title: Calmodulin Binding to Death Receptor 5-mediated Death-Inducing Signaling Complex in Breast Cancer Cells.
abstract: Activation of death receptor-5 (DR5) leads to the formation of death-inducing signaling complex (DISC) for apoptotic signaling. TRA-8, a DR5 specific agonistic antibody, has demonstrated significant cytotoxic activity in vitro and in vivo without inducing hepatotoxicity. Calmodulin (CaM) that is overexpressed in breast cancer plays a critical role in regulating DR5-mediated apoptosis. However, the mechanism of CaM in regulating DR5-mediated apoptotic signaling remains unknown. In this study, we characterized CaM binding to DR5-mediated DISC for apoptosis in TRA-8 sensitive breast cancer cell lines using co-immunoprecipitation, fluorescence microscopic imaging, caspase signaling analysis, and cell viability assay. Results show that upon DR5 activation, CaM was recruited into DR5-mediated DISC in a calcium dependent manner. CaM antagonist, trifluoperazine (TFP), inhibited CaM recruitment into the DISC and attenuated DISC formation. DR5 oligomerization is critical for DISC formation for apoptosis. TFP decreased TRA-8 activated DR5 oligomerization, which was consistent with TFP's effect on DR5-mediated DISC formation. TFP and Ca2+chelator, EGTA, impeded TRA-8-activated caspase-dependent apoptotic signaling, and TFP decreased TRA-8-induced cell cytotoxicity. These results demonstrated CaM binding to DR5-mediated DISC in a calcium dependent manner and may identify CaM as a key regulator of DR5-mediated DISC formation for apoptosis in breast cancer. J. Cell. Biochem. 118: 2285-2294, 2017. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Biomedical Engineering, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
authors.forename: Romone M
authors.initials: RM
authors.lastname: Fancy
authors.affiliation: Department of Radiology, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
authors.forename: Harrison
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Medicine, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Department of Radiology, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
authors.forename: Kurt R
authors.initials: KR
authors.lastname: Zinn
authors.affiliation: Department of Radiation Oncology, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Buchsbaum
authors.affiliation: Department of Biomedical Engineering, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Song
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D053418:Death Domain Receptor Signaling Adaptor Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D014268:Trifluoperazine; D004533:Egtazic Acid
keywords: APOPTOSIS; BREAST CANCER CELLS; CALMODULIN; DEATH RECEPTOR 5; DISC; DR5 OLIGOMERIZATION