title: Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate.
abstract: Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p<0.05) in the HL-60 cell proteome after treatment with 2.5microM methotrexate for 72h. We found that three structural alpha4, alpha5, alpha7 proteasome subunits, a non-catalytic beta3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-kappaB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-kappaB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.
authors.affiliation: Department of Nephrology and Rheumatology, Center of Internal Medicine, Georg-August University Göttingen, D-37075, Göttingen, Germany.
authors.forename: Nitin Kumar
authors.initials: NK
authors.lastname: Agarwal
authors.forename: Gerhard Anton
authors.initials: GA
authors.lastname: Mueller
authors.forename: Claudia
authors.initials: C
authors.lastname: Mueller
authors.forename: Jan-Henrick
authors.initials: JH
authors.lastname: Streich
authors.forename: Abdul Rahman
authors.initials: AR
authors.lastname: Asif
authors.forename: Hassan
authors.initials: H
authors.lastname: Dihazi
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D008727:Methotrexate / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits; D040901:Proteomics; D054875:Ubiquitination / Q000187:drug effects; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: D000964:Antimetabolites, Antineoplastic; D016328:NF-kappa B; D009363:Neoplasm Proteins; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex; D008727:Methotrexate