilization to mitochondria, cleave and release AIF to mediate nuclear chromatin cleavage, c) HQ17(3)-induced autophagy may be implicated cell death. This study shows agents that are capable of eliciting an intricate effector network in therapy-induced cytotoxicity will have potential as adjuvants controlling the VHR-Ph+-ALL cells refractory to conventional high dose chemotherapies and TKI regime.
Session topic: 1. Acute lymphoblastic leukemia - Biology
Keyword(s): Cytotoxicity, Cell line, Ph+ ALL