title: Proteasomes are a target of the anti-tumour drug vinblastine.
abstract: Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.
authors.affiliation: Department of Experimental Medicine and Oncology, Section of Biochemistry, University of Turin, Via Michelangelo 27/B, 10126 Turin, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Piccinini
authors.forename: O
authors.initials: O
authors.lastname: Tazartes
authors.forename: C
authors.initials: C
authors.lastname: Mezzatesta
authors.forename: E
authors.initials: E
authors.lastname: Ricotti
authors.forename: S
authors.initials: S
authors.lastname: Bedino
authors.forename: F
authors.initials: F
authors.lastname: Grosso
authors.forename: U
authors.initials: U
authors.lastname: Dianzani
authors.forename: P A
authors.initials: PA
authors.lastname: Tovo
authors.forename: M
authors.initials: M
authors.lastname: Mostert
authors.forename: A
authors.initials: A
authors.lastname: Musso
authors.forename: M T
authors.initials: MT
authors.lastname: Rinaudo
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018922:HL-60 Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D014747:Vinblastine / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D014747:Vinblastine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex