HEME OXYGENASE 1 (HO-1) PROTECTS MYELOMA CELLS AGAINST BORTEZOMIB THROUGH NUCLEAR TRANSLOCATION AND REGULATION OF ER STRESS AND AUTOPHAGY PROTEINS
(Abstract release date: 05/19/16)
EHA Library. Tibullo D. 06/09/16; 132797; E1248
Dr. Daniele Tibullo
Contributions
Contributions
Abstract
Abstract: E1248
Type: Eposter Presentation
Background
Despite recent advances in proteasome inhibitor and immunomodulatory drug-based therapies, MM remains largely incurable, primarily owing to acquired resistance. HO-1 is a cytoprotective microsomal enzyme that catalyzes the degradation of heme. We have recently shown that the protective effect of HO-1 on drug-induced cytotoxicity in leukemic cells does not involve its enzymatic byproducts, but rather its nuclear translocation following proteolytic cleavage. It has been recently described that Bortezomib (BTZ) is able to increase HO-1 expression.
Aims
We investigated about the role of BTZ-induced HO-1 in MM cell lines (U266, SKM-M1).
Methods
Cell Proliferation was performed by ATPlite one step assay (Perkinelmer) and using flow cytometry Annexin V/ iodure propidio. ROS formation was evaluated by flow cytometry. Endoplasmic reticulum (ER) associated proteins, autophagy-related proteins and HO-1 protein were evaluated by western blot assay. The fluorescent images were obtained using a Confocal Laser Scanning Microscopy (CLSM, Zeiss LSM700, Milan, Italy).
Results
As expected, we observed that Btz (15 nM) induced apoptosis after 24h (p<0.001). Flow cytometric analysis revealed increased levels of ROS after 1h (p<0.0001) of treatment with a peak after 3h (p<0.001). Btz was able to induce a significant increase in HO-1 mRNA levels after 3h (p<0.0001) of treatment with a maximum peak after 6h (p<0.0001). Since HO-1 is one of the major endoplasmic reticulum (ER) associated heme protein, we analyzed the ability of BTZ to induce ER stress. BTZ was able to induce the expression of ER stress proteins (Bip, IRE1α, Ero1, PERK and CHOP) in MM cells after 6h (p<0.001) with a peak after 24h (p<0.0001). Moreover, we observed that BTZ was able to induce autophagy-related genes such as ATFG5 and BENC1 in U266 cell lines. Silencing HO-1 using siRNA, we observed reduction of proteins described above indicating that induction of ER stress and then autophagy pathways by BTZ is HO-1 mediated.Furthermore, by confocal microscope we observed that HO-1 localized both in the cytoplasm and in the nucleus of MM cells. Interestingly, blockage of nuclear translocation by E64, a selective inhibitor of the protein cleavage, induced MM cells to become more sensitive to BTZ (p<0.001). No change in cell viability was observed inhibiting HO-1 enzymatic activity by using TIN (zync protoporphirin, 10 uM). Since nuclear HO-1 it has been reported to be a regulator of DNA repair activities, we also explored its role in genomic instability of MM cells. Using the cytochinesis-block micronucleus (CBMN) assay, we observed that pre-treatment of U266 with E64 for 24h led to a significant reduction of the percentage of micronuclei (p<0.01) and nucleolasmic bridges (p<0.05) observed in binucleated cells. Next, we evaluated U266 ability to activate G2/M checkpoint after UV damage using CBPI (cytochinesis block proliferation index) assay. The percentage of monucleated cells (G2/M checkpoint activated) was higher in cells pre-treated with E64 than control (p<0.05).
Conclusion
Our data suggest that BTZ-induction of HO-1 is probably linked to the activation ER stress and autophagy pathway by BTZ through HO-1 activation. HO-1 nuclear translocation may be involved in MM BTZ resistance. In addition, nuclear HO-1 may be involved in genomic instability of MM cells.
Session topic: E-poster
Keyword(s): Bortezomib, Drug resistance, Multiple myeloma, Resistance
Type: Eposter Presentation
Background
Despite recent advances in proteasome inhibitor and immunomodulatory drug-based therapies, MM remains largely incurable, primarily owing to acquired resistance. HO-1 is a cytoprotective microsomal enzyme that catalyzes the degradation of heme. We have recently shown that the protective effect of HO-1 on drug-induced cytotoxicity in leukemic cells does not involve its enzymatic byproducts, but rather its nuclear translocation following proteolytic cleavage. It has been recently described that Bortezomib (BTZ) is able to increase HO-1 expression.
Aims
We investigated about the role of BTZ-induced HO-1 in MM cell lines (U266, SKM-M1).
Methods
Cell Proliferation was performed by ATPlite one step assay (Perkinelmer) and using flow cytometry Annexin V/ iodure propidio. ROS formation was evaluated by flow cytometry. Endoplasmic reticulum (ER) associated proteins, autophagy-related proteins and HO-1 protein were evaluated by western blot assay. The fluorescent images were obtained using a Confocal Laser Scanning Microscopy (CLSM, Zeiss LSM700, Milan, Italy).
Results
As expected, we observed that Btz (15 nM) induced apoptosis after 24h (p<0.001). Flow cytometric analysis revealed increased levels of ROS after 1h (p<0.0001) of treatment with a peak after 3h (p<0.001). Btz was able to induce a significant increase in HO-1 mRNA levels after 3h (p<0.0001) of treatment with a maximum peak after 6h (p<0.0001). Since HO-1 is one of the major endoplasmic reticulum (ER) associated heme protein, we analyzed the ability of BTZ to induce ER stress. BTZ was able to induce the expression of ER stress proteins (Bip, IRE1α, Ero1, PERK and CHOP) in MM cells after 6h (p<0.001) with a peak after 24h (p<0.0001). Moreover, we observed that BTZ was able to induce autophagy-related genes such as ATFG5 and BENC1 in U266 cell lines. Silencing HO-1 using siRNA, we observed reduction of proteins described above indicating that induction of ER stress and then autophagy pathways by BTZ is HO-1 mediated.Furthermore, by confocal microscope we observed that HO-1 localized both in the cytoplasm and in the nucleus of MM cells. Interestingly, blockage of nuclear translocation by E64, a selective inhibitor of the protein cleavage, induced MM cells to become more sensitive to BTZ (p<0.001). No change in cell viability was observed inhibiting HO-1 enzymatic activity by using TIN (zync protoporphirin, 10 uM). Since nuclear HO-1 it has been reported to be a regulator of DNA repair activities, we also explored its role in genomic instability of MM cells. Using the cytochinesis-block micronucleus (CBMN) assay, we observed that pre-treatment of U266 with E64 for 24h led to a significant reduction of the percentage of micronuclei (p<0.01) and nucleolasmic bridges (p<0.05) observed in binucleated cells. Next, we evaluated U266 ability to activate G2/M checkpoint after UV damage using CBPI (cytochinesis block proliferation index) assay. The percentage of monucleated cells (G2/M checkpoint activated) was higher in cells pre-treated with E64 than control (p<0.05).
Conclusion
Our data suggest that BTZ-induction of HO-1 is probably linked to the activation ER stress and autophagy pathway by BTZ through HO-1 activation. HO-1 nuclear translocation may be involved in MM BTZ resistance. In addition, nuclear HO-1 may be involved in genomic instability of MM cells.
Session topic: E-poster
Keyword(s): Bortezomib, Drug resistance, Multiple myeloma, Resistance
Abstract: E1248
Type: Eposter Presentation
Background
Despite recent advances in proteasome inhibitor and immunomodulatory drug-based therapies, MM remains largely incurable, primarily owing to acquired resistance. HO-1 is a cytoprotective microsomal enzyme that catalyzes the degradation of heme. We have recently shown that the protective effect of HO-1 on drug-induced cytotoxicity in leukemic cells does not involve its enzymatic byproducts, but rather its nuclear translocation following proteolytic cleavage. It has been recently described that Bortezomib (BTZ) is able to increase HO-1 expression.
Aims
We investigated about the role of BTZ-induced HO-1 in MM cell lines (U266, SKM-M1).
Methods
Cell Proliferation was performed by ATPlite one step assay (Perkinelmer) and using flow cytometry Annexin V/ iodure propidio. ROS formation was evaluated by flow cytometry. Endoplasmic reticulum (ER) associated proteins, autophagy-related proteins and HO-1 protein were evaluated by western blot assay. The fluorescent images were obtained using a Confocal Laser Scanning Microscopy (CLSM, Zeiss LSM700, Milan, Italy).
Results
As expected, we observed that Btz (15 nM) induced apoptosis after 24h (p<0.001). Flow cytometric analysis revealed increased levels of ROS after 1h (p<0.0001) of treatment with a peak after 3h (p<0.001). Btz was able to induce a significant increase in HO-1 mRNA levels after 3h (p<0.0001) of treatment with a maximum peak after 6h (p<0.0001). Since HO-1 is one of the major endoplasmic reticulum (ER) associated heme protein, we analyzed the ability of BTZ to induce ER stress. BTZ was able to induce the expression of ER stress proteins (Bip, IRE1α, Ero1, PERK and CHOP) in MM cells after 6h (p<0.001) with a peak after 24h (p<0.0001). Moreover, we observed that BTZ was able to induce autophagy-related genes such as ATFG5 and BENC1 in U266 cell lines. Silencing HO-1 using siRNA, we observed reduction of proteins described above indicating that induction of ER stress and then autophagy pathways by BTZ is HO-1 mediated.Furthermore, by confocal microscope we observed that HO-1 localized both in the cytoplasm and in the nucleus of MM cells. Interestingly, blockage of nuclear translocation by E64, a selective inhibitor of the protein cleavage, induced MM cells to become more sensitive to BTZ (p<0.001). No change in cell viability was observed inhibiting HO-1 enzymatic activity by using TIN (zync protoporphirin, 10 uM). Since nuclear HO-1 it has been reported to be a regulator of DNA repair activities, we also explored its role in genomic instability of MM cells. Using the cytochinesis-block micronucleus (CBMN) assay, we observed that pre-treatment of U266 with E64 for 24h led to a significant reduction of the percentage of micronuclei (p<0.01) and nucleolasmic bridges (p<0.05) observed in binucleated cells. Next, we evaluated U266 ability to activate G2/M checkpoint after UV damage using CBPI (cytochinesis block proliferation index) assay. The percentage of monucleated cells (G2/M checkpoint activated) was higher in cells pre-treated with E64 than control (p<0.05).
Conclusion
Our data suggest that BTZ-induction of HO-1 is probably linked to the activation ER stress and autophagy pathway by BTZ through HO-1 activation. HO-1 nuclear translocation may be involved in MM BTZ resistance. In addition, nuclear HO-1 may be involved in genomic instability of MM cells.
Session topic: E-poster
Keyword(s): Bortezomib, Drug resistance, Multiple myeloma, Resistance
Type: Eposter Presentation
Background
Despite recent advances in proteasome inhibitor and immunomodulatory drug-based therapies, MM remains largely incurable, primarily owing to acquired resistance. HO-1 is a cytoprotective microsomal enzyme that catalyzes the degradation of heme. We have recently shown that the protective effect of HO-1 on drug-induced cytotoxicity in leukemic cells does not involve its enzymatic byproducts, but rather its nuclear translocation following proteolytic cleavage. It has been recently described that Bortezomib (BTZ) is able to increase HO-1 expression.
Aims
We investigated about the role of BTZ-induced HO-1 in MM cell lines (U266, SKM-M1).
Methods
Cell Proliferation was performed by ATPlite one step assay (Perkinelmer) and using flow cytometry Annexin V/ iodure propidio. ROS formation was evaluated by flow cytometry. Endoplasmic reticulum (ER) associated proteins, autophagy-related proteins and HO-1 protein were evaluated by western blot assay. The fluorescent images were obtained using a Confocal Laser Scanning Microscopy (CLSM, Zeiss LSM700, Milan, Italy).
Results
As expected, we observed that Btz (15 nM) induced apoptosis after 24h (p<0.001). Flow cytometric analysis revealed increased levels of ROS after 1h (p<0.0001) of treatment with a peak after 3h (p<0.001). Btz was able to induce a significant increase in HO-1 mRNA levels after 3h (p<0.0001) of treatment with a maximum peak after 6h (p<0.0001). Since HO-1 is one of the major endoplasmic reticulum (ER) associated heme protein, we analyzed the ability of BTZ to induce ER stress. BTZ was able to induce the expression of ER stress proteins (Bip, IRE1α, Ero1, PERK and CHOP) in MM cells after 6h (p<0.001) with a peak after 24h (p<0.0001). Moreover, we observed that BTZ was able to induce autophagy-related genes such as ATFG5 and BENC1 in U266 cell lines. Silencing HO-1 using siRNA, we observed reduction of proteins described above indicating that induction of ER stress and then autophagy pathways by BTZ is HO-1 mediated.Furthermore, by confocal microscope we observed that HO-1 localized both in the cytoplasm and in the nucleus of MM cells. Interestingly, blockage of nuclear translocation by E64, a selective inhibitor of the protein cleavage, induced MM cells to become more sensitive to BTZ (p<0.001). No change in cell viability was observed inhibiting HO-1 enzymatic activity by using TIN (zync protoporphirin, 10 uM). Since nuclear HO-1 it has been reported to be a regulator of DNA repair activities, we also explored its role in genomic instability of MM cells. Using the cytochinesis-block micronucleus (CBMN) assay, we observed that pre-treatment of U266 with E64 for 24h led to a significant reduction of the percentage of micronuclei (p<0.01) and nucleolasmic bridges (p<0.05) observed in binucleated cells. Next, we evaluated U266 ability to activate G2/M checkpoint after UV damage using CBPI (cytochinesis block proliferation index) assay. The percentage of monucleated cells (G2/M checkpoint activated) was higher in cells pre-treated with E64 than control (p<0.05).
Conclusion
Our data suggest that BTZ-induction of HO-1 is probably linked to the activation ER stress and autophagy pathway by BTZ through HO-1 activation. HO-1 nuclear translocation may be involved in MM BTZ resistance. In addition, nuclear HO-1 may be involved in genomic instability of MM cells.
Session topic: E-poster
Keyword(s): Bortezomib, Drug resistance, Multiple myeloma, Resistance
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