Contributions
Abstract: PB1609
Type: Publication Only
Background
Acute lymphoblastic leukemias (ALLs) harboring t(9;22)(Ph+ -ALL) is classified as very high risk (VHR) ALL due to 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 in 24 hours at micromolar concentration on several ALL cell lines, including Imatinib-refractory Ph+-ALL SUP-B15 cells, spared normal PB leukocytes, and non-toxic in experimental rats. HQ17(3) presents as a potential anti-leukemic agents and a model for designing anti-leukemic regimen. We previously showed HQ17(3)-induced cell demise was characterized by oxidative stress, mitochondrial membrane potential (MMP) loss and nuclear DNA fragmentation. HQ17(3)-induced cell death is caspase-independent, and is different from the RIP1-mediated necroptosis or lysosomal permeabilization-mediated cell death since inhibitors to caspases, RIP-1K or lysosomal cathepsins/proteases failed to protect SUP-B15 cells from death. HQ17(3) enhanced autophagic flow and autophagy inhibitors attenuated the cell death. Interestingly, the ER stress markers (chaperon Grp78 and phosphorylated-eIF2a) were found upregulated as early as 5hr after HQ17(3) treatment.
Aims
To illustrate the characters of the HQ17(3)-induced non-classical death on Ph+-SUP-B15 cells, focus on ER stress and mitochondrial Ca2+ homeostasis.
Methods
Cell growth inhibition in response to HQ17(3) or inhibitors was analyzed by ACP assay. Annexin V/PI and flow cytometry analysis detected cell death. Autophagy was revealed by aggregation of ectopically expressed EGFP-LC3. MFN1/2, OPA1 (mitochondrial markers) were analyzed by western blot. Nuclear accumulation of apoptosis inducing factor (AIF), colocalization of mitochondrial COX-IV and LC3-II (mitophagy) were revealed by immunofluorescence stain and confocal microscopy. Mitochondrial Ca2+ [Ca2+m] accumulation was shown by fluorescent Rhod-2 probe, mitochondrial superoxide was measured after Mitosox stain.
Results
We showed [Ca2+m] accumulation at the time ER stress occurred, accompanied by mitochondrial superoxide elevation, followed by MMP loss and nuclear translocation of apoptosis-inducing factor (AIF). However, inhibition of AIF cleavage by calpain inhibitor PD150606 reduced the HQ17(3)-induced cell death slightly. Further, Ca2+ chelator Bapta-AM prevented [Ca2+m] overload and rescued cell death more effectively, indicating [Ca2+m] participated in other aspects in cell death. HQ17(3) treatment lead to decreased mitochondrial proteins MFN1/2 and OPA1, while Mitotracker green stain showed significant loss of mitochondrial mass preceded cell death, indicating damaged mitochondria succumbed to mitophagy, which was confirmed by the presence of COX IV and LC3B co-localization.
Conclusion
In Ph+-ALL SUP-B15 cells HQ17(3) induces ER stress by yet-defined mechanism, mobilizes Ca2+ to mitochondria, results in cleavage and release of AIF to mediate chromatin fragmentation, Ca2+-overload leads to oxidative stress in turn and perturbs mitochondria integrity. Damaged mitochondria induce extensive mitophagy and cell death ensues. Therefore, agents that help elicit similar intricate effector network associated with ER/mitochondria stress will have potential as adjuvants controlling the VHR-ALL cells refractory to conventional chemotherapies and TKI regime.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Calcium, Cytotoxicity, Mitochondria, Ph+ ALL
Abstract: PB1609
Type: Publication Only
Background
Acute lymphoblastic leukemias (ALLs) harboring t(9;22)(Ph+ -ALL) is classified as very high risk (VHR) ALL due to 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 in 24 hours at micromolar concentration on several ALL cell lines, including Imatinib-refractory Ph+-ALL SUP-B15 cells, spared normal PB leukocytes, and non-toxic in experimental rats. HQ17(3) presents as a potential anti-leukemic agents and a model for designing anti-leukemic regimen. We previously showed HQ17(3)-induced cell demise was characterized by oxidative stress, mitochondrial membrane potential (MMP) loss and nuclear DNA fragmentation. HQ17(3)-induced cell death is caspase-independent, and is different from the RIP1-mediated necroptosis or lysosomal permeabilization-mediated cell death since inhibitors to caspases, RIP-1K or lysosomal cathepsins/proteases failed to protect SUP-B15 cells from death. HQ17(3) enhanced autophagic flow and autophagy inhibitors attenuated the cell death. Interestingly, the ER stress markers (chaperon Grp78 and phosphorylated-eIF2a) were found upregulated as early as 5hr after HQ17(3) treatment.
Aims
To illustrate the characters of the HQ17(3)-induced non-classical death on Ph+-SUP-B15 cells, focus on ER stress and mitochondrial Ca2+ homeostasis.
Methods
Cell growth inhibition in response to HQ17(3) or inhibitors was analyzed by ACP assay. Annexin V/PI and flow cytometry analysis detected cell death. Autophagy was revealed by aggregation of ectopically expressed EGFP-LC3. MFN1/2, OPA1 (mitochondrial markers) were analyzed by western blot. Nuclear accumulation of apoptosis inducing factor (AIF), colocalization of mitochondrial COX-IV and LC3-II (mitophagy) were revealed by immunofluorescence stain and confocal microscopy. Mitochondrial Ca2+ [Ca2+m] accumulation was shown by fluorescent Rhod-2 probe, mitochondrial superoxide was measured after Mitosox stain.
Results
We showed [Ca2+m] accumulation at the time ER stress occurred, accompanied by mitochondrial superoxide elevation, followed by MMP loss and nuclear translocation of apoptosis-inducing factor (AIF). However, inhibition of AIF cleavage by calpain inhibitor PD150606 reduced the HQ17(3)-induced cell death slightly. Further, Ca2+ chelator Bapta-AM prevented [Ca2+m] overload and rescued cell death more effectively, indicating [Ca2+m] participated in other aspects in cell death. HQ17(3) treatment lead to decreased mitochondrial proteins MFN1/2 and OPA1, while Mitotracker green stain showed significant loss of mitochondrial mass preceded cell death, indicating damaged mitochondria succumbed to mitophagy, which was confirmed by the presence of COX IV and LC3B co-localization.
Conclusion
In Ph+-ALL SUP-B15 cells HQ17(3) induces ER stress by yet-defined mechanism, mobilizes Ca2+ to mitochondria, results in cleavage and release of AIF to mediate chromatin fragmentation, Ca2+-overload leads to oxidative stress in turn and perturbs mitochondria integrity. Damaged mitochondria induce extensive mitophagy and cell death ensues. Therefore, agents that help elicit similar intricate effector network associated with ER/mitochondria stress will have potential as adjuvants controlling the VHR-ALL cells refractory to conventional chemotherapies and TKI regime.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Calcium, Cytotoxicity, Mitochondria, Ph+ ALL