title: Combined treatment with bortezomib plus bafilomycin A1 enhances the cytocidal effect and induces endoplasmic reticulum stress in U266 myeloma cells: crosstalk among proteasome, autophagy-lysosome and ER stress.
abstract: Bortezomib (BZ), a first line 26S proteasome inhibitor, induces a potent cytocidal effect with caspase-3 activation in multiple myeloma (MM) cell lines. Since IκBα is a substrate of the proteasome, the initial rationale for using BZ in MM has been to inhibit NF-κB. However, BZ rather activated NF-κB activity in U266 cells. BZ induces autophagy as well as endoplasmic reticulum (ER) stress in various cell lines tested. Inhibition of initial autophagosome formation by treatment with either 3-methyladenine or siRNA for LC3B in U266 cells and knockdown of the atg5 gene in a murine embryonic fibroblastic cell line all resulted in attenuation of BZ-induced cell death. In contrast, combined treatment with BZ and bafilomycin A1 (BAF), which is a specific inhibitor of vacuolar-ATPase and is used as an autophagy inhibitor at the late stage, resulted in synergistic cytotoxicity, compared with that by either BZ or BAF alone. BAF treatment also induced ER stress, but the kinetics of inductions of ER stress-related genes [e.g. CHOP (GADD153) and GRP78] completely differed between BZ- and BAF-treatments: BZ induced these ER stress markers within 8 h, whereas treatment with BAF required more than 48 h in U266 cells. In order to synchronize ER stress, we pre-treated U266 cells with BAF for 48 h, followed with BZ for 48 h. The sequential treatment with BAF and BZ induced a further enhanced cytotoxicity, compared with the simultaneous combination of BAF and BZ. These data suggest crosstalk among the ubiquitin-proteasome system, the autophagy-lysosome system, and ER stress. Controlling these interactions and kinetics appears to have important implications for optimizing clinical cancer treatment including MM-therapy.
authors.affiliation: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Miyazawa
authors.forename: Shota
authors.initials: S
authors.lastname: Moriya
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ohtomo
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Che
authors.forename: Munekazu
authors.initials: M
authors.lastname: Naito
authors.forename: Masahiro
authors.initials: M
authors.lastname: Itoh
authors.forename: Akio
authors.initials: A
authors.lastname: Tomoda
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D001897:Boronic Acids / Q000008:administration & dosage* / Q000494:pharmacology; D000069286:Bortezomib; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D004721:Endoplasmic Reticulum / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D018922:HL-60 Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D018942:Macrolides / Q000008:administration & dosage* / Q000494:pharmacology; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects* / Q000378:metabolism; D011719:Pyrazines / Q000008:administration & dosage* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013312:Stress, Physiological / Q000187:drug effects*; D020298:U937 Cells; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: D001897:Boronic Acids; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D018942:Macrolides; D011719:Pyrazines; D000069286:Bortezomib; C040929:bafilomycin A1; D046988:Proteasome Endopeptidase Complex